SLEEP-WAKING STATES AND mE ENDOGENOUS OPIOID SYSTEM
(HET SLAAP-WAAK PATROON EN HET ENDOGENE OPIOIDEN SYSTEEM)
Proefschrift
TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE
GENEESKUNDE
AAN DE ERASMUS UNIVERSITEIT ROTTERDAM
OP GEZAG VAN RECTOR MAGNIFICUS
PROF. DR M.W. VAN HOF
EN VOLGENS BESLUIT VAN HET COLLEGE VAN DEKANEN.
DE OPENBARE VERDEDIGING ZAL PLAATSVINDEN OP
WOENSDAG 28 MEl 1986 OM 14.00 UUR
DOOR
OTASOWIE EGHE UKPONMWAN
GEBOREN TE BENIN CITY
PROMOTIECOMMISSIE
PROMOTOR:
PROF.
DR.
I.L.
BONTA
CO-PROMOTOR: DR M.R.
DZOLJIC
OVERIGE LEDEN: PROF.
DR.
E.L.
PROF.
DR.
Ch.D.
PROF.
DR.
P.R.
NOACH
KAPLAN
SAXENA
CONTENTS
page
Scope of this thesis
Part I:
1
General Introduction
Chapter 1 .. Neurobiology of sleep-waking states
3
Chapter 2 •. Endogenous opioid system
46
Part II:
Mutual interactions between normal sleep and endogenous
opioid system
Chapter 3 .. Endogenous opioid peptides and sleep
75
Chapter 4 .. Stages of vigilance and enkephalin-induced seizure
83
Part III:
Mutual interactions between disturbed sleep and opiates/
opioid peptides
Chapter S .. REM sleep deprivation decreases the antinociceptive
88
property of enkephalinase inhibition, morphine and
cold-water-swim
Chapter 6 .. An analgesic effect of enkephalinase inhibition is
98
modulated by monoamine oxidase B and REM sleep
deprivation
Chapter ? .. REM sleep deprivation antagonized morphine-
108
induced akinesia and catalepsy
Chapter B •. REM sleep deprivation decreases the grooming and
121
shaking behaviour induced by enkephalinase inhibitor
or opiate withdrawal
Chapter 9 .. Enkephalinase inhibition antagonizes the increased
135
susceptibility to seizure induced by REM sleep
deprivation
Chapter 10.REM sleep deprivation inhibits nitrous oxide withdrawal
744
Concluding Remarks and Summary
150
Samenvatting
155
List of Publications
161
Curriculum Vitae
763
Acknowledgement
164
SCOPE OF THIS THESIS
In the general introductory part of this
review
thesis
(Chapters
and
2)
of some pertinent literature related to sleep-waking states and opioid
peptides is offered.
theories
of
A global
functions
of
view
sleep,
of
the
neurochemical
mechanisms
and
as well as the physiological and possible
clinical consequences of total or selective REM sleep deprivation is given
Chapter
a
1.
In
Chapter
2,
which
in
is concerned with the role of endogenous
opioid peptides, particular attention is paid to the possible modulatory
role
of endogenously released opioid peptides in the regulation of some behavioural
states in physiological and pathological conditions.
It is generally known that exogenously
peptides
can
alter
interaction between
sleep
pattern
sleep-waking
and
administered
decrease
states
and
opiates
and
sleep.
A
REM
endogenous
opioid
opioid
possible
system
is
suggested by the report that the episodic release of plasma humoral endorphins
during sleep is associated with
1.1 .7).
In
addition,
the
the
REM
sleep
concentrations
phase
(Chapter
1,
of opioid peptides in some brain
nuclei of the rat which are known to be important in sleep-waking
are
highest
(Chapter
section
endogenously released
pattern,
we
opioid
studied
1 .3.2).
peptides
the
effects
propensity
that
to
sleep
is
at
in
of
the
regulation
phosphoramidon,
of
an
sleep-waking
inhibitor
Several clinical
epileptic
the sleep-waking cycle may modulate the occurence of some types
some
studies
phenomena
Therefore,
of
studies
of epileptic phenomena in human subjects (Chapter 1, section 1 .5.2, iii).
addition,
its
Thus in order to clarify the effect of
enkephalinase A on sleep-waking states (Chapter 3).
suggest
regulation,
in the dark (active) phase, during which wakefulness is high and
lowest in the light (rest) phase, when the
highest
section
we
and
studied
indicate
petit
the
a
mal
similarity
epilepsy
effects
of
enkephalin-induced epileptic phenomena using
between
(Chapter
different
In
enkephalin induced
4
discussion).
sleep
electrophysiological
stages
on
parameters
(Chapter 4).
These initial studies (Chapters 3 and 4) suggested an interaction
sleep-waking
states
and
endogenous opioid system.
sleep deprivation (REMSD) reduced the pain
stimulation
(Chapter
threshold
between
The observation that REM
to
noxious
electrical
1 section 1 .5.2d i) was an indication of the importance
2
of REM sleep in the regulation of nociception.
the
experiments
were
designed
to
explore
Therefore in Chapters 5 and 6,
a direct effect of REMSD on the
analgesic effects of morphine, an enkephalinase inhibitor
phosphoramdion
and
cold-water-swim.
The profound antagonistic effect of REMSD on opiate/opioid peptide induced
analgesia
REMSD
stimulated further interest to investigate the relationship between
and
other
Therefore
the
opiate/opioid
following
akinetic-cataleptic
convulsions,
(Chapters 7-9).
(Chapter
10)
opioid
syndrome,
grooming,
peptide
modulated
modulated
spontaneous
wet-dog-shakes
Additional experiments
and
with
behavioural
behaviours
vertical
morphine
nitrous
were
phenomena.
investigated:
motor
withdrawal
oxide
were
activity,
symptoms
performed
since it is known that this anaesthetic agent can stimulate the
release of endogenous enkephalins and induce opiate-like
withdrawal
symptoms
(Chapter 2, section 2.5.2, iv).
Finally, the possible clinical consequences of our findings are
in the relevant chapters.
described
3
PART I: GENERAL INTRODUCTION
CHAPTER 1
NEUROBIOLOGY OF SLEEP-WAKING STATES
Sleep is a heterogenous process
cycles
of
organised
into
rhythmically
occurring
different stages which are characterised by specific behavioural,
electrophysiological, autonomic and endocrine
changes.
Broadly,
sleep
is
divided into non-rapid-eye-movement (NREM) sleep and rapid-eye-movement (REM)
sleep, also called paradoxical sleep (PS)
REM
sleep
occupies
a
(Aserinsky
and
Kleitman,
1953).
large part of neo-natal mammalian sleep (Roffwarg et
al., 1966) suggesting an involvement of REM sleep in brain maturation (Corner
et al.
1980).
In adult mammals REM sleep appears to regulate adaptivity and
plasticity of waking behaviour (McGrath and Cohen, 1978).
1.1
Sleep-wakefulness phenomenology
Sleep and wakefulness, like many life
bird
migration
and
hibernation,
processes
from
to
follow a certain pattern of oscillations,
which are regulated by a strict space-time system as well as
In
heart-beating
body
position.
nocturnal rodents such as rats, sleep occupies 80% of the light phase and
20 %the dark period.
time
in
adult
rats,
REM sleep, which occupies 15-20% of
shows
a
slight
the
total
sleep
prepondrance during the light phase
(Borbely, 1982).
1.1.1
Behaviour
Pre-sleep behavioural repertory is characterised by the searching
safe
niche
for
a
and preparing the body (grooming) for sleep (Parmeggianni, 1980;
Cooper, 1979).
Behaviourally, sleep onset is characterised by the suspension
of active contact with the environment (Parmeggianni, 1980).
A close relationship between electrographic recordings and behaviour
been
observed
in
most
animals so far studied.
has
However there is a growing
4
body of evidence that behaviour and the usual electrophysiological correlates
may become dissociated (Sakai, 1985).
1 .1.2
Motor functions
Sleep-wakefulness
activity.
In
cycle
is
accompanied
by
feature of sleep.
electromyogram
During
loss
of
the
NREM
antigravity
postural support (Ursin, 1968;
phasic
changes
in
motor
With the onset of sleep there is a progressive decrease in
(EMG).
sleep
muscle
attenuated form, whereas when the animal enters
complete
phasic
general, quiescience of motor and postural support is a basic
motoric
phenomena
tonus
into
REM
is present in an
sleep,
there
is
muscle tonus with consequent failure of
Timo-Iaria et al.,
associated
with
REM
Other
important
include,
myoclonic
1970).
sleep
twitching and burst of rapid eye movements.
1.1.3
Neuronal activity
The pattern and intensity of neuronal activity in
often
related
to
the
state
of
vigilance.
some
Bioelectric
is
of the
geniculate
other
studied extensively during sleep-wakefulness
areas
have
been
reticular
areas
rhythms
neocortex, hippocampus, lateral
brain
body,
brain
formation
and
cycle (Steriade et al., 1977).
i) Neocortex
The neocortical EEG patterns of the rat during different vigilance states
has
been
1978).
well
documented (Timo-Iaria et al.
1970;
Terrier and Gottesman,
Based on these reports the EEG of sleep-wakefulness cycle of the
rat
can be differentiated into four distinct stages as follows:a) Wakefulness in rat is characterised by desynchronised (fast) waves (30 -40 Hz
and 30
~v
amplitude).
b) Light slow wave sleep (LSWS) in the rat consists of spindles (6-12 Hz
20-40
msec. 50-300
~V),
range,
K-complexes and some delta waves in frontal-parietal
cortex EEG.
c) Delta sleep:
This stage of NREM sleep consists of predominantly delta
waves
(>50%).
d) REM sleep in the rat is characterised by desynchronised (fast) waves (20
40
~V)
Hz,
in the frontal-parietal cortex.
In the cat
sleep-wakefulness
is
differentiated
into
five
stages
as
5
follows:
active
and
quiet
wakefulness,
light slow sleep, deep slow wave
sleep and REM sleep (Ursin, 1968).
ii) Hippocampus and entorhinal cortex
The electrical activity of the
undergo
distinct
oscillations
hippocampus
REM
the
During
states in the sleep-wakefulness continuum.
and
and
cortex
exploratory
wakefulness
sleep, the dorsal hippocampal EEG show a regular theta wave pattern
(4-6Hz) in both rats and cats (Jouvet et al., 1959;
In
entorhinal
which correlate well with specific vigilance
Monmaur et al.,
1979).
NREM sleep the hippocampus and the entorhinal cortex present an irregular
EEG pattern consisting of high amplitude sharp waves intermingled
waves
(Jouvet
et
al .•
1959).
In
the
ventral
(100-300 #V, 50-100 msec duration) are most frequent during
are
rare
during
with
slow
hippocampus spikes waves
NREM
sleep
wakefulness and REM sleep (Jouvet et al., 1959;
and
Hartse et
al.. 1979).
bo~
iii) Reticular formation and lateral geniculate
Phasic EEG spike activity during REM
occur
in
the
cat
sleep
was
cortex
demonstrated
by
Mouret
and
(1961)
co-workers (1963).
and
they
were
recorded.
(Stern et al., 1974).
subjects
PGO
waves
in
occipital
These spikes have been
designated ponto-geniculo-occipital {PGO) waves according to
which
to
pontine reticular formation by Jouvet and Michel (1959).
lateral geniculate body by Mikiten and co-workers
(visual)
first
the
loci
from
have not been demonstrated in rats
They have not been unambiguously demonstrated in human
(Gaillard,
The phasic PGO spike waves occur a few minutes
1980).
prior and during REM episode and are generally absent during wakefulness
and
NREM sleep (Hartse et al., 1979).
1.1.4
Respiration
Cyclic
changes
sleep-wakefulness
in
cycle
respiration
(Parmeggiani,
have
McGinty
During NREM sleep breathing is generally slower,
than
in
wakefulness.
In
both
and
shallow
and
during
Beahm,
more
the
1 984).
regular
Only small changes in ventilatory response to carbon
dioxide and mechanoceptive reflex
wakefulness.
observed
been
1980;
occur
experimental
during
respiratory rate is irregular, highly variable
brief apneas during REM sleep.
NREM
sleep
compared
and
human
subjects,
animals
and
may
be
with
accompanied
the
by
Arousal thresholds in response to hypoxia and
6
hypercapnia are increased during REM sleep
compared
vith
NREM
sleep.
In
addition, response to laryngeal stimulation during REM sleep could consist of
apnea instead of the usual coughing.
1 .1.5
Cardiovascular system
The heart rate and arterial blood pressure are decreased in both man
animals
during
NREM
The largest drop in heart rate and systemic arterial blood
was
observed
and
sleep compared with wakefulness (Snyder et al., 1964).
pressure
during REM sleep (Guazzi and Zanchetti, 1965).
in
cat
However in man
heart rate and blood pressure are increased during REM sleep with respect
to
NREM sleep (Snyder et al., 1964).
1.1.6
Thermoregulation
An ultradian rhythm of
homeostasis-poikilostasis
which
correlate
specific vigilance state has been documented (Parmeggiani, 1980;
Beahm, 1984).
lower
During NREM sleep, the core
temperature
set point compared with wakefulness.
is probably
processes
due
such
to
as
a
combination
reduction
in
of
the
is
thermoregulatory
motor action.
and
behavioural
symptoms
vasodilation, polypnea, sweating and
ambient
or
hypothalamic
temperature
regulated
animals
a
and
passive
In contrast to NREM sleep,
such
thermogenesis
during
as
REM
shivering,
in
response
sleep.
thermal
to
either
changes disappeared during REM sleep.
The lack of shivering during REM sleep may not be related to
since
at
The decrease in body temperature
hypothalamic thermoregulatory mechanisms are inactivated
Physiological
with
McGinty and
muscle
atonia,
which exhibited REM without atonia after pontine lesion still
failed to show shivering (McGinty and Beahm, 1984).
1.1.7
Endocrine and neuropeptide secretions
Sleep-related growth hormone secretion occur during the NREM sleep
(Takahashi
et al., 1968;
Mitsugi and Kimura, 1985).
of prolactin is triggered by sleep onset (Parker et
secretion
of
cortisol
phase
The episodic secretion
al.,
1980),
while
the
and thyroid-stimulating-hormone are decreased during
sleep compared with wakefulness (Parker et al., 1980;
Mitsugi
and
Kimura,
Recently it was demonstrated that the episodic release of plasma
humoral
1985).
7
endorphin
is associated with REM sleep (Oksenberg et al., 1980).
together suggest that
sleep
processes
might
play
an
These data
important
role
in
regulating the functional reactivity of hormonal and neuropeptide systems.
1.1.8
Genital response
Nocturnal penile tumescence (NPT) during sleep has been
during
REM
sleep
in man (Karacan et al., 1966).
shown
pressure
are
Rogers et al., 1985 ).
The
highly predictable occurence of NPT during REM sleep (80-90%) has
to
distinguish
organic
from psychogenic impotence.
impotency associated with diabetes mellitus the
greatly
diminshed,
whereas
in
occur
The female counterpart of
nocturnal NPT, clitoral erection and increase in vaginal pulse
associated with REM sleep (Karacan et al., 1970;
to
been
used
For example in organic
REM
sleep
related
NPT
is
psychogenic impotency there is little or no
reduction in NPT (Karacan et al., 1978).
1.1.9
Circadian sleep-wakefulness phenomena
Biological cycles with a period of approximately one lunar
and
that
persist
in
the
absence
of
external
day
h),
(24
environmental
cues
(-zeitgebers-). have been termed circadian oscillations (Aschoff, 1964).
animals
human
and
isolated
subjects
from
sleep-wakefulness cycle continues to show
a
period
from
only
is
different
slightly
external
clearly
24
time
defined
h
(Groos,
In
cues,
rhythm
the
whose
1984).
The
sleep-wakefulness cycle is therefore a true circadian rhythm which
has
demonstrated
Circadian
in
rats
(Borbely,
1982) and in man (Wever, 19?9).
been
influences on sleep-wakefulness in rats affect phase timing and not the daily
amount
of
sleep.
Lesior.
of
the
suprachiasmatic
nucleus
sleep-wakefulness circadiar. rhythm but not the daily amount of
and
Kawamura,
demonstrated
1975).
to
be
abolished the
sleep
(Ibuka
Recently the hypothalamic paraventricular nucleus was
involved
in
the
maintaince
of
REM
sleep
rhythm
(Piepenbrock et al., 1985).
1 .2
Neural basis oF sleep-wakefulness
The study of the pathology of viral encephalitis and the associated sleep
disorders
led
von
Economo
(1929)
to
differentiate
two
syndromes,
a)
8
hypersomnia related to lesions
hypothalamus
and
b)
of
mesencephalic
sleeplessness
in
which
forebrain and related striate structures.
tegementum
the
These
lesion
and
posterior
affected
observations
led
basal
to
the
notion of -neural determinism of sleep-waking states-.
1 .2.1
Neural substrates of wakefulness
The demonstration of ocular and EEG signs of sleep after the
of
the
brain
stem
the level of the occulomotor nuclei -cerveau isole-
at
(Bremer, 1935), suggested the presence of an arousal center in
third
of the pons.
the
Magoun
(1949)
anterior
Further lesion studies in the rat by Nauta (1946) placed
the -waking center- at the mesencephalo-hypothalamic junction.
and
transection
described
an
arousal
system
Later Moruzzi
located in the reticular
formation of the rostral segments of the pons and the midbrain.
Neural elements rostral to the brain stem reticular formation contributed
to wakefulness mechanism.
One such brain area was localised in the posterior
hypothalamus (Moruzzi, 1964).
that
in
cerveau
This observation is supported by
the
finding
isole- preparations in which the locus coerulus and raphe
nuclei were completely isolated from the forebrair. of the animals, an initial
period
of
hypersomnia
was
followed
by
behavioural
and
EEG patterns of
wakefulness (Hanada and Kawamura, 1981 ).
1.2.2
Neural mechanism of non-rapid-eye-movement (NREM} sleep
The initial report of Hess (1927), that electrical stimulation of midline
thalamic
nuclei
induce
behavioural
and
experimental indication of the existence
Other
workers
EEG
of
an
signs of sleep was the first
active
1961 ) .
center.
have since demonstrated that stimulation of other brain areas
can induce behavioural and electrophysiological signs
al.,
hypnogenic
Three
hypnogenic
brain
areas
may
of
be
sleep
(Favale
et
differentiated
ponte-bulbar raphe system, hypothalamic and thalamo-cortical.
i) Ponte-bulbar raphe system
The existence of medullary sleep centers was suggested
medullary
anesthesia,
or
cooling,
converted
awake, pattern (Berlucchi et al., 1964).
reports
that
sleep EEG into an activated,
EEG synchronisation and behavioural
sleep were elicited by low frequency stimulation of then.
(Favale et al., 1961 ).
by
tractus solitarus
9
The lesion
by
partial
raphe complex induced, initially, total
o~
sleep
recovery
in
cats (Jouvet, 1974).
failed to implicate raphe nuclei in sleep regulation.
raphe
followed
However other workers
For
example
midbrain
lesions in rats failed to alter sleep-wakefulness (Bouhuys and van den
Further~ore
Hoofdakker, 1977).
in
insomnia
the
dorsal
raphe
resulted
that
neurons
fire less during slow wave sleep compared with waking
(McGinty and Harper, 1976) and
nucleus
unit discharge studies, showed
in
that
electrical
stimulation
arousal (Jacobs et al., 1973).
the raphe nuclei in the
regulation
of
sleep
and
of
the
raphe
Thus the exact role of
awake
vigilance
states
remains to be clarified.
ii) Hypothalamic system
The sleep modulating effect of the hypothalamic area was first
by
the
study
preoptic area.
of
suggested
Hess (1944) in which he elicited sleep by stimulating the
Profound insomnia was demonstrated in rats after
inducing
a
lesion in the preoptic area (Nauta, 1946).
iii) Thalamo-cortical NREM sleep system
Medial thalamic stimulation induce behavioural and
(Akert
et
al.,
1952).
However,
has
also
been
elicited
signs
of
sleep
the complete destruction of the thalamus
eliminated only sleep spindles but not slow
Sleep
EEG
by
waves
(Naquet
stimulation
et
al.,
1965).
of the basal forebrain and
orbital cortex (Sterman and Clemente, 1962)
1.2-3
Neural mechanisms of REM sleep
The pontine brain structures are
However
a
large
body
of
essential
sleep, such as ponto-geniculo-occipital (PGO)
neocortical
desynchronisation
(Sakai, 1985).
reticularis
for
REM
sleep
generation.
evidence indicate that different features of REM
are
spikes,
The pontine-medullary brain nuclei,
magnocellularis
are
postural
atonia
and
regulated by different neural substrates
essential
for
locus
coerulus
and
n.
postural atonia during REM
sleep (Sakai, 1985).
PGO generators are
pontine
tegmental
located
area
in
such
the
as
caudal
brachium
mesencephalic
conjunctivum,
and
rostral
rostral
n.
parabrachialis lateralis, locus coeruleus and n.
laterodorsalis, whereas EEG
activation
reticularis magnocellularis
during
(Sakai, 1985).
REM
sleep
involves
the n.
10
1.3
Neurochemical basis oT sleep-wakefulness
endogenous
Several
(noradrenaline,
substances,
dopamine,
such
as
the
biogenic
amines
serotonin and $-phenylethylamine), acetylcholine,
gamma-amino-butyric acid and
many
neuropeptides
(enkephalins,
endorphins,
sleep peptide) have been proposed as regulators o~ different vigilance
delta
states.
1.3.1
Biogenic amines. acetylcholine and GABA
i) Noradrenaline (NA)
Inhibition of tyrosine hydroxylase with a-methyl-p-tyrosine (dMT) or
blockade
of
dopamine-~-hydroxylase
with
disulfiram
reduced waking and decreased the concentrations of NA in the
1974).
brain
(Jouvet,
These data suggested an involvement of NA in the regulation of waking
and REM sleep.
al.,
the
suppressed REM sleep,
1971 ).
In contrast
~-MT
enhanced REM sleep in the rat
(Hartmann
et
However, Kafi and co-workers (1977) have demonstrated that only
high doses of
~-MT,
which induced an almost total inhibition of NA synthesis,
suppressed REM sleep while lower doses facilitated this sleep stage.
The possible involvement of NA in regulating the sleep-waking
also
suggested
by
states
is
the finding that the electrolytic or surgical lesions of
noradrenergic neurons of the locus coerulus which decreased brain
suppressed REM sleep and reduced wakefulness (Jouvet, 1974).
presence of REM sleep was demonstrated inspite low
brain
NA
levels
In contrast the
NA
concentrations
(Jones et al., 1977).
In
summary,
transmission
in
inspite
NA
of
neurons
some
is
contradictory
involved
the
data
it
that
appears
modulation of REM sleep and
wakefulness.
ii) Dopamine (DA)
Destruction of the
tegmentum
decreased
ascending
DA-fibers
behavioural
of
the
ventral
mesencephalic
not
did
wakefulness
alter
electroencephalographic signs of arousal (Jones et al., 1973).
In rats the administration of spiroperidol,
a
DA
receptor
antagonist,
produced a dose-dependent increase in total sleep and a decrease of REM sleep
(Kafi
and
Gaillard,
1976).
Apomorphine
(Apo)
in
doses
sufficient
to
11
stimulate
post-synaptic
DA
receptors
decreased
total sleep and increased
waking, whereas low doses of Ape which stimulate DA
REM
sleep
time
(Kafi
and
Gaillard,
enhanced REM sleep (Hartmann
involvement
of
et
al.,
1976).
1975).
that
increased
Similarly small doses of DA
In
addition,
the
possible
various DA receptors in the modulation of alertness has also
been suggested (Dzoljic and Godschalk,
suggest
autoreceptors
the
activation
of
D-1
1978).
However.
more
recent
data
receptors induced arousal, while the
stimulation of D-2 receptors was associated with sedation and sleep (Gessa et
al.. 1985).
It
thus
appears
that
the
dopaminergic
system
is
involved
in
the
regulation of behavioural wakefulness and REM sleep.
iii) 5-Hydroxytryptamine (5-HT, serotonin)
Destruction of the raphe system, which contain the
neurons
induced
insomnia
which
levels in cats (Jouvet, 1974).
neurons
in
the
rostral
was
associated
cell-bodies
of
the
raphe
5-HT
with decreased brain 5-HT
This finding indicated that
part
of
5-HT
containing
system are involved in sleep
mechanisms.
The involvement of serotonin in NREM sleep induction was
by
also
supported
the finding that para-chlorophenylalanine (PCPA) which depleted brain 5HT
decreased both NREM and REM sleep stages in
Borbely,
1982).
The
sleep
cats
and
rats
(Jouvet,
1974,
suppressant effect of PCPA was reversed by the
administration of 5-HT precursors, tryptophan and 5-hydroxytryptophan.
The serotonin theory of sleep has been
During
chronic
studies,
sleep
challenged
by
several
reports.
in raphe-lesion animals tended to return to
pre-lesion baseline even though 5-HT levels remained low (Morgane and
1974).
alter sleep (Bouhuys and van den Hoofdakker,
chronic
Stern,
Brain lesions in rats which reduced brain 5-HT concentrations did not
tryptophan-deficient
1977).
Similarly
PCPA
or
a
diet. both of which can reduce brain 5-HT, had
no effects in rats (Rechtschaffen et al., 1973;
Clancy et
al.,
1978).
In
addition, serotonin neurons were most active during wakefulness compared with
any sleep stage (McGinty and Harper, 1976).
Similarly, the release
of
5-HT
was highest during wakefulness than when the animals were asleep (Puizzillout
et al .• 1979).
the
dorsal
al., 1973).
In addition, electrical stimulation of 5-HT rich
raphe
neurons
of
nucleus reduced both NREM and REM sleep stages (Jacobs et
Although these data are inconsistent with the general
postulate
12
that
5-HT
is a hypnogenic neurotransmitter, 5-HT neurons may play some role
in the PGO spikes -gating mechanisms- For example a decreased brain
associated
with
the
release
of
5HT
vas
PGO spikes into the NREM and awake states
(Jouvet, 1974).
Although the role of 5-HT in the regulation of vigilance
clear
is
not
has been recently proposed that 5-HT, released as neurotransmitter
it
during waking, might also act as a neurohormone
and/or
states
the
liberation
in
of hypnogenic factor(s).
inducing
the
synthesis
These would be stored, and
later influence SWS and PS (Jouvet, 1984).
iv) p-phenylethylamine (PEA}
P-Phenylethylamine is
mammalian
brain.
in
the
It is the substrate for monoamine oxidase B (MAO-B).
an
endogenous _occurring
The
structural similarity between PEA and amphetamine
has
led
to
the
suggestion
(Sandler and Reynolds, 1976).
brain
results
in
that
polygraphic
(a-methylphenylethylamine)
Increasing the concentrations of
desynchronised
studies
present
PEA may act as an endogenous amphetamine
EEG
pattern
several mammalian species (Sabelli et al.,
Sleep
amine
also
and
1975;
demonstrated
PEA
in
the
behavioural arousal in
Dzoljic
et
al.,
1977).
that both NREM and REM sleep
stages were suppressed by inhibitors of MAO-B (Cohen et al., 1982).
PEA
may
therefore be considered as one of the neuromodulators of wakefulness.
v) Acetylcholine {Ach)
Several experiments in animals and human subjects
have
demonstrated
an
involvement of acetylcholine (Ach) in the induction of REM sleep.
Both blockade of Ach receptors and
inhibition
of
Ach
synthesis
using
atropine, scopolamine or hemicholinium is generally accompanied by a decrease
in REM sleep.
(Domino et al., 1968;
Domino and Stawisk, 1970)
and
reduced
the
muscarinic
agonist
and
wakefulness at high
the frequency of PGO spikes {Henriksen et al., 1972).
The cholinesterase inhibitor, physostigmine and
arecoline,
facilitated
REM
sleep
at
low
concentrations (Sitaram and Gillin, 1980).
doses
In cats, physostigmine
prolonged
REM sleep periods with increase in rapid eye movements, PGO spikes and atonia
(Domino et al., 1968).
vi) Gamma-aminobutyric acid (GABA)
GABA and it's metabolite gamma-hydroxybutyrate (GHB), appear to possess a
sleep
enhancing
effect
in
several
mammalian species.
GHB stimulated REM
13
sleep-like state in cats (Stock, 1982).
supported
the
by
al., 1975).
finding
A hypnogenic role for GABA, is
also
that GHB induced EEG synchronisation (Dzoljic et
Recently L-cycloserine, a substance
GABA increased NREM and REM sleep stages.
which
can
increase
brain
However REM sleep was decreased by
high doses of this substance (Scherschlicht, 1985).
1.3.2
Neuropeptides and hormones
i) Endogenous opioid peptides
Endogenous opioid peptides appear
to
play
an
important
role
regulation of sleep-wakefulness cycle in both man and animals.
yet.
Many
the
However, many
aspects of the relationship between opioid peptides and sleep have
elucidated
in
not
been
suggestions related to sleep and opioid peptides are
derived from stimulation
~~d/or
blockade of opioid receptors
with
drugs
as
morphine and naloxone.
Morphine is an opium alkaloid named by Serturner in 1803
the
Roman
god
of
dreams.
name is not appropriate.
produced
a
The intravenous
dose-dependent
increase
in
while delta sleep, REM sleep (dreaming
decreased
in
human
after
Morpheus
Electrophysiological studies indicate that this
subjects
period)
et
(Kay
(iv)
administration
of
morphine
wakefulness, and muscular tension,
al.,
and
sleep
1969 ).
efficiency
(1970), demonstrated that heroin also reduced REM sleep, delayed sleep
wakefulness.
increased
Thus,
stimulation
were
Lewis and co-workers
of
opioid
onset
receptors
consistently induced insomnia in human subjects.
Studies in animals are also supportive of a stimulatory role
peptides.
Diurnal
variations
in enkephalins,
~-endorphin
for
opioid
and dynorphin in
brain areas important for the regulation of sleep-wakefulness correlate
the
basic
light
(rest)-dark
methionine-enkephalin
hypothalamus,
preoptic
(active)
concentrations
area
and
cycle in rodents.
in
the
anterior,
medial
1984).
~-Endorphin
phase
(Kerdelhue
et
Tang et
also reached a peak concentration in the preoptic
area, pons, medulla oblongata, cerebellum and anterior pituitary
dark
basal
stratium occur during the dark period and
decline to lowest levels during the light phase (Kumar et al., 1982;
al.,
with
Thus the peak of
al.,
1983).
during
the
Similarly immunoreactive dynorphin
concentrations were highest during the dark phase
in
the
hypothalamus
and
14
pituitary (Przewlocki et al., 1983).
The diurnal variation in opioid peptide
concentrations, with peaks during dark periods, suggest that opioid
might
play
an important role in priming animals for wakefulness as the dark
period is kno~ to suppress sleep in rats (Inoue et al., 1984;
Other animal experiments have demonstrated
receptors
REM
peptides
or
that
1985).
stimulation
of
opioid
opiates is accompanied by an increased
Administration of $-endorphin or morphine decreased
sleep
al., 1967;
al.,
opioid
with
wakefulness.
and
peptides
both
NREM
stages and increased wakefulness in rats and cats (Khazan et
1981;
Scherschlicht et
King et al.
2
(D-Ala )-Met5-enkephalinamide (DALA) and
Echols and Jewett, 1972;
Microinjection
1982).
of
$-endorphin and des-Y-endorphin into the ventral
tegmental
area
stimulated
behavioural wakefulness manifested as increased locomotor activity (Broekkamp
and
Phillips,
Stinus
1979;
et
al.,
The
1 980).
lack
a
of
clear
sleep-influencing effect after intracerebroventricular administration of metor leu- enkephalin (Riou et al., 1982) could be explained by
these
peptides
are
rapidly
cerebrospinal fluid (Dzoljic
resistant
analog
of
metabolised
et
al.,
by
S~udies
DALA
was
found
high voltage slow waves
that
with
DALA
a
more
have demonstrated an increase in both
behavioural and EEG wakefulness (Tortella et al., 1978;
1 979).
fact
enkephalinases present in the
1985).
met-enkephalin,
the
Dzoljic
and
Crucq,
to induce biphasic effects, an initial stupor with
with
eyes
open
and
arousal
with
activated
EEG
pattern.
Collectively these data indicate that endogenous opioid
important
play
an
The
mechanism of the arousal effects of opiates and opioid peptides is still
not
It
in
system
the maintaince of wakefulness in human and animals.
clear.
role
does
not
5-hydroxytyptophan or
appear to involve 5-HT or NA, since pretreatment with
~-methyl tyrosine
did not alter the
arousal
effect
of
morphine (Echols and Jewett, 1972).
An additional point of
period
is
preceded
by
interest
intensive
is
that
diurnal
physiological
sleep
grooming which declines prior to diurnal
arousal phase (Bolles, 1960;
Cooper, 1981 ).
Following the administration of
morphine
or
enkephalin
783030
extended
and
increased
the
synthetic
sleep
sleep
reduced.
(Echols
and
Naloxone
Jewett,
RX
however
1972;
naloxone failed to alter sleep-wakefulness (King
grooming
decreased
Cooper, 1981 ) .
et
al.,
episodes were
grooming
and
In contrast,
1981 ).
However,
15
besides
this
and
the
suppress sleep is not
fact
fully
that the mechanism (s) by which opioid/opiates
understood,
has
it
been
proposed
that
the
activation of endogenous opioid system impaired the mechanism responsible for
switching from grooming to sleep (Cooper, 1981 ).
ii) Endogenous sleep-waking factors
The concept of sleep factors (hypnotoxins) was initiated by the classical
experiments
of
Lengendre and Pieron (1910) in which the cerebrospinal fluid
{CSF) or serum of sleep deprived donor
recipient
dogs.
Since
then
dog
several
induced
sleep
sleep
promoting
in
non-deprived
factors
have been
extracted from various tissues and fluids from different animals.
approaches
have
been
utilised
in
the
search
for
the
promoting factors:- a) that sleep promoting factor should
prolonged
waking
and
Two
basic
endogenous sleep
accumulate
during
b) that sleep promoting factors should be extractable
during spontaneous sleep.
1) Foetor S
Sleep inducing Factor S is derived from CSF and brains of sleep
animals
(Pappenheimer et al .• 1967).
deprived
The icv administration of FactorS was
able to increase delta sleep in rabbits and rats (Pappenheimer et al .•
Fencl
et
al .•
1971 ).
from the urine of sleep deprived subjects as
administration
of
the
1967;
Recently Krueger (1983) identified FactorS derived
synthetic
a
muramyl
peptide.
The
icv
analog of muramyl peptide such as muramyl
dipeptide (MDP) induced s:eep and fever in rat in a similar fashion to Factor
S (Krueger. 1983).
A slight and slow cumulative increase of SWS in rats were
observed only after icv administration of MDP during the dark
et
198~).
al .•
urine.
period
(Inoue
MDP is present in the cell membrane of bacteria found in the
Whether MDP is truely the endogenous sleep factor S or
derived
from
brain
stem
bacteria floral in the urinary tract remains to be clarified.
2) Sleep-promoting-substance (SPS)
The accumulation of sleep-promoting-substances (SPS) in
from
the
24 h sleep deprived rats has been well documented (Inoue et al.
The crude brain stem extracts have been demonstrated consistently
locomotor
activity
and
increase
total
to
1985).
reduce
SWS and REM sleep in mice and rats
(Inoue et al .• 1985).
Four active components
extract
SPS.
A
fraction
have
been
which
separated
from
the
original
crude
appears to be identical to the nucleoside
16
uridine, increased both SWS and REM sleep in mice (Komoda et al.,
rats
(Inoue
et
al., 1985).
This
is
in
o~
An interesting aspect
SPS £acilitates sleep during the dark period but
not
1983)
and
these studies is that
in
the
light
phase.
line with the concept that an endogenous sleep substance should
not induce additional sleep when the physiological demand for sleep has
been
satisfied (Inoue et al., 1985).
3) REM sleep factor
REM sleep deprivation appears to induce the
The
factor.
icv
administration
accumulation
of
REM
sleep
of CSF from REMSD cats restored REM sleep
during PCPA induced insomnia in cats (Sallanon et al., 1982) and reversed the
REM
sleep
deficit
induced
by
~-adrenergic
blockade (Adrien and Dugovic,
1984).
4) Delta sleep-inducing peptide
Delta sleep-inducing peptide (DSIP) is derived from the brain and GSF
animal
kept
asleep
by
electrical stimulation of the intralaminar thalamus
(Monnier et al., 1963).
(Schoenenberg
and
of
This sleep factor has been identified as nonapeptide
Monnier,
1977).
Central administration of DSIP enhanced
both NREM and REM sleep stages (Ursin and Larsen, 1983;
Inoue et al., 1984).
However,
tended
DSIP
was
ip
injected
effective
not
mostly
detected
at
low
doses,
( Scherschlict et al., 1984).
increased
REM
drug
while
In clinical
high
doses
trials
:)SIP
given
withdrawal
and
No
sleep
(Schneider-Helmert,
the
naloxone-precipitated
(Scherschlicht et al., 1984).
of
DSIP
in
increase
DSIP
not
were
rebound
1985).
was
effective
intravenously
human
and
withdrawal
was
demonstrated
DSIP appear to interact
with opioid receptors since it could antagonized stress
insomnia
to
of
sleep, SWS, spindle sleep, sleep efficiency and decreased the
frequency of awakening in insomniacs.
after
and
The hypnogenic effect
wakefulness (Tobler and Borbely, 1980).
or
in
morphine-induced
dependent
animals
It thus appear that the pharmacological action
animals depends on environmental conditions and the
pathophysiology of the organism.
5) Mesencephalic reticular formation perfusate
Drucker-Colin (1973) found that the perfusate collected via
cannula
a
push-pull
from the mesencephalic reticular formation (MRF) of a sleeping donor
cat decreased sleep latency and increased SWS, without affecting
Antibodies
produced
to
MFR
REM
sleep.
perfusate peptides collected during REM sleep,
17
decreased REM sleep in cats.
6) Sleep and waking factors from cerebrospinal fluid (CSF)
CSF collected from rats during the light period (sleep
dark
phase)
inhibited
time locomotor activity, while CSF obtained during dark period enhanced
locomotor activity in the light phase (Sachs et al., 1976).
are
of
These
findings
interest since opioid peptides which are possibly neuromodulators of
wakefulness, reached peak concentrations during the dark phase (Kumar et al.,
1982;
Kerdelhue et al., 1983;
Tang et al., 1984).
7) Growth hormone (GH)
GH induced a dose-related increase in REM sleep but decreased SWS in both
animals
and
man
(Drucker-Colin
et
al.,
1975a;
Mendelson et al., 1980).
These data support the suggestion that SWS induced GH
release
is
essential
for REM sleep induction (Stern and Morgane, 1977).
Functions of sleep
1.4
It is generally known that one night
annoying
symptoms
such
if
activities.
distinct
sleep
was
was
is
followed
monophasic
and
that
accompanied
NREM
and
REM
by
very
Sleep is often
Such simplistic a concept would
However the discovery
states
loss
as fatigue and decreased vigilance.
assigned a restorative function.
teneable
sleep
have
been
by a near halt of all
sleep
stages
were
a tacit indication they may serve different functions.
Attempts to formulate the roles of sleep in the life of an organism has given
rise to many theories of the function of sleep.
1.4.1
Ontogenetic function
One important observation during ontogenesis is the preponderance of
sleep
(active
al., 1966;
sleep)
in
Jouvet-Mounier et al., 1970).
Thus in human neonates
REM
occupies 50 % of the total time and even higher in premature infants.
sleep
In rat
pups, kittens and foetal guinea pigs, REM sleep occupy about 60-80 % of
total
time.
which
the
These reports indicate that REM sleep may provide the necessary
endogenous stimuli for brain maturation and development at the critical
during
REM
human neonates, rat pups and kittens (Roffwarg et
neural
elements
are
being
rapidly organised.
time
This line of
reasoning forms the basis for the -ontogenetic theory" of REM sleep function.
18
This
hypothesis
is supported by the £act that £rom birth to young adulthood
human NREM sleep was reduced by 25 % while REM sleep
was
reduced
by
75
%
(Roffwarg et al., 1966).
Suppression of active sleep in rat pups
altered
neural
growth
with
chlorimipramine
not
and DNA content of some brain areas but also induced
increased anxiety level and a deficiency in sexual activity of adult
(Corner
et
al.,
only
1980).
Although
several
reports
animals
are supportive of the
ontogenetic theory of REM sleep, the persistence of this sleep state in adult
animals
is
still
a puzzle.
It has been suggested that REM sleep in adults
may regulate adaptive behaviours (McGrath and Cohen, 1978).
1 .4.2
Cognitive function
In adult animals
learning,
memory
and
humans
REM
sleep
appear
and intellectual functions.
to
be
essential
for
A recent comprehensive review
showed a consistent positive relationship between learning and an increase in
REM sleep (Smith, 1985).
a functional
cognitive
~ole
theory
Studies based on REM sleep deprivation also suggest
in unprepared learning
(McGrath
and
Cohen,
1978).
The
of function of REM sleep is also supported by the clinical
observations that REM
sleep
was
reduced
in
mentally
retarded
patients,
polypeptide
synthesis
(Petre-Quadens, 1966).
1.4.3
Synthetic function
REM
sleep
(Drucker-Colin
provide
a
condition
for
and Valverde-R, 1982).
increased
The peptide synthesis function of REM
sleep appear to be related it's role in learning and memory.
increases
disrupts retrival and memory consolidation (Rogers et al.,
al.,
Whilst learning
protein synthesis, substances which can decrease protein synthesis
1974;
Flood
1975) and suppress REM sleep (Drucker-Colin and Valverde-R 1982).
REM sleep in
adult
animals
might
allow
the
high
turnover
of
et
Thus
proteins
necessary for neural reprogramming.
1.4.4
Restitutive function
Hartmann
restitution
(1973)
function
and
Oswald
(1974)
for REM sleep.
that REMSD in human subjects reduced
proposed
a
cerebral
or
brain
This view is supported by the findings
the
ability
to
cope
with
stressful
19
events
(Greenberg
et
al., 1972).
Since REMSD is associated with increased
motivational behaviour, such as hypersexuality and aggressiveness, REM
sleep
has also been considered to reduce waking drive behaviours (Vogel, 1979).
The main theory of the function of SWS is that serves bodily
and
musculoskeletal
recovery
(Hartmann,
1973;
Oswald,
1974).
behaviourally active day increased SWS stages without altering
and
REM
sleep
uptake of
(Horne
amino
acid
and Minard, 1985).
into
tissue
restitution
sleep
A
length
In addition GH, which stimulates
(Korner,
1965),
is
released
by
SWS
TSD in rats and man was followed by sleep rebound.
An
increase
in
SWS
(Takahashi et al., 1968).
1.5
Sleep deficiency
1.5.1 Total sleep deprivation (TSD)
being the most pronounced effect {Borbely, 1982).
Changes in hormones, enzymes, proteins and
been
observed after TSD.
al.,
Steriods
such
dihydrotestosterone
and
pituitary
prolactin,
hormone
thyroid
hormones,
activity {Sinha et al .• 1973:
1979).
hormones
and
the
neurotransmitters
have
For example TSD induced an increase in the release
of thyroid stimulating hormone,
hydroxylase
some
adrenal
testosterone,
were
follicle
cortex
and
Parker et al •. 1976;
as
estradiol
melatonin
decreased
hormone,
Palmbald et
androstenedione,
after
stimulating
TSD,
hormone,
cortisol
tyrosine
were
while
the
luteinizing
not
affected
{Cortes-Gallegos et al., 1983).
In rats, TSD increased cerebral GABA levels but decreased glutamic
glycine,
alanine and lysine (Godin and Mandel, 1965).
Comparing the effects
of TSD and REMSD, Panov (1982) found that TSD increased the RNA
the
locus
coerulus
and n.
acid,
contents
of
raphe pontis, while REMSD was associated with a
decrease in RNA in these brain areas.
There were no changes in 5-HT
levels
following
TSD
(Wesemann
and
5-hydroxyindoleacetic
and
Weiner,1982),
acid
there
was an
increase in 5-HT and 5-HIAA after 1 h sleep recovery (Borbely et al.,
1980).
Profound behavioural changes have
there
a
al., 1974;
been
whereas
(5-HIAA)
demonstrated
after
TSD.
Thus
was decrease in speed of performance, alertness, accuracy (Lubin et
Loveland
and
Williams.
1963),
motivation,
and
induced
mood
20
deterioration
1969).
(Johnson,
depersonalization,
(60-90
h)
disorganisation
TSD
and
Prolonged
hallucination,
cognitive
induced
loss
of
thought train (Morris et al., 1960).
Sleep deprivation
phenomena
(Pratt
et
has
also
al.,
been
1968).
demonstrated
Little
is
to
known
increase
of
the
epileptic
biological
consequences of selective SWS deprivation.
1.5.2
REM sleep deprivation (REMSD)
1.5.2a
Methodological aspects of REM sleep deprivation
In human REMSD is induced by using the forced arousal technique
1960).
The
same method can be used in animals.
In this method animals are
implanted with EEG/EMG electrodes for sleep stage monitoring
arousal
with
sensory
stimulus
Selective REMSD using the forced
practical problem.
(Dement,
and
subsequent
any time the animal entered into REM sleep.
arousal
technique
presents
very
serious
Using this technique in rats Morden and co-workers (1967)
found that the number of awakenings from REM sleep, rose rapidly from 135/8 h
on
first
day
to 350 on the third day.
It became practically impossible to
arouse animals from REM sleep without curtailing NREM
method
was
sleep.
An
devised by Jouvet and co-workers (1964) which involves placing a
cat on an inverted -flower pot- surrounded by a pool of water.
is
based
on
differences
in
The technique
muscle tone during NREM and REM sleep stages.
While sitting on the flower pot (=platform, island or pedestal),
can
ingenious
obtain
NREM
sleep
since
animal
muscle tone is present, but during REM sleep
muscle tone is abolished which causes the animal to wet
into the water.
the
it's
nose
or
fall
The -flower pot- technique was adapted for rats by Cohen and
Dement (1965) and for mice by Fishbein
(1970).
REMSD
by
the
flower
pot
technique has obvious advantages over forced arousal:
a) several animals can be REM sleep deprived at the same time
b) the experimenter is not required to monitor the sleep
polygraph
Both of these. help to reduce the over all cost of
are
however
the
experiments.
There
other practical problems associated with using -flower pot- for
REMSD:
a) the extent of REMSD
21
b) the technique introduces stress factors such as dampness
and confinement
b) there is the additional problem of cleaning the tank and
animals daily
Several studies have demonstrated
that
diameter)
deprived
were
more
REM
sleep
rats
(11.5-14 em diameter) (Morden et al., 1967;
studies
revealed
that
animals although to
monitored
REM
less
sleep
extent.
was
on
than
et
al.,
These
1969).
these
studies,
1974)
sleep
polygraph
of
In
in
sleep
was
The
which
they
rats on platforms continously for 4 days have
these
reports
rat
weighing
200-225
g,
and
with diameters 6.5 em (REMSD) and 11.5 em (stress group) were used.
Under these conditions, there was no essential difference in
REMSD
em
for a few hours rather than the for the whole 4-5 day period.
clarified this problem.
platform
(4-7
reduced in the large platform
in
elaborate studies of Mendelson and co-workers (1973;
followed
platforms
animals on large platforms
Mouret
also
However
small
in
the
the
small and large groups during the first 24 h.
degree
of
However in the
fourth 24 h rats on large platforms has no REM or NREM sleep loss, whereas in
the small platform group REM sleep was reduced by 50
~
but NREM sleep was not
different from baseline.
(100
g
rat
body
The work of Mendelson's group sets a good standard
2
weight/14.7-15.6 cm platform area) for using the -flower
pot- technique to selectively deprive rats of REM sleep.
be
maintained
for
at
However
rats
must
least 48 h on platforms to achieve REMSD selectivity
A corresponding stress group (large platform. 100g rat body
54-61 cm2 platform area) should be added to the experimental design.
(Vogel, 1975).
weight/
The selectivity of the pedestal technique for REMSD in
rats
recently
et
validated
electrophysiologically
(Hilakivi
has
al.,
discrepancies often encountered in studies using the -flower
are
probably
also
been
1984).
pot-
The
technique
due to the uncritical attitude of some workers in disregarding
This
the ratio of animal size to platform diameter or area
problem
has
been fully discussed (Hicks et al., 1977).
A second confounding factor in the -flower pot- technique of REMSD is the
stress
(dampness
and isolation) which is associated with the procedure.
has been reported that rats on small platforms
initial
body
al., 1967).
weight
However,
lost
about
10
~
of
It
their
compared with control animals in home cages (Dement et
Selye·s
indices
of
stress
(weight
loss,
adrenal
22
hypertrophy and thymus atrophy) and stomach ulceration were not
different between small (REMSD) and large platform (stress)
et
al., 1 974;
Levental et al. , 1975;
addition there were
histology
between
no
differences
REMSD
signi~icantly
rats
(Mendelson
Coenen and van Luij telaar, 1 985).
in
blood
counts
and
lympoid
In
tissue
and stressed animals (Drucker-Galin et al., 1974).
Another procedure has also been used to control for stress factors associated
with
REMSD
by
- flower pot- technique.
In this rats are forced to swim in
water of 18-19 °C, 10 em deep. for 1-2 h daily and thereafter allowed to have
undisturbed
Adrenal
sleep.
hypertrophy
different between -swimming- rats and
-flower
pot
method
(Stern
et
deprived
by
the
new
animals
al.,
Seyle's stress indices were not
and
REM
1971;
essentially
pendulum
body
weight
sleep
or
were not
by
the
Mendelson et al., 1974).
The
different
procedure
loss
the
deprived
in
rats
classical
REM
sleep
-flower pot-
technique (Coenen and van Luijtelaar, 1985).
Studies in mice (25 g body
placed
on
platforms
(1-3
weight)
em
also
diameter)
indicate
that
these
animals
were more REM sleep deprived than
animals on large platforms (8-25 em diameter) (Fishbein, 1970;
Kitahama
and
Valatx, 1 980).
In conclusion, these data indicate that the
still
a
reliable
procedure
for
REMSD
-flower
in rodents.
pot-
technique
The large platform or
forced swimming are adequate controls for the stress factors associated
the
flower
pot
technique
for REMSD.
confinement, animals in both small and
is
with
In order to reduce the str_ess due to
large
platforms
should
be
allowed
daily free locomotion in home cages.
1.5.2b
Biochemical changes after REM sleep deprivation
i) Serotoninergic system
It has been demonstrated that REMSD increased
(5-HT)
turnover
confirmed
that
(Pujol
REMSD
et
al.,
increased
1968).
the
rat
brain
serotonin
Weiss and co-workers (1968) also
5-hydroxyindoleacetic
acid
(5-HIAA)
but
decreased 5-HT in the brains of rats deprived of REM sleep. Following REMSD
there was also an increase in the formation of [ 3 H]-serotonin
from
3
[ H]-tryptophan in the brainstem-mesencephalon in rats (Hery et al., 1970).
In cats there were no alterations in
the
CSF
5-HIAA
levels
(Radulovacki,
23
a£ter REMSD, but Livrea and co-workers (1977) demonstrated an increase
1973)
in 5-HIAA in the lumbar
However
CSF
of
human
subjects,
deprived
of
REM
sleep.
no changes in 5-HT 2 binding sites in the frontal cortex of REM sleep
deprived animals have been demonstrated (Farber et al., 1983).
changes
in
5-HT
levels
in
some
brain regions.
decreased in the locus coerulus and A5 but increased in
ventral
REMSD induced
5-HT concentrations were
frontal
cortex
and
tegmentum area without alterations in 5-HIAA concentrations in these
brain areas (Mattiace et al., 1983).
ii) Dopaminergic system
Biochemical analysis shows that REMSD for 4-10
days
increased
striatal
dopamine concentrations in rats (Ghosh et al., 1976). without altering lumbar
CSF concentrations of homovanillic acid (HVA) in human
al.,
1977).
Farber
and
co-workers
(Livrea
et
(1983) found that REMSD increased the
concentration of dihydroxyphenylacetic
frontal
subjects
acid
(DDPAC)
in
the
striatum
and
cortex of rats compared with controls but not with the stress group.
There were no changes in the binding of pre- or post-synaptic DA receptors.
iii) Noradrenergic system
REMSD did not alter noradrenaline (NA) turnover brain of rats
al.,
1968b).
reported
However
(Pujol
et
1968b).
Changes
(MHPG),
demonstrated (Mattiace et al., 1983 ).
decrease in NA and MHPG levels in
affecting NA concentrations in
raphe.
REMSD
et
increases in NA turnover after REM sleep rebound was
al.,
3-Methoxy-4-hydroxyphenylglycol
dorsal
(Pujol
did
A5~
the
not
in
after
NA
and
REMSD
It was found
it's
have
that
metabolite,
recently
REMSD
been
induced
and an increase in MHPG levels, without
striatum, ventral
alter [ 3 H]clonidine
tegmental
( o:2 )
area
bin ding
and
sites
(Mogilnicka and Pile, 1981 ), but decreased$ receptor binding (Mogilnicka
al.,
1980).
a
et
However other workers found no changes in$ receptor binding in
the brain of REMSD animals (Abel et al., 1983).
iv) Cholinergic system
A decrease in Ach concentration was
REMSD
rats
(Tsuchiya
et al., 1969).
observed
in
the
telencephalon
of
There were no alterations in striatal
concentration of Ach after 96 hr REMSD, whereas a decrease was observed after
10 days of REMSD (Ghosh et al .• 1969).
v) Amino acids
In cats deprived of REM sleep GABA concentrations were increased
in
the
24
~ormation,
reticular
thalamus
and
frontal
cortex,
while
decreases were
observed in the colliculi and caudate nucleus (Micic et al., 1969;
et al., 1971 ).
Karadzic
Whole brain GABA, glutamic acid, glutamine and threonine were
not changed in the brain of REM
sleep
deprived
animals
(Himwich
et
al.,
have
been
1973).
vi) Peptides
Alterations in peptides in some brain areas and the pituitary
observed
after
REMSD.
Thus substance P was reduced in locus coerulus (LC),
central grey area (CG), medial and dorsal raphe (MR, DR).
(VTA),
substantia
nigra
ventral
caudate areas, whereas somatostatin levels were reduced in DR,
VTA.
Increases
after
REMSD
decreased
of
these
(Mattiace
in
the
tegmental
zona compacta (SNC), medial hypothalamus (HMC) and
CG,
SNC
and
peptides were seen in caudate and preoptic areas
et
al.,
pituitary
1981 ).
$-Endorphin
concentrations
were
but increased in the hypothalamus of REM sleep
deprived rats (Przewlocki, 1984).
vii) Nucleic acids and protein metabolism
The neuronal
RNA
supraoptic
nucleus,
Rubinskya,
1974).
content
was
of
Similarly,
concentrations in then.
the
brain
stem
nuclei,
including
the
decreased in REM sleep deprived animals (Demin and
Panov
(1982)
raphe dorsalis, n.
found
a
decrease
in
RNA
raphe pontis and locus coerulus
after REMSD.
Protein
synthesis
telencephalon,
cerebrum
{Bobillier et al., 1974;
in
some
and
brain
areas,
brainstem,
was
such
as
inhibited
the
by
cerebellum,
REMSD
in rats
Shapiro and Girdwood, 1981 ).
1.5.2c Electrophysiological changes after REM sleep deprivation
i) Neuronal excitability
Several studies have demonstrated that REMSD lowered
in
both
animals
and
man (Cohen and Dement, 1965;
have
assessed by
modulate
neuronal
Thus REMSD facilitated recovery of cortical potentials evoked
by external auditory stimuli (Dewson et al., 1967).
(1971)
thresholds
Bergonzi et al., 1973).
Studies of evoked potentials also indicate that REMSD can
excitability.
seizure
Satinoff and
co-workers
reported that REMSD increased the paleocortical excitability as
evoked
potentials,
but
decreased
evoked
activities
in
the
25
hindbrain
It was
sensory
then
inhibiting
areas induced by stimulation of hindbrain sensory nuclei.
suggested
that
internally
REMSD
generated
amplifies
signals
with the concept that REMSD increased
Drucker-Galin
(1982)
cortical
responsiveness
(Satinoff et al., 1971 ).
cortical
excitability,
by
In line
Bowersox
and
demonstrated that the amplitude of entorhinal cortical
evoked potentials, following prepyriform cortex stimulation, was increased by
REMSD.
In
contrast,
photic
decreased after REMSD (van
evoked
Hulzen
potentials
and
Coenen,
in
the visual cortex was
1984).
In
general
REMSD
increased neuronal excitability.
1.5.2d Behavioural changes after REM sleep deprivation
i) Nociception
REM sleep appears to be essential
Thus
it
has
been
demonstrated
noxious electrical stimulation.
for
that
the
regulation
REMSD
of
nociception.
reduced the pain threshold to
The decrease in
pain
threshold
was
evident 96 hr after the termination of REMSD (Hicks et al., 1978;
still
1979a).
ii) Waking motor and motivated behaviours
An increase in the number of cage crossings has been demonstrated
the
10-15
1981 ).
min
REMSD
after
REMSD
reduced
(Albert
neophobia,
et al., 1970;
increased
exploration,
rearing (Hicks et al., 1979b;
Moore et al., 1979;
Shock provoked aggression was
facilitated
in
REM
during
van Hulzen and Coenen,
ambulation
and
Mogilnicka et al., 1985).
sleep
deprived
animals
(Hicks et al., 1979).
Hypersexuality,
by
manifes~ed
as compulsive mounting behaviour was
provoked
chronic REMSD in cats (Dement et al., 1967).
REMSD
lowered
stimulation
(ICSS)
the
at
threshold
and
frequency
for
intracranial
self
the medial forebrain bundle site in rat (Steiner and
Ellman, 1972), whereas ICSS of the lateral hypothalamic area was not
altered
(Marti-Nicolovius et al., 1984).
Further
motivational
support
for
the
concept
that
REM
sleep
is
involved
in
behaviours, comes from the report that REMSD also enhanced food
competition between male rats (Hicks et al., 1981 ).
iii) Learning and memory
Several authors have studied the effect of REMSD on learning and
memory,
26
however most of the results are conflicting (Smith, 1985).
For example Stern
(1971) reported that REMSD induced a clear learning deficit in one way active
and
passive
confirm this
avoidance
avoidance
tests
observation.
response
but
Albert
Similarly,
and
co-workers (1970) could not
retention
of
a
condition
passive
was disrupted by REMSD (Leconte and Bloch, 1970).
conflicting findings appear to be due to the lack of proper
control
These
of
rat
body weight to platform size, so that there may have been no great difference
in REMSD between small (REMSD) and large (control) platform animals (Hicks et
al.,
1977).
However
another
group
found no alteration in the two way
using a different procedure for REMSD
shuttle
avoidance
response
in
animals
deprived of REM sleep (van Hulzen and Coenen, 1979).
Contradictory effects of
reported.
REMSD
REMSD
on
memory
in
humans
decreased
also
been
had no effect on memory (Chernik, 1972) whereas a decrease
in memory was reported after REMSD (Fowler et al., 1973).
REMSD
have
creativity
(divergent
or
In
another
study
flexible thinking) but improved
serial memory task (Lewin and Glaubman, 1975).
Although some inconsistency exist in literature, the weight
suggests
that
REMSD
of
evidence
may disrupts complex and unprepared forms of learning,
especially those with emotional components (McGrath and Cohen, 1978).
1.5.3
Clinical aspects of REM sleep deprivation
i) Depression
Sleep pattern is generally altered in depressive conditions.
disturbance
in
The
major
depression involves shortened REM latency, an extended first
REM period, increased REM density/activity and
a
decrease
in
delta
sleep
(Kupfer et al., 1984).
It has been demonstrated that REMSD can improve some forms of
but
not
reactive
depressions (Vogel et al., 1975;
endogenous
Vogel, 1980).
In these
elaborate clinical studies, endogenous depressives that were not improved
REMSD
did
not
respond
to
imipramine.
by
Summarizing the recent literature,
Vogel (1983) outlined evidence for the therapeutic efficacy
of
subtype of endogenous depression:
a) REMSD and imipramine have similar therapeutic efficacies
b) The usefulness of antidepressant was related to their
REMSD
in
a
27
ability to induce substained and large REMSD
c) Drugs such as barbiturates, alcohol, diphenylhydantoin,
opiates and amphetamines can induce short-lasting REM
sleep reduction.
In addition tolerance to REM sleep
inhibiting properties of these drugs develops rapidly,
usually within one week or less.
These drug possess no
antidepressant properties.
d) The behavioural effects of REMSD in animals, such as
increased sexuality and aggressiveness, are opposite to
the behavioural alterations present in human depression.
In addition, wide range of typical and atypical antidepressants,
selective
REM
induced
sleep suppression with little or no alterations in NREM sleep
stage (Scherschlicht et al., 1982).
It has become generally accepted that REMSD can
endogenous
depression
antidepressants
exert
and
may
their
in
fact
improve
some
forms
of
be part of the mechanism by which
therapeutic
action
(Vogel,
1983).
The
antidepressant action of REMSD was long lasting up to 21 days.
ii) Schizophrenia
Unlike depression, REMSD did not alter schizophrenic symptoms (Vogel
Traub,
1968;
Gillin et al., 1974).
It was also demonstrated that REM sleep
rebound, following REMSD, was exaggerated in
suggested
that
active
and
schizophrenia
schizophrenics.
Thus
it
was
was associated with a decrease in REM
sleep need (Gillin et al., 1974).
iii) Epilepsy
Sleep
disturbances
epileptics.
For
and
seizure
example,
sleep, whereas NREM sleep
patients
stage
2
phenomena
often
co-exist
in
human
with grand mal seizure had reduced REM
was
increased
(Besset,
1982).
Sleep
pathology in epileptics with cortical or deep temporal foci was mainly in the
form of a decrease in NREM stages 3 and 4 (Montplaisir et al., 1982).
In addition
seizure
to
sleep
disturbances
activities
occur
preferentially
associated
in
with
sleep
epilepsies,
while
some
others occur in
wakefulness
The paroxysmal discharges in petit mal
sleep
onset
and
awakening.
classified according to
the
Two
epilepsies
subgroups
distribution
of
of
were
petit
epileptic
facilitated
by
mal seizure exist,
discharges
during
28
sleep.
In
one
group epileptic discharges occur during REM sleep. while in
the second group paroxysmal discharges were suppressed during REM sleep (Ross
et al., 1966;
Billiard, 1982).
REMSD has been demonstrated consistently to lower the
in
experimental
animals
facilitated seizure
(see
activity
(Bergonzi et al., 1973).
section 1 .5.2c).
during
the
night
seizure
threshold
In human epileptics, REMSD
after
REMSD
was
stopped
In another report selective SWS deprivation, rather
than REMSD, was more effective in provoking epileptic attacks in
children (Becket al., 1977).
pycnoleptic
29
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46
CHAPTER 2
ENDOGENOUS OPIOID PEPTIDES
2.1
Nomenclature and classification
The discovery that electrical stimulation of the periaqueductal gray area
induced
morphine-like analgesia (Akil et al., 1972) led to the search for an
endogenous
opiate
an
isolated
properties
endogenous
similar
morphine-like
Later
ligand.
to
Hughes
compound
that
of
from
and
the
morphine.
co-workers
brain
They
(1975;
with
termed
1975a)
pharmacological
this
endogenous
compound enkephalin (from en kephalos, in the head).
From the
initial extract named enkephalin two related pentapeptides which differ
in
the
only
carboxyl terminal amino acid were identified (Hughes et al., 1975b),
namely methionine-enkephalin (met-enkephalin, met-ENK) and leucine-enkephalin
(leu-enkephalin,
leu-ENK).
Since the initial discovery of endogenous opioid
pentapeptides by Hughes and co-workers, several
other
ligands
with
opioid
activity have been isolated from various tissues in different animals (Hughes
et al., 1980;
Bloom, 1983).
Other peptides with opioid activity
designated
~-endorphin
al., 1976;
Ling et al., 1976).
were
(Li and Chung, 1976),
found
~.
in
the
pituitary
Opioid peptides which are extended leu-ENK sequences have been
from
the
pituitary.
Goldstein
and
andY-endorphin (Bradbury et
extracted
and co-workers (1979) isolated and named a
pituitary opioid peptide as dynorphin 1 _
.
In the same year another opioid
13
peptide, a decapeptide described as -big- leu-ENK was named ~-nee-endorphin
(Kangawa
and
dynorphin 1 _ 17
et al., 1981 ).
Matsuo,
and
1979).
Other
~-nee-endorphin
peptides
include
(Minamino et al., 1980;
Several other morphine-like
from various animal tissues for example,
substances
~-casomorphins
have
dynorphins _ ,
1 8
1981; Goldstein
been
isolated
derived from £-casein
(Brantl et al., 1979) and humoral (H) endorphin (Sarne et al., 1978).
47
2.2
Metabolism of endogenous opioid peptides
At
present
described :
three
classes
~-endorphin,
of
endogenous
opioid
enkephalins and dynorphin.
peptides
have
been
There is now sufficient
evidence that these opioid peptides originate from three different precursors
(Hughes et al., 1980).
2.2.1
Biosynthesis
i)
~-endorphin
Biochemical evidence in the
other
literature
such
non-opioid
indicate
as
that
$-endorphin
and
corticotropin,
$-melanocyte-stimulating hormone (MSH), p-lipotropin and adrenocorticotrophic
hormone (ACTH) are formed from a common precursor termed pro-opiomelanocortin
(POMC)(Hughes et al., 1980;
Bloom, 1983).
The
POMC
derived
peptides
are
produced by proteolytic cleavage of the lipotropin.
ii) Enkephalins
Several studies have demonstrated that enkephalins are
ribosomally
synthesized
al., 1980).
This pro-enkephalin contains one copy
protein
produced
from
a
precursor termed pro-enkephalin (Hughes et
of
leu-ENK
and
six
of
met-ENK (Noda et al., 1982).
Several synthetic analogues of enkephalins have
example,
D-Ala2 -met 5 -enkephalinamide
(DALA)
and
been
produced
for
2
D-Ala -D-Leu5 -enkephalin (DADLE).
Structures of enkephalins
Met-enkephalin:- Tyr-Gly-Gly-Phe-Met
Leu-enkephalin:- Tyr-Gly-Gly-Phe-Leu
iii) Dynorphin
Dynorphin and
~-nee-endorphin
are derived from a protein precursor termed
prodynorphin (Kakidani et al., 1982).
48
2.2.2
Biodegradation of enkephalins
Opioid
peptides
are
inactivated
by
several
peptidases
present
in
mammalian tissues.
i) Soluble aminopeptidases
Aminopeptidases degradate enkephalins by cleavage of the Tyr-Gly
bond.
The
major
metabolite
enkephalins is tyrosine (Hambrook
following
et
al.,
aminopeptidases
1976).
peptide
hydrolysis
of
These
enzymes
can
be
Enkephalinase A is a dipeptidyl-carboxypeptidases which
cleaves
Gly-Phe
inhibited by bestatin.
ii) Enkephalinase A
peptide
bond
in enkephalins.
Enkephalinase A is sensitive to inhibition by
thiorphan, phosphoramidon and kelatorphan (Hudgin et al., 1981;
Waksman
et
al., 1985).
iii) Enkephalinase B
Enkephalinase
enkephalins
B
is
a
dipeptidyl~aminopeptidase
inactivates
which
by cleavage of Gly-Gly amide bond (Gorenstein and Synder, 1979).
No selective inhibitor of this enzyme has been demonstrated.
iv) Angiotensin-converting-enzyme (ACE)
Similar
to
enkephalins,
enkephalinase
but
does
not
A,
ACE
appear
situations (Erdos et al., 1978).
to
hydrolyses
play
Gly-Phe
amide
bond
of
essential role in in vivo
an
Captopril which is
an
inhibitor
of
this
enzyme, does not modulate the enkephalinergic system in vivo.
Enkephalins are inactivated in vitro by all four enzymes mentioned above.
However,
the
soluble
amino
peptidases
and
enkephalinase
A appear to be
involved in the biotranformation of synaptic enkephalins in vivo.
Knowledge of the biological mechanisms involved in
the
inactivation
of
$-endorphin and dynorphin is still poor.
2.3
Opioid receptors
The existence of specific opioid receptors in
the
mammalian
brain
demonstrated simultaneously by three groups (Pert and Snyder, 1973;
al .. 1973;
on
the
opioid
Terenius, 1973).
effects
of
receptors.
opiates
The
Later studies of Martin and
in
was
Simon et
co-workers
(1976)
spinal dogs indicated the heterogeneity of
three
different
opiates,
morphine,
49
ethylketocyclazocine
distinct syndromes.
different
opioid
(EtKGZ)
and N-Allylnorphenazocine (SKF 10047) produced
Based on this behavioural study the existence
receptors
have
been
~
suggested:
of
three
(mu) for morphine, x
(kappa) for EtKCZ and o (sigma) for SKF 10047 (Martinet al., 1976).
The concept of multiple opioid receptors was also confirmed and
in
studies
autoradiography (Lord et al., 1977).
co-workers
that
morphine
These authors concluded like Martin and
showed
preference
for
However the
~-receptors.
enkephalins, especially leu-ENK, interacted mainly with the opioid
designated
6-receptors.
£-Endorphin
More recently two subtypes of
~
~
2
~,
x-receptors
et al., 1983).
and
equipotent at
designated
~,
Enkephalin bind to
morphine bind to both
(Garzon
was
receptor
suggested (Pasternak et al., 1983).
sites.
Dynorphins
~
~
and
~,
Table I:
Ligands
2
have
been
and 6 sites, while
show
preference
for
The relative affinities of some opiates
Relative affinity oT ligands Tor opioid receptors
(modified oTter Hughes et al.
receptors
and 6-receptors.
and opioid peptides for opioid receptors are shown in table I.
1980, Garzon et al., 1983)
Receptors
"
6
'
met-ENK
++
+++
+
leu-ENK
+
+++
+
~-endorphin
+++
+++
+
mer hine
+++
+
+
dynoruhin(1-17)
++
+
+++
EtKCZ
+
+
+++
Naloxone
+++
+
+
Key:
extended
utilizing, guinea-pig ileum and mouse vas deferens bioassays and
+=low, ++=moderate, +++=high
50
Similarly a heterogeneity of opioid receptors has
the
human
brain
receptors
for
peptides
is
(Maurer
et al .• 1983)-
physiological
not
yet clear.
functions
been
demonstrated
in
The importance of multiple opioid
of
endogenously
released
opioid
The opioid receptor types possible involved in
the pharmacological effects of opiate and opioid peptides
are
indicated
in
table III
2.3.1
Opioid receptor distribution in the brain
The idea that the various opioid receptors are differentially distributed
in
various
brain areas is supported by considerable body of evidence (Atweh
and Kuhar. 1983)-
the
rat
brain
was
assayed
[ 2 ]-Tyr-D-Ala-Gly-NMet-Phe-Gl-ol
by
using
(DAGO)
(DTLET)
1983b).
of~
Recently the distribution
and o-opioid
highly
selective
for
#-receptor
6-receptors
for
receptors
~
Table II show the relative preponderance of
ligands
(Quirion
and
in
and
et
al.,
6-receptors
in
some brain regions.
The distribution of x-receptors in the rat brain is similar
to
that
of
#-receptors (Quirion et al., 1983a).
2.4
Physiological
implications
of
regional
distribution
opioid
of
peptides
2.4.1
Analgesia
Opioid peptides are present in high concentrations
in
the
brain
areas
(periaqueductal gray area, intralaminal thalamic nuclei and raphe nuclei) and
spinal cord (laminae I and II of dorsal horn) related to pain
(Hokfelt
et
al.,
1977;
Simantov et al., 1977).
the PAG and pituitary has been demonstrated
analgesia (Akil et al., 1972;
release of opioid peptides.
8-endorphin
levels
in
the
neurointermediate lobe
of
threshold
al.,
noxious
(Millan
electric
8-endorphin
et
shock
analgesia
induce
naloxone
reversible
Yanagida et al., 1985) possibly by causing the
A lesion of the arcuate nucleus,
hypothalamus,
the
pituitary
1980).
(chapter
concentrations
to
and
Electrical stimulation of
periventricular
was
REMSD
1,
to
reduced
decrease
pain
decreased the pain threshold to
section
(Przewlocki.
reported
which
area and in the
1 .5.2b)
1984).
and
The
the
pituitary
involvement
of
51
Table II:
Relative preponderance oT opioid receptors in some
brain areas (modified after Quirion et al.
Brain region
1983b)
Receptors
++++
++
frontal cortex
++
++
layers II IV of cortex
+++
layers V IV of cortex
+
+++
caudate putamen
++++
+++
olfactory tubercle
+
+++
se tum
+
thalamus
+++
am
+++
n.
h
accumbens
dala
othalamus
+
++
+
habenula
++++
+
interpeduncular nucleus
+++
+
central
++
+
+++
+
us
hi
r
cerebellum
n.
tractus solitarius
locus coerulus
+++
dorsal horn (suinal cord)
+++
Key:
+
-=absent, +=low, ++=moderate, +++=high, ++++=very high
52
endogenously released opioid peptides in the
also
supported
by other reports.
regulation
of
system
(Hugdin
et
Rupreht et al., 1983).
induced
both
in
to
potentiate
the
al., 1981) increased the pain threshold
(Carenzi et al., 1981;
analgesia
is
The icv administration of enkephalinase A
inhibitors, thiorphan and phosphoramidon which are known
enkephalinergic
nociception
animals
and
~-Endorphin
given
centrally
human subjects (Loh et al., 1976;
Hosobuchi and Li, 1978).
2.4.2
Locomotor activity
Opioid peptides are present in high concentrations
putamen,
globus
in
important
in
the
regulation
activities (Chambers et al., 1971 ).
the
pallidum,
putamen,
of
skeletal
in
the
These
muscle
brain
tone
areas
and locomtor
In Parkinsonism a deficiency of
ENK
in
substantia nigra and the ventral tegmental area has
Agid and Javoy-Agid, 1985).
been demonstrated (Taquet et al., 1983;
receptors
nucleus,
pallidus (basal ganglia), amygdala, substantia nigra and in
the ventral tegmental area (Khachaturian et al., 1983).
are
caudate
substantia
nigra
and
striatum
Opioid
have been implicated in
opiate-induced muscular rigidity and catalepsy (Turski et al., 1983).
The administration of $-endorphin or
morphine-like
catalepsy
in
met-enkephalin
rats (Bloom et al., 1976;
icv
can
Chang et al., 1976).
Similarly, the administration of an enkephalinase inhibitor.
induced
hypotonic
preoptic
area,
hypothalamus
immobility
n.
are
in
accumbens,
particularly
sensitive
to
opiates and opioid peptides (Tseng et al.,
However
the
administration
gray
area,
rats
(Wei
et
al.,
1977;
also
The medial
anterior
and
the cataleptogenic effects of
1980;
Winkler
et
al.,
1982).
of low doses of morphine or opioid peptides icv
stimulated locomotor activity, wet-dog-shakes (WDS) and
morphine, enkephalins and
thiorphan
mice (Chaillet et al., 1983).
periaqueductal
stimulate
Brady
and
Holtzman, 1981 ).
into
~-endorphin
body
ventral
scratching
in
Microinjection of
tegmental
area
or
n.
accumbens produced behavioural changes characterised by sniffing and grooming
interrupted by bursts of locomotor
opiates
and
Kelly et al., 1980;
WDS,
activity.
These
stimulant
actions
of
opioid peptides were naloxone-reversible (Pert and Sivit, 1977;
grooming
and
Stinus et al.,
body
1980).
scratching
were
Similarly
also
naltrexone-sensitive
observed
following
icv
administration of the enkephalinase inhibitor phosphoramidon (Rupreht et al.,
53
1983).
2.4.3
Temperature
A
role
endogenously
~or
released
opioid
peptides
in
temperature
regulation is suggested by the report that administration of an enkephalinase
inhibitor,
or
thiorphan
naloxone-reversible
met-enkephalinamide
hypothermia.
induce
could
endogenously
released opioid peptides involves the preoptic and the anterior
hypothalamic
(Stanton
al.,
et
1985).
Similarly
induced hyperthermia in low
~-endorphin
hypothermia
due
a
to
areas
This
central
concentrations
administration
and
hypothermia
of
in
high doses (Tseng et al., 1980).
2.4.4
Feeding
Endogenously
(Jalowiec
et
released
al.,
morphine,
~-endorphin
et
1977;
al.,
opioid
1981 ).
peptides
Systemic
or
appear
to
regulate
intracerebral
or enkephalins facilitated food consumption
Jalowiec et al., 1981;
feeding
administration
(Grandison
Tepperman and Hirst, 1983).
peptides in the hypothalamic brain area are particularly
important
of
Opioid
for
the
regulation of feeding behaviours (Przewlocki et al., 1983).
2.4.5
Respiratory and cardiovascular system
Local application
surface
of
the
of
pons
morphine
in
cats
and
enkephalins
selectively
to
the
dorsa-rostral
decreased
the
frequency
respiration, whilst tidal volume or the response to carbon dioxide were
unchanged
or
increased
(Hurle
et al., 1983).
Morphine was less effective
than enkephalin in reducing the frequency of respiration.
can
easily
reverse
hypoventilation (Bowman
depression
both both
0~
~
morphine-induced
nnd
r~spiration
and
6-receptors
Rand,
1980)
respiratory
but
not
(Pazos and Florez, 1983).
are
involved
in
of
left
the
the
However,
naloxone
depression
nnd
enkephalin-induced
It was suggested that
respiratory
depressant
actions of opioid peptides and opiates.
A decrease in blood pressure after the application of
the
ventral
surface
and Mediavilla, 1977).
brain
produced
met-enkephalin
to
of the brain stem in the cat has been reported (Florez
The microinjection of met- or leu-enkephalin into the
either hypertension or hypotension, depending on the site of
54
injection.
the
It has been suggested that
A
medulla.
responses
and
naloxone-resistant
naloxone-sensitive
t~o
types of opioid receptors exist in
receptor
receptor
which
which
mediate
mediates
vasopressor
vasodepressor
responses (Fuxe et al., 1979).
2.4.6
Sexual behaviour
The
possible
behaviour
involvement
of
is suggested by several authors.
narcotic addicts has been reported
opiate
endogenous
withdrawal
opioid
in
sexual
A decrease in sexual function in
(Crowley
and
Simpson,
1978).
Whereas
may be associated with premature ejaculation, spontaneous
erection in men and sexual arousal in women (Parr,
opiate-dependent
peptides
rats
1976).
Pretreatment
of
with enkephalinase inhibitors thiorphan or phelorphan
stimulated penile-licking during naloxone-precipitated withdrawal (Dzojlic et
al .•
in
preparation).
following
the
phosphoramidon
A similar activation of penile licking was observed
administration
of
another
enkephalinase
inhibitor
REMSD animals (unpublished observation). However central
administration of ~-endorphin or D-Ala2 -met 5 -enkephalinamide reduced mounting
behaviour
in
to
normal,
Gessa et al .• 1979).
behaviour
were
non-dependent male rats (Meyerson and Terenius, 1977;
These inhibitory actions of opioid peptides
blocked
by
naloxone
and
naltrexone.
on
sexual
Further studies are
necessary for the clarification of the role of different opioid receptors
in
sexual behaviours.
2.4.7
Neuronal excitability
Accumulating evidence indicate
peptides
are
that
both
opium
alkaloids
and
opioid
capable
of exerting an inhibitory or a facilitatory action on
cerebral excitabiltiy.
The administration of large doses of morphine, icv or
systemically.
can induce convulsive behaviours in mice and rats (Gilbert and
Martin, 1975;
Snead and Bearden,
1982).
Pretreatment
with
subconvulsant
doses of morphine can block the convulsant effect of morphine and enkephalins
(Urea and Frenk, 1983;
$-endorphin
and
Dzoljic, 1982). Similar pretreatment with morphine,
[D-Ala2 -D-Leu 5 ]enkephalin
(DADLE)
also
antagonised
electroshock-induced seizures (Puglishi-Allepra et
Alder,
1984).
al.,
1984;
Berman
and
This anticonvulsant action of opiates and opioid peptides was
blocked by naloxone (Berman and Alder. 1984).
Recent data indicate that
the
55
6-opioid
~-opioid
receptors
the
receptors are involved
peptides
epileptic
in
the
actions
Siggins, 1980;
et
both
6
and
1984).
opioid
~
opioid
on
the
However
receptor
anticonvulsants (Tortella et al., 1985).
depending
of
opioid
Haffmans and
in
the
limbic
al., 1978), particularly in the hippocampus (French and
Haffmans et al., 1983;
test
administered
actions
Frenk. 1983;
The target area of this action appear to be
(Henriksen
seizure
of enkephalins, whilst
anticonvulsant
(Dzoljic and vd Poel-Heisterkamp, 1982;
Dzlojic, 1983).
area
mediate
It
in
the
agonists
does
appear
fluorothyl
appear
that
to
be
exogenously
peptides may have both pro- and anti-convulsant actions
experimental
conditions.
The
role
of
pro-
and
anti-convulsant opioid systems in human epilepsies is still unknown.
2-4-8
Tolerance and dependence
Similar to morphine the development of tolerance to opioid
been
reported
(Wei
and Loh, 1976;
animals tolerant to the
Tseng et al., 1977).
sufentanyl,
~agonist
a
peptides
has
Interestingly, in
6-opioid
receptor
agonist
DADLE was still able to induce analgesia and catatonia (Schulz et al., 1981 ).
This might indicate the lack of cross-tolerance
agonists.
It
also
dependence
to
endogenously
precipitated
an
appears
that
opiate-like
1984).
Social
in
interaction
an
organism
opioid
abstinence
enkephalinase inhibition (Christie
resulting
an
released
appear
opioid-like
and
to
-social
~
between
can
syndrome
receptor
develop tolerance and
peptides.
Namely
after
Chester,
6
and
1 982;
naloxone
chronic stress or
Bean
and
Vaught,
activate endogenous opioid peptides
dependence-.
Isolation
or
separation provoked symptoms such as vocalisation and irritability.
induced by social isolation could be reduced by morphine and
social
Symptoms
potentiated
by
naloxone (Panksepp, 1981 ).
The possible involvement of changes in endogenous opioid peptides in
process
of
opiate
dependence
is
not
clear.
However,
the
chronic morphine
treatment increased the activity of a high affinity enkephalinase (Malfroy et
al.,
1978).
Enkephalinase
inhibiton
attenuated
some
symptoms of opiate
withdrawal in morphine dependent animals (Dzoljic et al., in preparation).
An increase in protein synthesis also
mechanism
of tolerance to opiates.
appears
to
be
involved
in
the
Several drugs with the common ability to
inhibit protein synthesis reduce the development of tolerance and
dependence
56
to
In addition, it has been reported that
morphine (Bowman and Rand, 1980).
the development of opiate dependence is associated with an
increase
in
the
synthesis of secretory proteins in the pons-medulla and striatum-septum (Retz
and Steele, 1983).
tolerance
These brain areas are
functionally
involved
in
opiate
The hippocampal CA~ area also appear
and dependence (Herz. 1978).
to be modulate both opioid and opiate-withdrawal WDS (Isaacson and
Lanthorn.
1981).
Some pharmacological actions
of
opiates
or
opioid
peptides
possible brain areas involved have been described in this section.
summarises some pharmacological actions of opioid
and
the
Table III
peptides/opiates
and
the
receptor types involved.
Drugs and behavioural states affecting endogenous opioid peptides
2.5
2.5.1
Neuropeptidase inhibitors
Potentiation of enkephalinergic activities following
the
inhibition
of
neuropeptidases have been demonstrated under several experimental conditions.
For
example kelatorphan a
5
met -enkephalin
(met-ENK)
biodegradation of
levels
exogenously
Similarly
1985).
potent
thiorphan
inhibitor
in
in
vivo
striatal
and
striatum
bestatin
midbrain
another
and
(see
leucinal
chapter 3).
increased
blocked
the
al.,
A (Hudgin et al.
(peptidase
met-ENK
et
levels
Yaksh and
inhibitor),
and
Harty,
induced
1982).
potent inhibitor of enkephalinase A (Hudgin et al.,
1981 ), induced naltrexone-sensitive analgesia
insomnia
and
increased
(Waksman
inhibitor
naloxone-reversible analgesia (Zhang et al., 1982;
Phosphoramidon,
enkephalinase
enkephalins
enkephalinase
1981 ), given alone, or in combination with
elevated
rat
administered
an
of
(Rupreht
et
al.,
1983)
and
Other neuropeptidase inhibitors such as bestatin
brain
analgesia (Waksman et al., 1985;
met-ENK
levels
and
Davis et al., 1983).
~-endorphin-stimulated
In addition, naloxone
precipitated some behaviours characteristic of the opiate abstinence syndrome
in
rats
after
chronic inhibition of enkephalinase (Bean and Vaught, 1984).
These reports indicate that inhibition of
increases
some
neuropeptidases
induce
in opioid peptides with concomitant alterations in behaviour.
effect of peptidase inhibitors on the brain concentrations of
such as
can
~-endorphin
other
and dynorphin, still remains to be established.
The
opioids
57
Table III:
involved
Pharmacological effects of opiates and receptor subtypes
after
(modified
Trends
in
Pharmacol.
Sci.,
vol
6
centre'fold)
Pharmacological actions
Rece12tors
"
SU:Qras:Qinal
analgesia
stress-induced analgesia
eu haria
d
..
s haria
sedation
0
X
"1
0
..
?
?
?
?
?
catale s
••
increased locomotion
decreased locomotion
..
..
withdrawal signs
ictal EEG S:Qiking
anticonvulsant
erthermia
h
othermia
?
?
tolerance
h
?
..
..
?
?
?
res:Qirator;y de:Qression
Key:
**"'direct
established
involvement,
-=not involved
*=possible
involvement,
?=not
58
2.5.2
Centrally acting drugs
i) Narcotic analgesics
Morphine elicited a decrease
concentration
in
ascribed,
However,
enkephalin
reduction in
The analgesic
prolonged
levels
~-endorphin
effect
of
in
the
morphine
the
morphine
striatum
administration
and
has
to
demonstrate
A similar
septum,
midbrain
Plasma concentration of 8-endorphin-like
material was reduced in heroin addicts (Ho et al., 1980).
failed
(>1month)
pituitary.
contents were demonstrated in the
and pituitary (Herz et al., 1980a).
studies
elevated
partly, to the release of endogenous opioid peptides (Schlen
and Bentley, 1980).
decreased
but
~-endorphin
of this peptide in whole brain (without the cerebellum) and in
the pituitary (Bruni et al., 1985).
been
plasma
changes
In contrast, other
in brain enkephalin concentrations
after acute or chronic morphine treatment or naloxone-precipitated withdrawal
(Fratta
et
al.,
Wesche et al., 1977).
1977;
Chronic (10 days) treatment
with morphine increased enkephalinase activity and
~-opioid
receptors in
the
haloperidol
and
striatum (Takashi et al., 1981 ).
ii) Neuroleptics
Chronic
administration
chlorpromazine
of
increased
des-Tyr 1 -Y-endorphin in brain
phenobarbital
and
the
antipsychotic
the
formation
slice.
promethazine
did
opioid peptides (Davis et al., 1984).
reported
to
increase
plasma-
and
schizophrenics (Emrich et al., 1980;
Other
not
drugs
of
central
Y-endorphin
depressants
alter the concentrations of these
However, neuroleptic therapy has
CSF-
as
such
endorphin
activity
in
been
chronic
Naber et al., 1984).
Met-enkephalin levels in the striatum and nucleus accumbens were elevated
after
chronic
haloperidol
treatment
and
with
reserpine.
the
The
antipsychotic
drugs such as clozapine,
non-cataleptogenic
neuroleptic
such
as
clozapine was less effective in elevating met-ENK levels (Hong et al., 1980).
The clinical relevance of
the
neuroleptic
induced
alterations
in
opioid
peptides is not yet clear.
iii) Antidepressant drugs
Antidepressant drugs such as cloimipramine, desimipramine, amitriptyline
provoked
iprindole
a selective increase in met 5 -enkephalin-like
immunoreactivity in the striatum and nucleus accumbens
(De
Felipe
et
al.,
59
1985).
~-endorphin
An increase in
contents in the pituitary and hypothalamus
has also been reported after treatment with some antidepressants
(Pzewlocki,
1984).
Interestingly,
depressive
antidepressant
patients
Bongiorno, 1982;
drugs
decreased
~-endorphin
plasma
in
in correlation with therapeutic activity (Rapisarda and
Genazzani et al., 1984).
iv) Anaesthetic agents
The analgesia induced by nitrous oxide has been
endorphinergic
transmission
since
naltrexone (Yang et al., 1980).
naloxone
attributed
to
enhanced
be attenuated by naloxone and
addition,
morphine
attenuated
whilst
Recently nitrous oxide has been demonstrated to increase met-ENK
in the CSF
o~
rats (Quack et al., 1985).
o~
decreased the release
o~
In
could
potentiated nitrous oxide withdrawal convulsions in mice {Manson et
al., 1983).
levels
it
6-opioid
Chronic ethanol consumption
enkephalin in the striatum inducing supersensitivity
receptors,
but
(Lucchi et al., 1984).
af~inity o~
reduced the
The
role
of
endogenous
~-opioid
the
opioid
receptors
peptides
in
pharmacological actions of anaesthetic agents is not fully understood.
the
There
is however the idea that the analgesic and eurphoric effects of nitrous oxide
are due partially to release
o~
endogenous opioid peptides.
v) GABA and benzodiazepins
GABA and muscimol were found
striatal
met-ENK.
In
a
to
parallel
decrease
in
potassium-evoked
release
of
vivo study, acute administration of
benzodiazepines such as diazepam decreased met-ENK levels in the striatum but
increased
them
in
the
The
hypothalamus.
drug
induced
enhancement
of
enkephalin was rapid in onset (2-5 min) (Herz et al., 1980b).
vi) Drugs modulating 5-HT system
~en~luramine
The serotonin releaser,
contents
in
the hypothalamus but not in the
brain stem (Harsing et
depletes
5-HT
al.,
1982).
met-ENK
~rontal
Conversely,
and
B-endorphin
cortex, hippocampus and
PCPA
and
5-7-DHT
which
levels in the brain reduced B-endorphin concentrations in the
hypothalamus, thalamus, and brain stem
administration
increased
of
these
substances
but
did
not
not
in
the
pituitary.
Acute
alter brain contents of this
peptide (Harsing et al., 1982).
The clinical signi£icance
o~
changes in opioid peptides induced by
interfering with GABA and 5-HT system is not clear.
drugs
60
2.5.3
Stress
Abundant evidence suggests that endogenous opioid peptides are
by
various
stress
regimens.
Electroshock
induced
modulated
a naloxone-reversible
analgesia and motor inhibiton (Nabeshima et al., 1985).
A parallel
increase
in brain enkephalin levels and the pain threshold has been demonstrated after
electric shock (Madden et al., 1977).
Electroconvulsive
~hich
shocks,
are
associated with a naloxone-reversible postictal analgesia and catalepsy (Urea
et al., 1981 ), elevated preproenkephalin mRNA and
in
the
hypothalamus
and
limbic
area
enkephalin
(Yoshikawa
stimuli, such as arthritis or injecting formalin
enkephalin
1981 ).
levels
in
the
brain
in
the
enhanced dynorphin in
pituitary
gland
et al., 1983;
released
cortex
these
rat
and
brain
did
s~imming
not
paws,
acute
al.,
stress
Painful
increased
Kuraishi et al.,
alter
dynorphin
hypothalamus, whereas tail-pinch stress
regions
(Morley
et
al.,
1982).
contains high concentrations of opioid peptides
Kerdelhue et
during
et al., 1985).
into
(Cesselin et al., 1980;
Immobilisation stress or forced
concentrations
concentrations
1983;
exposure
Tang
et
al.,
The
(Prze~locki
1984),
which
are
(Guillemin et al., 1977), probably
indicating an involvement of pituitary opioid peptides. along
~ith
ACTH,
in
the stress response.
2.6
Role of opioid peptides in psychopathology
Several
suggested
behavioural
possible
psychopathology.
and
limbic
actions
implication
of
of
opioid
peptides
endogenous
studied
opioid
peptides
In addition opioid peptides are present in the
areas
where
they
are
well
placed
to
modulated
in
animals
in
human
brain
stem
vigilance,
motivation and emotions.
2.6.1
Opioid peptides and depressive states
In several studies an increase in plasma and CSF
$-endorphin
depressive conditions have been demonstrated (Risch, 1982;
1984), while in other reports no changes in the plasma or CSF
of
$-endorphin
were
found
(Naber et al., 1982).
~-.
1979).
in
concentrations
Patients with endogenous
depression were also more tolerant to pain than normal volunteers
Antidepressant
levels
Genazzani et al.,
(Davis
et
drugs decreased plasma $-endorphin in parallel
61
with their therapeutic activity (Rapisarda and Bongiorno, 1982;
al..
These
1984).
data
indicate
that
excess
Genazzani et
of opioid peptides may be
involved in the pathogensis of some forms of endogenous depression.
Opioid peptides in schizophrenia
2.6.2
The icv administration
~-endorphin
elicited
rigid
immobility
in
rats.
This observation led Bloom and co-workers (1976) to propose that an excess of
central
opioid
schizophrenia.
peptides
might
be
involved
in
the
may have a neuroleptic-like therapeutic action, since
extrapyramidal-like
These
ridigity.
propositions
schools of thought ie i) that excess and ii)
peptides,
pathophysiology
underlie
schizophrenia.
that
Increased
this
have
a
of
~-endorphin
In contrast, Jaquet and Mark (1976) proposed that
peptide
induced
given rise to two
de~iciency
~-endorphin
o~
opioid
levels in CSF of
schizophrenic and manic patients were reported by some workers (Rimon et al.,
1980),
and
1978).
However other authors could not
1981 ).
naloxone
appeared
to reduce psychotic symptoms (Watson et al.,
con~irm
these findings (Naber et al.,
Direct biochemical evidence that excess opioid peptides are secreted
in schizophrenia or that opiate antagonist helps in this disorders
best
tenous.
~-Endorphin
is
at
either slightly accentuated (Gerner et al., 1980)
or
allevated some symptoms of schizophrenia (Kline
et
aL
1977)1
Des-Tyr -Y-endorphin (DTYE) decreased psychotic symptoms in schizophrenics
(Verhoeven et al., 1979).
therapeutic
In
following
effects
antipsychotic drugs such as
formation
of
Y-endorphin
slice (Davis et al., 1984).
schizophrenics
contrast
DTYE
other
workers
(Emrich
et
did
al.,
not
1980).
any
~ind
However
haloperidol and chlorpromazine increased the
and des-Tyr 1 -Y-endorphin in vitro in animal brain
Plasma and CSF
~-endorphins
were
inceased
in
, although there was no correlation between changes in plasma
opioid peptides and
the
(Emrich et al., 1980;
Evidently the
role
therapeutic
e~~ects
of
the
antipsychotic
drugs
Naber et al., 1984).
of
endogenous
disorders needs further clarification.
opioid
peptides
in
the
psychotic
62
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1947-1949
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Neuropharmacol., 21:
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1137-1144
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body
temperature
and
72
metabolic
rate
following
preoptic/anterior
microinjection of met-enkephalinamide in the
hypothalamus
of
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Peptides,
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333-343
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infusion
of
endorphins
into the ventral tegmental area:
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Evidence for
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1005-1014
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[D-Ala2 ,D-Leu5]enkephalin on feeding
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a
synaptic
Pharmac.
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by
and
~
activation
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6
antagonists,
opioid
receptors:
Life
Sci.,
37:
497-503
Tseng L-F, Loh H, Li CH (1977) Human $-endorphin:
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Kuschinsky
Pharmacal., 318:
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of
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in
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143-147
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79:
K
into the nucleus accumbens induces akinesia and catalepsy, but
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W
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content
625-630
and
75
,PART II: MUTUAL INTERACTIONS BETWEEN NORMAL SLEEP AND ENDOGENOUS OPIOID
SYSTEM
:S
CHAPTER
ENDOGENOUS OPIOID PEPTIDES AND SLEEP
ABSTRACT
Intracerebroventricular
inhibitor
phosphoramidon
(i.c.v.)
{25-100
administration
induced
~g)
a
non-rapid eye moevement sleep (NREMS) and rapid
of
the
enkephalinase
dose-related decrease in
eye
movement
sleep
(REMS)
time, with a corresponding increase in wakefulness.
~g)
The local application of phosphoramidon (1-25
(LC)
periventricular
or
into the locus coerulus
gray (PVG) substance also inhibited both NREMS and
REMS, and increased wakefulness.
Pretreatment
significantly
with
naltrexone
(0.1
mg/kg,
i.p.
application of naltrexone (10 #g/brain
area)
also
min
15
reduced the phosphoramidon-induced insomnia.
prior)
Similarly, local
decreased
the
insomnia
induced by the administration of phosphoramidon into the PVG or LC.
These findings indicate that endogenous
opioid
peptides
are
important
modulators of wakefulness.
INTRODUCTION
Few
studies
performed.
(Havlicek
on
this
subject
It has been found that
et
administration
al.,
of
1978).
0.5
#g
This
with
controversal
~-endorphin
was
confirmed
B-endorphin
et al.,
of either
1982).
corresponding
The
opioid
in
cats
(King et al., 1981 ).
authors did not find significant changes in
injection
results
have
been
had an arousal effect in rats
sleep
parameters
by
i.c.v.
However other
after
i.c.v.
#g B-endorphin, met-enkephalin or leu-enkephalin (Riou
doses,
species
peptide
interpretation of these results.
might
For
and
way
be
of
example,
of
administration
crucial
recent
importance
data
of
in
indicate
the
the
that
78
REM
sleep stages using the combined EMG, ECoG and hippocampal EEG parameters
(Fig 1 ).
ECoG
REM sleep episodes were identified by the appearance of low voltage
fast
waves, hippocampal theta pattern and EMG silence.
NREM sleep was
characterised by high voltage slow ECoG waves together with low
EMG.
Awake
stage was identified by high EMG and fast low voltage ECoG.
Drug
Phosphoramidon (Peninsula
i.c.v.
(maximum volume 2
Lab)
~1.
dissolved
i.c.v.
or 1
~1
in
saline
was
administered
local application).
hydrochloride (Endo Lab) was administered intraperitoneally (i.p.)
Naltrexone
dissolved
in saline.
Statistics
Statistical analysis was
done
using
paired
Student
t-test.
Statistical
significance was accepted at P-value of 0.05 or less.
RESULTS
Intraventricular administration of enkephalinase inhibitor
The administration of
decreased
The
sleep
the
phosphoramidon
increased
wakefukness
whilst
it
NREM and REM sleep stages in a dose-dependent manner (Fig 2).
suppressant
effect
of
phosphoramidon
was
associated
with
behavioural signs of excitation such as head shakes and body scratching.
The
opiate antagonist naltrexone administered in a dose range which did not alter
the
sleep-waking
pattern in the control animals (0.1-0.5 mg/kg) antagonized
phosphoramidon-induced insomnia (Fig 2).
Nicroinjection of enkepholinase inhibitor
Locus coerulus
Application
decreased
Naltrexone
NREM
(10
of
phosphoramidon
(1-25
~g)
into
the
locus
coeruleus
40-50% and REM 20-27% sleep stages in a dose-related manner.
~g.
5
min
prior)
decreased
the
phosphoramidon-induced
insomnia.
Central gray substance
Phosphoramidon (1-25
substance
of
~g)
injected into
the
periventricular
arousal effect of phosphoramidon was decreased by naltrexone (10
prior) (Fig 3).
gray
(PVG)
the brain induced a significant increase in wakefulness.
~g.
This
5
min
79
TT= 300 min
>>~
@:S(Saline 2 'f.l] icv)
(11.00-16.00 h)
80
CJ Ph ( Phosp horam idon 25 l-l9 icv)
f."ffi]Ph(
so f-!9 icv)
IS'::l Ph(
100 ll9 icv)
60
E nx(naltrexone 0.1 mg/kg ip)
E3nx{
O.t mg/kg +Ph 50 l-l9)
waking
100
NREM sleep
.§
.s"0"'
~
0
40
u
~
REM sleep
"
20
£
0
•
o,o
Figure 2:
Enkephalinase inhibition and wakefulness in
mean±S.E.M
administered
naltrexone.
ror
6
rats.
enkephalinase
Note
that
inhibitor
the
insomnia
phosphoramidon,
rats.
Each
induced
was
by
bar
is
i.c.v.
decreased
by
80
waking
)-
1 00
>-
TT= 300 min
(11.00-16.00 h)
NREM sleep
80
f2ZI
c:J
IT23
E:'ID
S(Sallne 1 ].!! icv)
Ph( Phosphor-amidon l ).lg/PVG)
Ph(
5 ).lg/PVGJ
Ph(
25 Jlg/PVG)
IB nx(naltrexone 10 )lg/PVG)
g nx{
10 J19 -.Ph 25 J19/PVG)
60
40
REM sleep
20
Figure 3:
mean
£nkephalinase inhibition and wakefulness in
±S.E.ro
for
3-5 rats.
rats.
bar
is
Note that the insomnia induced by intracerebral
local administered enkephalinase inhibitor phosphoramidon, was
naltrexone.
Each
abolished
by
81
DISCUSSION
The results
phosphoramidon
o~
this study indicate that specific enkephalinase
induces
both
NREM
and
REM
antagonized the sleep suppressive action
of
inhibitor
sleep suppression.
enkephalinase
Naltrexone
inhibitor
which
suggests that the increased wakefulness induced by phosphoramidon is mediated
by activation of opioid receptors.
The local application
of
phosphoramidon
into
specific
induced naltrexone sensitive insomnia in this study.
the fact that microinjection
behavioural
excitation
of
opiates
(Jacquet
and
into
Wolf,
these
brain
1981 ).
The
indicate that an endogenous opioid system, particularly
system
plays
an
important
role
in
the
brain
regions
This is consistent with
areas
enkephalinergic
the
maintaince
induces
above data might
of
wakefulness.
Furthermore, a number of pathological conditions such as stress, anxiety
other
psychic
disturbances,
in
which
symptom, are known to be associated with increased endorphin levels.
therefore
conceivable
that
and
an increase wakefulness is a common
activation
It
is
of the enkephalinergic system might
constitute the common mechanism underlying various sleep-waking disturbances.
REFERENCES
Havlicek V, Labella F, Pinsky C, Childiaeva R, Friesen H (1978)
In:
Characteristics and functions of opioids, JM
van
Ree
and
L
Terenius
(eds) Elsevier, Amsterdam pp 423-448
Hazato T, Shimamura
M,
Katayama
T,
Kasama
A,
Nishioka
S,
Kaya
K
Enkephalin degrading enzymes in cerebrospinal fluid, Life Sci., 33:
Hudgin RL, Charleson SE, Zimmerman M, Mumford R, Wood PL
selective peptide inhibitors. Life Sci., 29:
Jacquet
YF,
Wolf
G
(1981)
Morphine
and
(1981)
(1983)
443-448
Enkephalinase:
2593-2601
ACTH-24:
correlative
behavioral
excitations following microinjections in rat periaqueductal gray, Brain Res.,
219:
214-218
King C, Masserano JM, Codd
E,
Byrne
WL
(1981)
Effects
of
~-endorphin
morphine on the sleep-wakefulness behaviour of cats, Sleep, 4:
Riou F, Cespuglio R, Jouvet M (1982) Endogenous peptides and sleep
peptides
Neuropeptides, 2:
without
255-264
significant
effect
on
the
and
259-262
in
sleep-waking
the
rat
cycle,
82
Suda H, Aoyagi T, Takeuchi T, UmezaYa H (1973) A thermolysin inhibitor
by actinomycetes:
phosphoramidon, J.
Antibiotics, 31:
Umezawa S, Tatsuta K, Izawa 0, Tsuchiya T, Umezawa
H
metabolite phosphoramidon, Tetrahedron Letters, 1:
produced
621-623
(1972)
97-100
A
new
microbial
83
CHAPTER 4
STAGES OF VIGILANCE AND ENKEPHALIN-INDUCED SEIZURES
ABSTRACT
The effects of different sleep-wakefulness stages
seizure
phenomena
activities
of
were
studied
in
pari etc-frontal
the
the
rat
by
on
enkephalin-induced
recording the electrical
dorsal
cortex,
hippocampus
and
submandibular/nuchal muscle.
2
Administration of [D-Ala ]-Met-enkephalinamide (DALA, 10
induced
wakefulness.
hippocampus
The DALA-induced
were
sleep stage.
of
signs
electrographic
seizure
epileptiform
significantly
during
~g/2~1.
SWS,
activities
REM
in
i.c.v.)
sleep
and
EMG
and
ECoG,
higher during wakefulness compared with any
REM sleep significantly
inhibited
activity compared with SWS and wakefulness.
DALA-induced
ECoG
spiking
Similarly SWS decreased the ECoG
spiking activity compared with wakefulness.
It is suggested that an enkephalinergic system may
ethiopathogenesis
of
epilepsy
be
involved
in
the
of the -awaking type- particularly that with
petit mal characteristics.
INTRODUCTION
The clinical finding that some epileptic attacks
during
occur
more
frequently
the day and others at night suggests an influence of the sleep-waking
cycle on the occurence of epilepsy (Janz,
1962).
Although
some
types
of
epilepsy occur mainly during sleep, their occurence is not facilitated evenly
by different sleep stages.
Recent
data
indicate
that
intracerebroventricular
administration
of
enkephalin in rats induces electrographic and behavioural epileptic phenomena
(Dzoljic et al., 1979;
endogenous
opioid
Urea et al., 1977), and it has
peptides
play
a
modulatory
been
suggested
that
role in the pathogensis of
epilepsy (Dzoljic and Poel-Heisterkamp, 1982).
In
order
to
understand
better
the
relationship
between
stages
of
84
vigilance,
endopioids
epilepsy,
studied
we
effect
the
of
sleep-wakefulness cycle on enkephalin-induced seizures.
MATERIALS AND METHODS
Adult, male Wistar rats weighing 175-200 g were used.
of
the
electrocorticogram,
silver
recorded
electrical
bilaterally
provided
from
the
activities
implanted
the
neck
were
stage of vigilance, with
and
volume
from
the
region
~g./2 ~1)
means
administration
of
upon
the
rate
of
flow,
All rats were allowed at least a
Animals were
maintained
constant light/dark periods (light phase 06.00-22.00 h) and experiments
were carried out between 12.00-16.00 h to avoid variations due to changes
the
of
in unrestrained rat, in any
control
drug injection.
by
A new cannula system
electrodes.
external
(EMG)
Hippocampal
muscles.
CA1
7-day recovery period before experiments commenced.
-~~d~r
electromyogram
submandibular
steel
(DALA, 10
full
of
The
intracerebroventricular
of
[D-Ala2 -met]-enkephalinamide
frequency
and/or
recorded
stainless
possibility
recording
screw electrodes were threaded into the
bone overlying the frontal and parietal cortices.
was
For the
circadian
rhythm.
Student's t-test.
Results
were
analysed
in
statistically using paired
Statistical significance was accepted at P-value
of
0.05
or less.
RESULTS
DALA given during wakefulness induced
phenomena
such
or
of
seizure
rapid
However, the same dose of DALA given during slow wave
eye
movement
(REM)
electrographic signs of seizure (Fig 1 ).
resistant
signs
as cortical or hippocampal spikes and myoclonic contractions
in the muscles.
(SWS)
electrographic
sleep
produced
The REM
sleep
to DALA -induced seizure compared to SWS.
sleep
significantly
less
stage
more
proved
However, the inhibitory
effects of SWS and REM sleep on hippocampal spikes and myoclonic contractions
were
not
significantly
DALA-induced epileptic
Behavioural
phenomena
different
phenomena
such
as
from
were
each
observed
other.
during
-wet-dog-shakes-
and
The
most prominent
the
-fall
awake
stage.
down-
associated with the epileptic burst in the EEG and appeared only if DALA
were
was
85
"
AW
"0
0 10
10
••
0
.E
8
M
AW
-
-
0
£
6
J1
"
AW
-~
4
~
•"
0
~
~
M
0
EEGeortex
EEG hiPP.
-=xto
""
-~
l
~
EMG sub.m.
30
AW
AW
~ [h
"
·a. 20
~
AW
0 4
~
""
EEG cortex
EEG h•PP.
The effect of
~
vigilance
periods after drug administration.
respectively
spikes for 7 rats.
the
were
Note that sleep,
intensity
phenomena in the cortex,
of
the
AW
0
ii\
;
,o
0
~
EMGsub.m.
introcerebrovenrticularly)-induced
ECoG.
EMG sub.m-
"EX 10
40
0
~
(sub.m.).
EEG hipp.
EEG cortex
0
".
~ 8
~
"
AW
0
reduced
AW
8
,.
and
4
"0
;: 12
Figure 1:
6
-~
~
ENG
AW
8
EEG cortex
states
seizure
on
EMG sub- m.
EEG hipp.
enkephalin
phenomena
(DALA,
during
Only spikes up to 200 and
4
in
time
the
Each vertical bar is mean±S.E
counted.
particularly
REM
electrographically
hippocampus(hipp)
the
4DO~V
and
the
sleep
significantly
registered
submandibular
epileptic
muslces
86
given during wakefulness and not during the SWS and REM sleep.
DISCUSSION
It is known that REM sleep deprivation
(Cohen
and
Dement, 1965).
increases
normal REM sleep and to a lesser extent SWS decrease
electrographic
signs
of
neuronal
excitability
However, the results of this study indicate that
significantly
all
the
DALA-induced seizure phenomena in the hippocampus.
cortex and submandibular/neck muscle when compared to wakefulness.
The cause
of the inhibitory effect of sleep on enkephalin-induced seizure is not clear.
However the epileptic properties of enkephalin can
be
antagonized
only
by
anti-petit mal and not anti-grand mal drugs (Snead and Bearden, 1980).
Furthermore, petit mal
paroxysm
occurs
mainly
sleep-waking transition period (Janz, 1962).
during
wakefulness
or
This clinical observation is in
accordance with the inhibitory effects of sleep on enkephalin-induced seizure
demonstrated
in
this
study.
Therefore,
a
possible
involvement
of
an
endogenous opioid system in the ethiopathogenesis of epilepsy of the -awaking
type-,
particularly
considered.
those
with
petit
mal
characteristics
In addition, this experimental model can be used as
should
a
be
reliable
tool in future studies of the relationship between different vigilance states
and drug-modulated neuronal excitability.
REFERENCES
Cohen HB, Dement WC (1965) Sleep:
shock
in
rats
after
changes
deprivation
of
in
threshold
to
"paradoxical"
electroconvulsive
phase, Science .• 150:
1318-1319
Dzoljic MR, Lely AJvd, Mourik JBA (1979) Enkephalin-induced
blocked
myoclonic
twitches
by ergometrine and potentiated by haloperidol, Psychopharmacol., 66:
111-116
Dzoljic MR. Poel-Heisterkamp ALvd (1982) Delta opiate receptors are involved
the endopioid-induced myoclonic contractions, Brain Res.
Bull., 8:
in
1-6
Janz D (1962) The grand mal epilepsies and the sleep-waking cycle, Epilepsia, 3:
69-109
Snead OC, Bearden LJ (1980) Anticonvulsant specific
for
petit
mal
antagonize
87
epileptogenic effect of leucine enkephalin, Science, 210:
Urea G, Frenk H,
~~algesic
Liebeskind
JC,
Taylor
A
(1977)
and morphine properties, Science, 197:
Morphine
83-86
1031-1033
and
enkephalin:
88
PART III:
MUTUAL INTERACTIONS
BETWEEN
DISTURBED
SLEEP
AND
OPIATE/OPIOID
PEPTIDES
CHAPTER 5
REM
DEPRIVATION
SLEEP
THE
DECREASES
ANTINOCICEPTIVE
PROPERTY
OF
ENKEPRALINASE-INHIBITION, MORPHINE AND COLD-WATER-SWIM
ABSTRACT
1) In this study, the
stress
e~£ect
of REM
on
investigated
was
enkephalinase-inhibition
sleep
three
deprivation
analgesic
(phosphoramidon),
(REMSD)
procedures
and
morphine
or
as
chronic
follows:
cold-water-swim
induced antinociceptions.
2) REMSD (96 hr) completely abolished the analgesic effect of
~g.
(250
phosphoramidon
i.c.v.), morphine (20 ~g. i.c.v.) and 5 min cold-water-swim (5° C,
cold).
3) Rats exposed to chronic stress regimen did not show any tolerance
to
the
analgesic effect of phosphoramidon or CWS.
4) These data indicate that REMSD can decrease pain
threshold,
probably
by
altering enkephalinergic and other transmitter systems.
5) It is suggested that
conditions
which
pharmacological
manipulations
and/or
pathological
decrease REM sleep might affect the efficacy of opiate and
other analgesic procedures.
Additional clinical
studies
are
necessary
to
clarify the relationships of REMSD and pain threshold in human.
INTRODUCTION
The inhibitory action of opiates and opioid peptides on REM sleep is well
documented
(Khazan
et
al., 1967
King et al., 1981 ), while the effect of
REM sleep or REM sleep deprivation (REMSD) on opiate activity is less
However,
it
is
kno~
that
the
episodic
clear.
release of humoral endorphin is
associated with REM sleep (Oksenberg et al., 1980)
and
REM
sleep
inhib~ts
89
excitation
neuronal
exogenously
by
induced
(Ukponmwan and Dzoljic, 1983).
administered
enkephalins
These data suggest that phasic changes during
the sleep-waking cycle can modulate response to opiates and opioid peptides.
In order to clarify further the relationship between
and
sleep
disturbances
opiates, the effect of REMSD on the antinociceptive property of morphine
and enkephalinase inhibitor phosphoramidon was investigated.
potentiates
enkephalinergic
biotransformation
phosphoramidon
of
activity
enkephalins
in
the
(Hudgin
brain
et
al.,
by
Phosphor amidon
decreasing
1981).
the
using
By
and morphine, it was possible to study the effect of REMSD on
analgesic effects
induced
by
endogenous
opioid
pepides
and
exogenously
administered opiates.
Since it is known that REMSD can reduce the
electrical
stimulation
pain
threshold
to
noxious
(Hicks et al., 1979), we included in this study, the
relationship between REMSD and physically
induced
antinociception
such
as
cold-water-swim analgesia.
MATERIALS AND METHODS
All experiments were performed on male adult Wistar rats weighing between 150
and 175 gat implantation.
Surgery
For the intracerebroventricular (i.c.v.) administration of
steel,
guide
ventricle.
the
cannula
was
constant
drugs.
stainless
directed 1 mm above the lateral
The injection cannula protruded 1 mm below the guide cannula into
ventricle.
At
least
experiments were commenced.
a
stereotaxically
environment
5-7
day
recovery
period
was
allowed
before
Throughout this study, all animals were kept
in
chamber with a light-dark cycle 12 hr (light period
09.00-21.00 h) and a room temperature 22±1°C.
Food and clean drinking
water
were available ad libitum.
Three groups of
phosphoramidon,
experiments
(ii)
cold- water-swim (CWS)
divided
into
the
effect
analgesia.
were
of
The
three groups as follows:
and non-stressed (controls) rats.
performed
(i)
analgesic
effect
of
morphine on pain threshold, (iii) the
animals
in
each
experiment
were
REM sleep deprived, chronic stressed
Each group was submitted to CWS
or treated either with phosphoramidon or morphine.
procedure
90
REM sl&ep deprivation (RENSD)
REMSD was carried out
using
the
conventional
previously described (Mendelson et al., 1974).
of unequal
REMSD
corresponded
to
{Hicks
the
et
rat
al.,
sleep deprived for 96 hr continously.
free
assess
to
food
and
pot-
In order to avoid the problem
we
In the present set up, the animals had
clean drinking water.
Throughout the REMSD, the
water in the tank was changed regularly (11 .00-12.00 hr) once
During
the
period
of
technique
used platforms whose area
2
weights (14 cm : 100 g). Each rat was REM
body
1977),
-flower
every
24
hr.
cleaning the animals were allowed free locomotion in
individual cages, for 1 hr during which rats were kept awake manually.
Stress
The rats in this group were forced to swim in water 17-18 °C and 7 em for
2
hr
daily
(11 .00-13.00hr)
for
4
days.
The
animals were then allowed
spontaneous amount of sleep for the remaining period
Weight
loss
in
the
stress
and
of
the
day
(22
hr).
REMSD groups were not different from each
other.
Control
These animals were housed singly and allowed to have spontaneous
amounts
of sleep.
Cold-water-swim analgesia (CWS)
CWS was produced using the procedure
(1982),
with
temperature.
a
slight
modification
described
in
by
duration
Bodnar
of
and
swimming
The rats were forced to swim for 5 min in
water
5
Sperber
and water
°C.
Pain
threshold was determined before and then at 30, 60, 90 and 120 min after CWS.
The measurement of pain threshold was commenced 30 min
after
CWS
to
allow
administration
of
these
animals to become completely dry.
Phosphoramidon or morphine analgesia
This was
induced
by
intracerebroventricular
substances
Determination oT nociception
Pain sensitivity test was carried out between 13.00 and 16.00 h according
to
the
analgesiometric
method (Randall and Seltto, 1957).
The nociception
was expressed in the form of analgesiometric scores (AMS) g mm- 2 pressure.
The cut off value was maintained at 500 g mm- 2 to avoid damage to the paw.
Response to pain in this study
is
measured
by
squeak
or
paw-withdrawal.
91
Animals
which
above 150 g mm- 2 during control testing were not used
scored
for further experimentation.
In all three groups:
baseline
vas
pain
threshold
measured
phosphoramidon was administered.
af~er
REMSD, stress or controls
saline,
before
Nociception was
then
morphine
followed
for
2
or
hr
drug treatment.
Drugs
The following drugs were
(Merck)
and
saline.
A maximum of
used
phosphoramidon
2~1
in
this
(Peninsula
study,
morphine
hydrochloride
Lab) were administered dissolved in
was given i.c.v.
over a period of 10 sec.
Statistical analysis
The significance of differences between
after
different
treatments
analysis of variance
the
analgesic
scores
obtained
was evaluated by Student t-test. once a one way
(ANOVA)
had
revealed
that
different populations (Steel and Terrie, 1980).
the
samples
represented
Statistical significance was
accepted at P-values of 0.05 or less (two tailed).
RESULTS
The effect of REMSD on the analgesia induced by enkephalinase-inhibition
The intracerebroventricular
(250~g/~l)
in
threshold
during
control
or
the
(i.c.v.)
stressed
first
Phosphoramidon-induced
analgesia
administration
animals
caused
30
min
in
control
after
and
an
of
phosphoramidon
increase
treatment.
drug
stressed
animals
accompanied by signs of central excitation such as wet-dog-shakes,
grooming,
hypermotility.
The
in pain
was
excessive
antinociceptive action of phosphoramidon was
ccmpletely abolished by REMSD (Fig 1)
The effect of REMSD on morphine analgesia
Morphine (20
and
~g/2~1.
stressed animals.
i.c.v.) induced profound analgesia in
stressed group compared with control animals.
induced
by
2).
control
The
increase
pain
threshold
morphine lasted about 2 hr during which rats remained quiet with
decreased motility (not evaluated).
(Fig
both
The antinociceptive action of morphine was lowered in
REMSD completely
antagonized
analgesia
92
e---.11
Control + phosphoramidon (ph)
o---o Chronic stress
"'...cu
8
"'
u
o---o REMSD
+
ph
+ ph
200
-
·;:
cu
E
.2
"'
§,
100
2>----Q'i'-:r-
""'"'
0
0
t
15
Saline,
icv
Figure 7:
The
phosphoramidon
0
t
15
--<j!>----------<;:5!
30
60
120
Time (min)
ph 250 !J9, icv
inhibitory
(ph)
effect
induced
of
REM
analgesia.
control n=26, REMSD n=25 and stress
n=19
sleep
Each
deprivation
point
groups.
is
The
(R£MSD)
on
mean± S.£.M for
number
of
animals
receiving phosphoramidon in each group are as follows:- control+ ph=8, REMSD
+
and
stress
intracerebroventricularly
Note
+
induced
control and stressed animals.
completely abolished by REMSD.
an
phosphoramidon
that
increase
in
This analgesic effect
pain
of
given
threshold in both
phosphoramidon
was
93
o----e Control + morphine ( mo)
o---o Chronic stress + mo
500
.
o----c
REMSD
+
mo
400
~
,0
,
·;:
300
<11
1•
Q)
m
200
;;;
0
•
100
0
0
15
+
Saline,
0
15
30
60
t
120
Time {min)
mo 20 ]..lg, icv
icv
Figure 2:
(mo)
The inhibitory effect of REM sleep deprivation (REMSD) on morphine
analgesia.
stress n=19.
follows:
Each point is mean ± S.E.M for control n=26, REMSD n=25 and
The number of animals receiving morphine in each group
control
+
mo=10,
morphine induced long lasting
abolished by REMSD.
REMSD
+
increase
mo=10
in
and stress+ mo=B.
pain
threshold
was
are
as
Note that
completely
94
The effect of cold-water-swim (CWS) analgesia
The
pain
threshold
cold-water-swim
was
significantly
higher
in
compared to control or stressed animals.
rats
exposed
to
This CWS analgesia
was antagonized by REMSD (Fig 3).
e----e
Control +Cold Water Swim (CWS)
o---o Chronic stress
o--c REMSD + CWS
••
+
CWS
c
0
u
2QQ
VI
-~
c
"•
E
0
·;;
1 00
..Q'l
c•
•
0
t
Saline,
15 250
icv
Figure 3:
30
ti
60
90
cws
120
Time (min}
5'
The antagonistic
cold-water-swim
analgesia
n~26,
RENSD n=25 and stress
each
group
was
n=6.
effect
n~19.
Note
REM
of
(CWS).
Each
sleep
point
deprivation
(REMSD)
on
is mean± S.E.M for control
The number of animals submitted to CWS
in
CWS analgesia in both control and stress
that
groups was completely abolished in REM sleep deprived rats.
DISCUSSION
It
is
of
interest
enkephalinase-inhibitor
to
note
that
(phosphoramidon)
abolished in REM sleep deprived rats.
The
the
antinociceptive
and
morphine
activity
was
phosphoramidon-induced
of
completely
analgesia
95
(Rupreht
et al., 1983) is probably due to the known enkephalinase inhibition
and the consequent increase in the enkephalinergic activity (Hudgin
1981 ).
et
al.,
In addition it has been demonstrated that the enkephalinase inhibitor
thiorphan also possesses antinociceptive activity (Roques et al .• 1980).
The
mechanism
by
which
phosphoramidon is not clear.
REMSD
might
have
of the enkephalinergic system.
the known inhibitory effects of
Girdwood,
1981 ).
the
analgesic
effect
of
However, one possible explanation could be that
animals deprived of REM sleep
activity
antagonizes
REMSD
Consequently,
a
lowered
level
in
functional
This possibility is consistent with
on
peptide
synthesis
(Shapiro
and
the reduced activity of opioid peptides in
the brain may decrease pain threshold.
Previous
studies
have
demonstrated
that a decrease in peptide synthesis did not alter morphine analgesia (Loh et
al., 1969;
Tulunay and Takemori, 1974).
action
morphine
of
cannot
be
Hence the decreased antinociceptive
explained by a possible reduction in opioid
peptides.
Therefore,
another
neurotransmitter(s)
considered.
For
dopaminergic
explanation
which
are
example
activity
it
and/or
known
has
such
to
been
as
alteration
modulate
demonstrated
decrease
1983;
other
should
increase
and
be
of
cholinergic
Mogilnicka
et
Tsuchiya et al., 1969) could antagonize the analgesic activity of
opiates (McGilliard and Takemori, 1979;
al.,
that
serotoninergic
transmission which occur during REMSD (Farber et al.,
al., 1981;
in
nociception
1976).
Garlitz and Frey, 1972;
Tulunay
et
These data suggest that changes in biogenic amines might be an
important factor in the inhibitory action of REMSD on
analgesia
induced
by
exogenous and endogenous opioid peptides.
The physiological basis of CWS analgesia is not known,
but
due
to
the
lack of cross-tolerance with morphine produced antinociception, it appears to
be mediated by a non-opioid mechanism (Bodnar et al.
system
which
has
been
shown
to
play
1978) e.g the GABAergic
a role in analgesia in response to
environmental stress (Skerritt et al., 1981 ).
Changes
in
the
activity
of
GABA system were demonstrated in REMSD animals (Micic et al., 1967).
Relative to controls, no change in threshold to noxious paw pressure
observed
in
chronically
stressed
animals.
play an important role in the slight decrease in pain threshold
rats deprived of REM sleep.
was
Thus stress does not appear to
observed
in
96
Finally, although the mechanism by vhich REMSD decreases
is
pain
not certain, some clinical consequences should be considered.
should be
expected
accompanied
with
that
pathological
decrease
REM
conditions
sleep
could
and/or
drug
modify
the
effectiveness of opiates and other analgesic procedures.
studies
are
threshold
Namely, it
treatments
therapeutic
Additional clinical
required to clarify the significance of REM sleep in maintaince
of normal nociception in human.
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manipulation of GABA, Behav Neural Biol 36:
311-314
Bodnar RJ, Kelly DD, Steiner SS, Glusman M (1978) Stress-produced analgesia
morphine-produced
analgesia:
Lack
of
cross,
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and
Biochem Behav 8:
661-666
Farber J, Miller JD, Crawford KA, Mcmillen BA
receptor
sensitivity
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rat
brain
(1983)
Dopamine
metabolism
after REM sleep deprivation, Pharmacol
509-513
Gorlitz BD, Frey HH (1972) Central monoamines and antinociceptive
Eur J Pharamcol 20:
Hicks RA,
Okuda
A,
drug
Thomsen
D
(1977)
Depriving
rats
of
REM
sleep:
during
the
95-102
Hicks RA, Coleman DD, Ferrante F. Sahatjian M, Hawkins J (1979) Pain
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action,
171-180
identification of a methological problem, Amer J Physiol 90:
in
and
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recovery from REM sleep deprivation, Percept Mot Skills 48:
687-690
Hudgin RL, Charleson SE, Zimmerman M, Mumford R,
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N,
Weeks
electromyographic
JR,
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LA
Wood
2593-2601
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behavioural
Enkephalinase:
PL(1981)
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Electroencephalographic,
during
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Exp
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521 -531
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and
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cycloheximide,
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2711-2721
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AE
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The
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respiratory depression, Eur J Pharmacal 54:
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Mendelson WB, Guthrie RD, Frederick G, Wyatt RJ (1974) The flower pot
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Micic D, Karadzic
glutamic
V,
Rakic
LM
(1967)
Changes
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gamma-aminobutyric
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of paradoxical sleep, Nature 215:
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REM
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Roques BP, Fournie-Zaluski MC, Soroca E,
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JM,
Maleroy
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Schwartz JC (1980) The enkephalinase-inhibitor thiorphan shows antinocieptive
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R~preht
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Skerritt JH, Trisdikoon P, Johnston GAR (1981) Increased GABA binding
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867-873
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Sleep 82. pp 250-252, Karger, Basel.
and
enkephalin-induced
98
CHAPTER 6
ANALGESIC EFFECT
OF
ENKEPHALINASE
INHIBITION
IS
MODULATED
BY
MONOAMINE
OXIDASE B AND REM SLEEP DEPRIVATION
ABSTRACT
Both the MAO-B inhibitor deprenyl (2.5-10 mg/kg, i.p., 60 min prior)
the
MAO-B
i.c.v.)
~g.
substrate E-phenylethylamine (PEA, 40
analgesic action of
in
the
animals
enkephalinase
allowed
normal
inhibitor
sleep.
i.c.v.) potentiated the
phosphoramidon
Deprenyl
~g.
(250
The enhancing effect of PEA on
phosphoramidon analgesia was further potentiated by deprenyl (5 mgjkg,
pretreatment.
and
5mg/kg, i.p.) or PEA (40
~g.
i.p.)
i.c.v.) given alone
did not induce analgesia in animals allowed undisturbed sleep.
REM sleep deprivation (REMSD) decreased
abolished
the
analgesic
effect
of
the
basal
phosphoramidon.
pain
threshold
and
The administration of
deprenyl and/or PEA failed to restore the analgesic effect of
phosphoramidon
in REM sleep deprived animals.
The results indicate that excess PEA has
analgesic
activity
of
endogenously
a
stimulatory
effect
on
the
released
enkephalins
in rats allowed
undisturbed sleep but not in REM sleep deprived animals.
It is suggested that the failure of phosphoramidon
after
REMSD,
is
probably
due
to
a
functional
to
induce
analgesia
insufficiency
of
an
enkephalinergic system.
INTRODUCTION
Two forms of monoamine oxidase (MAO) are present in the mammalian
MAO-A
and
MAO-B.
Serotonin,
dopamine
substrates for MAO-A, while MAO-B shows
(PEA)
(Yang
evidence
and
Neff,
suggested
pharmacological
that
1974;
noradrenaline
for
Garrick and Murphy, 1980).
inhibition
effects/toxicity
animals (Iwamoto and Ho, 1972;
and
selectivity
of
of
MAO
activity
are
brain,
preferred
E-phenylethylamine
Several lines of
increased
the
opiates in patients (Taylor, 1962) and
Boden et al., 1984), although this effect was
99
only
seen
when both MAO-A and MAO-B were inihibited (Jounela et al., 1977).
Nevertheless some interactions between MAD-E
peptides
have
been reported.
of PEA (the
substrate
exogenously
administered
Garzon et al., 1980).
be
modulated
for
inhibitors
and
opiates/opioid
For example, an inhibition of MAO-B or excess
MAD-B)
potentiated
opiates/opioid
the
peptides
analgesia
(Fuentes
induced
et
al., 1977;
In addition, some pharmacological actions of
by opioid receptor blockade (Kubota et al., 1982;
Cooper, 1984) suggesting
a
possible
interaction
between
by
PEA
can
Dourish and
PEA
and
opioid
receptors.
This study was undertaken to clarify the relationship between
the
analgesic
effect
of
endogenous
(synaptic)
administration of an enkephalinase
inhibitor
sleep
REMSD.
and
animals
subjected
to
to
In
MAO-B
enkephalins
rats
allowed
these
following
undisturbed
experiments REM sleep
deprived rats were used because it has been shown that both MAO-B
and
inhibitors
REMSD possess antidepressant activity (Mann and Gershon, 1980;
al., 1980) and modulate
(Garzon et al., 1980;
the
analgesic
action
of
and
opiates/opioid
Vogel et
peptides
Ukponmwan et al., 1984a).
MATERIALS AND METHODS
Adult, male Wistar rats weighing 150-175
Drugs
g
were
used
in
this
study.
were injected intracerebroventricularly (i.c.v.), when required, via a
stainless
steel
placement
of
cannula
i.c.v.
implanted
cannula
was
described (Ukponmwan et al., 1985).
in
the
verified
A 4
day
lateral
ventricle.
using
Correct
the procedure recently
recovery
period
was
allowed
after cannula implantation before the experiments were commenced
REM sleep deprivation
REMSD(96 h) and the corresponding stress-control,
~reviously
described
(Ukponmwan
method of Mendelson (1974).
et al.
temperature
of
22±1°C
a
out
as
1984a) using a modification of the
constant
Throughout this study, all
environment
room
with
an
ambient
and automatically regulated light-dark cycle of 12 h
(light period 09.00-21.00 h).
ad libitum.
carried
This method is known to selectively deprive rats
of REM sleep after 96 h (Mendelson et al., 1974).
animals were maintained in
were
Food and clean drinking water
were
available
100
Assessment oT nociception
Pain sensitivity to noxious paw pressure was assessed between 13.00-16.00
h
using
to
the
analgesiometric
technique
of Randall and Selitto (1957).
Nociception was measured 15, 30, 60 and 120 min after drug administration and
expressed as analgesiometric scores (AMS) g mm- 2 pressure.
was measured by a squeak or paw-withdrawal.
The cut off value
Animals scoring above 150 g mm- 2
during control testing were not used for further experimentation.
Drugs
The following drugs were used in this study:
phosphoramidon
(Peninsula
Laboratories, San Carlos, CA. USA), deprenyl (Chinoin, Budapest, Hungary) and
B-phenylethylamine (PEA, Sigma, St.
i.p.
Louis, Mo, USA).
Drugs for
i.c.v.
or
administration were dissolved in physiological saline and administered
2~1
in volumes of
brain
is
known
or
500~1
respectively.
Since the half-life of PEA
in
tr.e
to be very short (Wu and Boulton, 1975), this substance was
given 5 min after phosphoramidon and the pain threshold was measured
10
min
later.
Statistics
The significance of differences between
the
analgesic
scores
obtained
after different treatments was evaluated by Duncan's new multiple range test,
once a one way
represented
analysis
different
of
variance
populations
(ANOVA)
had
revealed
(Steel and Terrie, 1980;
Statistical significance was accepted
at
P-values
of
0.05
that
samples
Saxena, 1985).
or
less
(two
tailed).
RESULTS
A significant difference in
groups
and
time
intervals
phosphoramidon (Fig 1 ).
and/or
the
was
analgesic
found
scores
after
across
the
Similarly the effect of pretreatment
E-phenylethylamine
(PEA)
on
phosphoramidon
the
various
administration
induced
with
of
deprenyl
analgesia was
significant across the treatment groups (Fig 2, p<0.01 ).
The eFFects oF deprenyl and(or E-phenylethylamine on phosphoramidon-
induced
analgesia in animals allowed normal sleep
The administration of the enkephalinase inhibitor phosphoramidon
et
al.
(Hudgi~
1981) (250 ~g. i.c.v.) significantly increased the pain threshold to
101
500
~
....._
000
~
~
''
~
SALINE
1.0 ml/kg ip (13)
OEPRENYL
2. S mg/kg ip
(9)
DEPRENYL
S. 0 mg/kg ip
'"
DEPRENYL
10.0 mg/kg ip (;)
300
w
"
0
u
~
u
"
t;;
200
~
0
~
u
"
~
z
~
,00
0
30
t
60
0
30
t
SALINE OR
60
f'HO$PH0RAM100N
DEPRENYL
TJME (MIN)-+
{250 J.l9 icv)
Figure 1:
The effect of d&pronyl on the analgesic action of the enkephalinase
inhibitor
phosphoramidon
in rots allowed undisturbed sleep.
determined by withdrawal of
Randall-Selitto
test).
hind
Each
paw
point
from
is
pressure
Nociception was
stimulation
number of animals per treatment group is indicated in parenthesis.
deprenyl
(2.5-10mg/kg,ip,
phosphoramidon
(250~
60
(modified
mean ± SEm at each time point.
Note
The
that
min prior) potentiated the analgesic effect of
icv) in a dose-related manner.
difference from saline pretreated group (p<O.OS).
(*) indicate significant
102
paw pressure.
The most prominent analgesic
effect
was
registered
between
15-30 min after drug administration (Fig 1)
Deprenyl
potentiated
(Fig
the
the
1 ).
MAO-B
Similarly
2. p <0.05).
(2.5-10
i.p.,
60
min
prior)
the
MAO-B
Pretreatment with
increased
substrate,
(40~g.
PEA,
i.c.v.,
5
min
also enhanced the phosphoramidon-induced analgesia (Fig
the
deprenyl
potentiating
(5
mg/kg,
effect
analgesic action of phosphoramidon (Fig
mg/kg,
mg/kg,
analgesic effect of phosphoramidon in a dose-related manner
post-phosphoramidon)
further
inhibitor,
2,
of
i.p.,
PEA (40
p<0.05).
60
~g.
min
prior)
i.c.v.) on the
Neither
deprenyl
(5
i.p.) (Fig 2, p>0.05) nor PEA (40 #g, i.c.v., data not shown) induced
analgesia in animals allowed undisturbed sleep.
The effects of
deprenyl
and
P-phenylethylamine
on
phosphoramidon-induced
analgesia in REM sleep deprived animals
The basal nociceptive threshold in REM sleep deprived rats was
but
significantly,
p<0.05).
the
lower
tha~
slightly,
in animals allowed undisturbed sleep (Fig 2,
Deprenyl (5 mg/kg. i.p .. 60 min prior) induced a slight increase in
pain
of REMSD animals.
threshold
the first 10 min after PEA (40
However,
the
analgesic
~g.
scores
A similar effect was observed during
i.c.v.) administration (data
REM
of
sleep
deprived
deprenyl (5 mg/kg, i.p., Fig 2) or PEA (40 Mg. i.c.v., not
different
from
those
of
not
shown)
were
REMSD
(Fig
2).
The
analgesic
subjected
score of REM sleep deprived rats after
administration of deprenyl (5 mg/kg, i.p., 60 min prior) and/or PEA
i.c.v.,
not
control animals (rats allowed undisturbed sleep).
Phosphoramidon (250 Mg, i.c.v.) had no analgesic action in animals
to
shown).
rats treated with
(40
Mg,
5 min post-phosphoramidon) plus phosphoramidon (250 Mg, i.c.v.), was
not different from those treated with deprenyl alone (Fig 2, p >0.05).
DISCUSSION
In the animals allowed undisturbed sleep, the antinociceptive
the
enkephalinase
inihibitor
phosphoramidon
(specific substrate for MAO-B) and
enyzme).
The
analgesic
effect
deprenyl
of
was
phosphoramidon
increase in endogenous enkephalins and the consequent
receptors
sensitive
to
naloxone
potentiated
(selective
and
naltrexone
effect
of
by both PEA
inhibitor
of
this
was probably due to an
activation
(Chaillet
of
opioid
et al., 1983;
103
500
D
ILL1
400
{n=6)
RATS ALLOWED UNDISTURBED SLEEP
REM SLEEP DEPRIVED ANIMALS
DEP"DEPRENYL (Smg/kg ip)
{n=6)
PH =PHOSPHORAMIOON (250 ~9 icv) ( n=9)
PEA=6-PHENYLETHYLAMINE (40 ).1.9 icv)
'e
E
IJl
300
w
~
0
u
~
!2
(n=l3}
~
~
w 200
~
0
{n=13)
;;
w
Q
~
<
z
<
(no:o56)
'"
ITJ
SAL
Figure 2:
DEP
DEP+PH
The effects of deprenyl (D£P), REM sleep
e-phenylethylamine
on
(PEA)
inhibitor phosphoramidon (PH).
hind
PH
paw
from
pressure
the
analgesic
deprivation
effect
of
Nociception was determined
stimulation
(modified
DEP+PEA+PH
PEA+PH
the
by
icv,SAL).
Each
bar
Randall-Selitto test).
mg/kg,ip)
and/or
PEA
(40
~g.
Note the following:
pain
threshold
undisturbed sleep;
analgesia by R£MSD.
and
or
of
The
saline
The number of
a)
D£P
(5
icv) significantly potentiated the analgesic
effect of PH in animals allowed undisturbed sleep;
basal
PH
is the mean analgesic score ± SEM.
rats per group is indicated in parenthesis.
and
enkephalinase
withdrawal
analgesic score was measured 60 min after DEP and 15 min after
(2;.d
(REMSD)
Ph-induced
analgesia
b)
R£MSD
compared
decreased
to
the
rats allowed
c) D£P and/or P£A did not alter the blockade of PH-induced
Tho levels of significance are given in the text.
104
We
Rupreht et al., 1983).
suggest
that
excess
of
PEA
analgesic action of enkephalins at the synaptic sites.
with reports indicating that MAO-B inhibition and/or
the
pharmacological
effects
peptides (Fuentes et al.;
of
exogenously
1977, Garzon et
facilitates
This is in accordance
excess
PEA
administered
al.,
the
potentiate
opiates /opioid
Ukporunwan
1980;
et
al.,
1983).
The mechanism by which MAO-B inhibition or
analgesic
possible
effect
that
of
PEA
excess
PEA
potentiates
endogenously released enkephalins is not clear.
enhances
the
receptors (Fuentes et al., 1977).
interaction
between
opiates
the
It is
and
their
Thus. the MAO-B system may be an important
regulator of the activity of opioids at the synaptic site in
animal~
allowed
undisturbed sleep.
The described facilitatory action of MAO-B inhibition on
transmission
in
enkephalinergic
animals allowed undisturbed sleep might be of relevance not
only in the physiology of nociception, but also in human disorders
the
alterations
in
MAO-B
have
been
reported.
demonstrated that endogenous depression is
brain
PEA
levels
(Wolf
and
Mosnaim,
in
which
For example, it has been
associated
with
a
decrease
in
1983), whereas an increase in MAO-B
activity is associated with the aging process (Benedetti
and
Keane,
1980).
In such cases an alteration in MAO-B activity and PEA levels could modify the
analgesic effects of endogenous opioid peptides.
However, the results of this study indicate that the possible alterations
in the bioavailability of the MAO-B substrate, PEA, may not play an essential
role in the failure of
deprived
rats.
did not alter
phosphoramidon
to
induce
analgesia
in
REM
sleep
This statement is based on the fact that deprenyl and/or PEA
the
inhibitory
effect
of
REMSD
on
phosphoramidon-induced
analgesia.
The basal pain threshold was lowered in rats deprived of REM sleep.
TCis
is in accordance with a previous study in which, using noxious electric shock
to assess pain sensitivity, it was established that REMSD decreased the
threshold
(Hicks
et
al.,
1979).
threshold in REM sleep deprived rats is not clear.
already
It might be
due
to
the
suggested functional insufficiency of enkephalinergic/endorphinergic
system during REMSD (Ukponmwan and Dzoljic, 1984b;
since
pain
The reason for the reduction in the pain
opioid
peptides
Ukponmwan et
al.,
1985)
play an important role in the regulation of the pain
105
threshold (Basbaum and Fields. 1984).
A functional insufficiency of an opioid system during REMSD (Ukponmwan et
al.,
1985)
might
partly explain why REM sleep curtailment is beneficial in
treating some forms of depression (Vogel et al., 1980),
opioid
activity
and
corresponding
observed in this affective disorder
decrease
(Risch,
since
an
increased
in pain sensitivity have been
1982;
Pickar
et
al.,
1982;
Further clinical experiments are necessary to clarify the roles of
MAO-B
Davis et al., 1979).
and
the enkephalinergic/endorphinergic systems in the regulation of the pain
threshold in human diseases.
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after
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to
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with
stimmuli
in
1148-1151
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~-phenylethylamine
and
and
1059-1064
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723-729
of
phenylethylamine,
Neuropharmacol
19:
106
Garrick NA, Murphy DL (1980) Species differences in the deamination of
and
other
substrates
for
monoamine
dopamine
oxidase in the brain, Psychopharmacol
72:27-33
Hicks RA, Coleman DD, Ferrante F, Sahatjian M, Hawkins J (1979) Pain
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rats
during
thresholds
recovery from REM sleep deprivation, Percept Mot Skills 48:
687-690
Hudgin RL, Charleson SE, Zimmerman M, Mumford R, Wood PL
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Enkephalinase:
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Iwamoto ET, Ho IK (1972) The effects of MAO inhibition on morphine analgesia
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Jounela AJ, Mattila, MJ, Knoll J (1977) Interaction of selective
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Kubota
K,
Matsuoka
Characteristic
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Sakuma
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1221-1224
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Mann J, Gershon S (1980) L-Deprenyl,
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selective
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RM
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the
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108
CHAPTER 7
REM SLEEP DEPRIVATION ANTAGONIZED THE MORPHINE-INDUCED AKINESIA AND CATALEPSY
ABSTRACT
An examination was made o£ the effect of REM sleep deprivation (REMSD) on
some forms of altered motor activity, such as akinesia and catalepsy, induced
by intraperitoneal (i.p.) or intracerebroventricular (i.c.v.)
of
morphine
in
adult,
male
Wistar rats .
administration
Administration of morphine (25
mgjkg i.p.) induced an akinetic-cataleptic syndrome and decreased spontaneous
vertical
motor
activity {SVMA) in animals allowed undisturbed sleep.
decreased the morphine-induced akinesia and catalepsy that are
mediated
by
an inhibitory
~-opioid
system.
i.c.v.
behaviour)
by REMSD.
as
increased
SVMA
~g)
was replaced by excitation in
It is suggested that REMSD is associated with a functional
an
inhibitory
explain
~-opioid
system,
the
morphine.
insufficiency
thus unmasking the excitatory morphine
The proposed insufficiency of an
an
and
Naltrexone (1 mg/kg,i.p.) blocked the
akinetic and cataleptic effects, but not the excitatory effects of
effects.
be
Similarly, decreased motor activity following
administration of morphine (25
form of jumping behaviour after REMSD.
of
to
The locomotor depressant action
of morphine was converted to excitation (manifested
hopping
REMSD
known
endogenous
opioid
system
might
increase in neuronal excitation during REMSD and the therapeutic
effect of REM deficiency in some types of depression.
INTRODUCTION
Interactions between
documented.
sleep (1 ).
For
sleep
example,
and
endogenous
activation
opioid
systems
have
been
of opioid receptor(s) .suppresses REM
REM sleep was associated with the
episodic
release
of
humoral
endorphin (2) and can decrease the neuronal excitation induced by exogenously
administered opioid peptides (3).
It has been suggested that alteration in REM sleep and endogenous
system
are
involved
in
some
psychosomatic
disturbances;
for
opioid
example,
109
increased
REM
endogenous
sleep
density
depression
and
opioid
In
(4-7).
activity
addition,
it
are
associated
has
been
with
shown
pharmacological and mechanical REM sleep deprivation can improve
that
~arms
some
of depression (8) and affect an opiate/opioid-induced analgesia (9).
These data taken together suggest a functional interaction between REM
sleep
and opioid system in both physiological and pathological conditions.
To further clarify the role of REM sleep
activity,
the
behaviours
in
the
regulation
of
opioid
effects of REM sleep deprivation (REMSD) on morphine- induced
were
studied.
attention
Particular
was
to
paid
some
morphine-induced effects such as akinesia and catalepsy, which are frequently
present in psychopathological conditions (10).
METHODS
Adult, male Wistar rats weighing
housed
in
groups
of
3-5
rats
1 50-200
cage
per
g
(40
were
used.
Animals
were
20 x 15 em) in constant
X
environment chamber with a light-dark cycle 14:10 h (light phase, 07.00-21.00
h) and ambient room temperature 22±1°C.
REMSD
Animals were deprived of
-flower
classical
pot-
REM
sleep
technique
continously
previously
for
described
96
h
using
( 11).
In
the
this
procedure, rats were placed on platforms (14±0.5 cm2 /100 g body weight
avoid
the
problem
of
unequal
water 0.5-1.0 em below the
selectively
deprive
REMSD (12).
island
surface.
rats of REM sleep (11 ).
The platform was surrounded by
This
procedure
is
known
to
In our experiment, the roof of
the REMSD tank was designed to permit free assess to food and clean
water.
to
drinking
The water in the tank was changed once every 24 h, during which time
the animals were subjected to 1-2 min of handling and then allowed to rest in
r.ome cages for 45-60 min and kept awake manually.
Stress control
To control for the unspecific stress factors associated with -flower pottechnique for REMSD, rats were placed on platforms large enough (57-59
cm2 /100 g body weight ) for them to curl up and have normal sleep without
falling
into
the
unspecific stress
water.
factors
The
large
(isolation
platform
and
is
dampness)
known to stimulate the
associated
with
the
11 0
Nflower
pot-
(11 ,1)).
technique
of
REMSD
without
affecting
REM sleep after 96 h
Otherwise, the stressed group of animals were treated as
in
REMSD
group.
Control
Animals in this group were housed in home cages (40x20x15 em) for
Like
the
96
h.
REMSD and stressed groups, rats in the control groups were handled
daily for 1-2 mins.
All animals had free assess to drinking water and food.
Behavioural observations
Behavioural observation and scoring were carried out in a quiet room with
a
temperature 22±2°C.
Changes in behaviours were monitored in a transparent
cylinder 18 em in diameter and 27 em height.
REM
dust.
sleep-deprived
rats
and
recording cage within 5-10 min after
REM
sleep
deprived
or
stressed
The floor was covered with
stressed
animals
discontinuation
animals
were placed in the
of
these
individually
after
30
min
procedures.
were first dried using absorbent
towel, since they were often wet on removal from platforms.
observed
saw
habituation
to
All
rats
the cage.
were
Morphine-
induced behaviours such as akinesia. catalepsy, or rearing were assessed
scored
as
present
and
or absent every 10 min for the first 30 min and every 30
min thereafter for another 90 min.
Akinetic and cataleptic behaviours
Akinesia was defined as loss of spontaneous locomotor
was
scored
as
A
activity.
rat
akinetic if it did not move for 5 min after placement at the
corner of the cage.
Catalepsy was determined using the bridge test and/or
reflex
(14,15).
In
this
least
min
were
of
righting
procedure rats were placed gently across a 10 em
wide bridge and/or on their backs.
at
loss
scored
Animals that maintained this position for
as
cataleptic.
All cataleptic animals were
akinetic, but not all akinetic rats displayed catalepsy simultaneously;
phenomena
were
seperately
evaluated.
both
Hopping behaviour was defined as the
sudden jump along a horizontal plane.
~easurement
of spontaneous vertical motor activity (SVMA) using an
automated
method
During preliminary experiments, it
morphine-induced
increased rearing.
locomotor
inhibition
was
into
observed
that
excitation,
REMSD
converted
characterised
This effect of REMSD was particularly evident 60-120
by
min
111
after
morphine treatment.
Therefore the SVMA was assessed quantitatively in
both REMSD and control groups.
Varimex
SVMA was recorded by means of a
computerized
(Columbus Instrument, Columbus, Ohio, USA), in which rearing animals
interrupted a magnetic field located between the floor
of
the
cage
and
a
plane 18 em above.
On the experimental
platforms,
weighed,
day,
rats
were
transferred
em) without sawdust and allowed 30 min habituation.
injected
with
was
home
All
animals
Then, morphine (25 mg/kg,
monitored
or
were
then
i.p.)
was
administered,
circadian
and
during the 60-120 min period after drug administration.
The effects of morphine on SVMA vere studied between 11.00-14.00 h
known
cages
saline (1 ml/kg,i.p. ), and motor activity was recorded for an
additional 30 min.
SVMA
from
and placed in transparent Plexiglas cages (40 x 20 x 15
to
avoid
variation in locomotion (16) and opioid receptor reactivity
(17).
Drug, dose and route of administration
Morphine
administered
hydrochloride
(Brocacef,
dissolved
physiological
in
The
Maar sen,
saline.
The
Netherlands)
locomotor inhibitory effects of morphine vere studied at a fixed dose
mg/kg,
i.p.,
since
it
has
been
shown
was
cataleptogenic and
of
25
that 20 mg/kg i.p.
induce robust
administration
morphine
catalepsy/ridigity in rats (18).
The
intracerebroventricular
~g/rat)
was
carried
out
(i.c.v.)
of
by means of chronically implanted cannulae.
dose vas selected because it is known to induce
the
inhibitory
(25
This
effects
of
opiates (19).
Statistical analysis
Results expressed as percentages were analyzed using a one-tailed
exact
probability
Kruskal
Wallis
one
Mann-Whitney test.
or less.
test.
way
Data
Fisher
presented as counts were analyzed using the
analysis
of
variance
(ANOVA)
followed
by
a
Statistical significance vas accepted at P-values of 0.05
112
RESULTS
Intraperitoneal (i.p.) administration of morphine
Effect of REMSD on morphine-induced akinesia.
significant
akinetic behaviour in
and stress, Fig.
and
stressed
1, p<0.01 ).
animals
animal~
Morphine(25 mg/kg)
This akinetic effect of
reached
induced
a
allowed undisturbed sleep (control
morphine
in
control
maximum intensity within first 30-60 min and
disappeared at 120 min after drug
However,
administration.
there
was
no
significant difference in the akinetic effect of morphine between control and
stressed animals.
REMSD significantly decreased morphine-induced akinesia compared with the
control
group
at 30 min and 60 min (Fig.
for each time period).
excitation
1, p<0.05, p<0.005, respectively,
Furthermore, REMSD converted the akinetic
characterised
by
rearing,
body
e££ect
to
jerks, and hopping behaviours.
Saline (1 ml/kg,i.p.) did not induce akinesia in control, stressed
or
REMSD
animals.
Effect of REMSD on morphine-induced catalepsy. The administration of morphine
(25 mg/kg) induced signi£icant catalepsy in animals
(control and stressed rats) 30 and 60 min
p<0.05).
The
morphine-induced
after
catalepsy
allo~ed
drug
~as
undisturbed sleep
treatment
(Fig.
characterised by a profound
state o£ muscular ridigity (evident in the bridge test) and loss of
re£lex,
disappeared
~hich
morphine-induced
di££erent
from
catalepsy
stressed
Naltrexone
(1
~hen
mg/kg,
akinetic-cataleptic
120
the
groups.
akinesia and catalepsy, i.e.
the control group only
at
in
min.
control
The
number
group
A close relationship
~as
a signi£icant catalepsy score
sho~ed
i.p.)
blocked
completely
in animals
allo~ed
of
not
~as
almost all animals
syndrome
2,
righting
rats
sho~ing
significantly
observed
~as
bet~een
registered in
akinesia.
the
morphine-induced
undisturbed sleep
(n~10
for
control and stress groups together).
REMSD signi£icantly decreased morphine-induced
control
animals
allo~ed
spontaneous
amounts
p<0.005 for 30 and 60 min, respectively).
o£
catalepsy
sleep
compared
(Fig.
~ith
2, p<0.02,
113
~
100
control (n=12)
REMSD ( n=13)
90
stress (n=13}
80
70
.~
•
60
c
"••
50
0
.§
c
•
40
~
30
20
0
GiiCI &:&0 CliO . . , . . ,
0
10 20 30
0 10 20 30
t
t
Sa!ine
Morphine
( lml/kg ip)
(25mg/kg ip)
of
RE~
sleep
60
90
120
time (min)
deprivation
(RE~SD)
Figure 1:
Effect
on
morphine-induced
akinesia.
Number of animals per treatment group is indicated in parentheses.
Note that REMSD significantly decreased the morphine-induced akinesia.
114
%
100
....._... control (n=12)
o--o
90
REMSD {n=13)
........_,._ stress (n=13)
80
70
-~
a.
60
•
50
:§•
.
u
•
E
·o
•
40
~
30
20
10
o"~~~~~~~
0 1 0 20 30
0 10 20 30
60
t
Saline
( lml/kg ip}
Morphine
( 25mg/kg ip)
oF
REM
90
120
time (min)
t
Figure 2:
EFFect
catalepsy.
Number of animals per treatment group is indicated in parentheses.
sleep
deprivation
(REMSD)
on
morphine-induced
Note that REMSD significantly decreased the morphine-induced catalepsy 50
after drug treatment.
min
115
Effect of REMSD on morphine-induced inhibition of SVMA. Kruskal Wallis
showed
a
significant
difference
across
the
treatment
groups
ANOVA
(Fig.
3,
p<0.001, H=20.39, df=5).
Morphine (25 mg/kg) decreased SVMA
morphine-induced
inhibition
in
control
However,
animals.
the
of SVMA in stressed rats was not different from
that in control animals (Fig.
3).
In the
REMSD
group,
morphine
had
the
opposite effect. which was manifested as a significant increase in SVMA (Fig.
3, p<0.02).
group
This morphine-induc-ed increase in SVMA in the REM sleep deprived
was
not
altered
by
pretreatment with naltrexone (1 mg/kg,i.p., not
shown).
Intracerebroventricular
undisturbed
decreased
sleep
(with
However,
of
administration
i.c.v.
locomotion
catalepsy).
administration
associated
the
morphine.
of
of
such
The
jumps/2h,
n~7)
intensity
of
jumping
behaviour
induced
akinesia
(25
such
allowed
~g)
(25
as
morphine
deprived of REM sleep provoked exctitatory behaviours
jumping.
animals
morphine
symptoms
administration
In
as
~g)
and
to animals
hopping
and
in the REMSD group (25±9.5
was significantly higher than in control (no
jumps
in
2
h,
n=5) and stressed animals (1.2±0.8 jumps/2h, n=5, p<0.01 ).
DISCUSSION
The results of this study indicate that the akinetic-cataleptic
and
locomotor
syndrome
suppressant effects of morphine were antagonised by REMSD and
replaced by excitatory behaviours manifested as increased SVMA:
hopping
and
jumping.
It has been
suggested
akinesia
and
sensitive
~-opioid
study,
since
catalepsy
a
Therefore,
a
REMSD might
sugge~t
the
decrease
of
effects
of
morphine
such
as
due to the activation of a naloxone/naltrexone
relatively
decreased
inhibitory
are
inhibitory
receptor (20-22).
naltrexone
the
that
low
This idea could be
dose
of
morphine-induced
~
receptor
supported
blocking
akinetic-cataleptic
by
this
substance
syndrome.
morphine-induced akinetic-cataleptic syndrome by
an insufficiency of an endogenous opioid system mediating
effects
of
opiates.
Similarly,
analgesic effects of opiates (9), which is supposed to
REMSD
be
antagonised
mediated
by
the
the
116
600
c
E soo
~
(n=6)
400
saline
(1 ml/kg, ip}
morphine
(25 mg/kg, ip)
300
200
(n=6)
100
(n=7)
0
LcoNTROL
Figure 3:
__j
L
REMSD
__j
L
STRESS
__J
EFfect of REN sleep deprivation (REMSD) on the inhibitory action of
morphine on spontaneous vertical motor activity (SVMA).
Number of animals per
treatment group is indicated in parentheses.
REMSD
locomotor
SV/f/A.
Note that
converted
the
inhibitory effect of morphine to excitation manifested as increased
117
same type of receptors (23,24).
Although morphine fails to induce akinetic/cataleptic syndrome
animals.
changes
acid
(GABA),
and
somatostatin
acetylcholine,
during REMSD (25-27) cannot
account for the inhibition of morphine-stimulated akinesia and
REM
sleep
REMSD
in other transmitter/modulators are probably not involved.
For example, the observed decrease in brain concentrations of
Y-aminobutyric
in
deprived
animals,
since
blockade
not
alter
opiate-induced
Similarly, increased serotonin release
facilitated akinesia and catalepsy (30), while an elevated brain turnover
serotonin
was
observed
during
REMSD (31).
that
clinically
effective
of
A number of studies indicate a
functional hyperactivity of dopaminergic system following REMSD (32).
known
in
of cholinergic and GABAergic
systems or the administration of somatostatin did
akinetic-cataleptic syndrome (28,29).
catalepsy
neuroleptics
dopamine receptors may induce catalepsy (33).
that
It
is
act as antagonists at
However, opiates do not act as
antagonists of dopamine receptors (34).
A second important
effects
of
finding
that
intracerebroventricularly
The reason for the conversion
excitation
was
by
REMSD
is
of
not
REMSD
converted
administered
the
clear.
mediates
One
is
the
effects
However,
morphine
analgesia,
naloxone-insensitive
naloxone-sensitive
akinesia
receptor
Activation or blockade of the
respectively,
the
~~opioid
opioid
catalepsy:
and
system
depressant
morphine to excitation.
depressant
inhibitory and excitatory behaviours. which are mediated
receptors.
the
of
morphine
can
by
induce both
two
receptor
groups
is
the
which mediates excitation (21 ,35,36).
receptor can inhibit
excitatory effect of morphine (36).
and
facilitate,
Thus. the conversion
of the motor inhibitory effects of morphine to excitation during REMSD
be
of
system which
other
the
to
explained by the suggested deficiency of an inhibitory
~-opioid
could
system in
REM sleep deprived animals and a corresponding predominance of the excitatory
opioid receptor system.
Therefore, in conclusion, we suggest that functional insufficiency
~-opioid
system
during
excitability in REM
sleep
REMSD
might
deprived
of ·a
be a background to increased neuronal
animals.
Namely,
it
is
known
that
endogenous opioids exert an inhibitory influence on the release of excitatory
transmitters (37).
blockade
This possiblity can
be
supported
by
the
report
that
of opioid receptors with naloxone facilitated epileptogenesis (38).
118
The proposed insufficiency of an opioid
explanation
for
system
during
REMSD
might
be
an
curtailmen~
a therapeutic and diagnostic value of REM sleep
in some cases of depression (8) and epilepsy (39), respectively.
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YF
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the
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the
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JT
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A
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facilitates
~asi
della
e cicli
121
CHAPTER 8
REM SLEEP DEPRIVATION DECREASES THE GROOMING AND SHAKING BEHAVIOUR INDUCED BY
ENKEPHALINASE INHIBITOR OR OPIATE WITHDRAWAL
ABSTRACT
administration
Intraventricular
phosphoramidon
(1
enkephalinase
of
syndrome consisting of excessive grooming with body scratching
symptom
prominent
wet-dog-shakes
and
phosphoramidon-induced
pretreatment
inhibitor,
10-S- 5.6 x 10- 7 moles, i.c.v.) induced a behavioural
X
Both the
naloxone-precipitated
and
bo~
The
scratching
were
as
the
frequency
phosphoramidon-induced
withdrawal
WDS
were
WDS
decreased
most
of
the
the
with the opioid receptor blocking agent, naltrexone (2.9 x 10- 6
moles/kg, i.p.).
WDS
(WDS).
in
naive
by
rats
and
decreased in REM sleep deprived
rats compared with animals allowed normal sleep (control and stress
groups).
The results are discussed in light of a possible functional insufficiency
of an endogenous opioid system during REMSD.
insufficiency might be a background to the
It has been suggested that this
increased
neuronal
excitability
during REMSD.
INTRODUCTION
Several
endogenous
substances
including
opioid
peptides
demonstrated
to induce grooming and wet-dog-shakes (5,6.9,12).
demonstrated
that
the
administration
of
have
enekphalinase
the
been
Recently, we
inhibitor,
phosphoramidon, induced behaviours such as grooming and wet-dog -shakes (WDS)
{28).
These behavioural phenomena. which can be induced by various drugs
naive
animals,
are
indications that both
part
ol
groom~ng
the
morphine withdrawal syndrome.
and WDS may share a
common
neural
in
There are
mechanism
( 1 0) .
It has been demonstrated that inhibition ol protein synthesis reduced the
severity
of
opiate withdrawal phenomena (19).
REM sleep deprivation (REMSD) can decrease
In addition it is known that
protein
synthesis
(30).
REMSD
122
also
inhibited
alterations
morphine
in
REM
pharmacological
induced
sleep
effects
can
of
analgesia
modulate
opiate
(38).
These data suggest that
protein
both
substances.
synthesis
Therefore, we analyzed the
relationship between REMSD and grooming and/or shaking behaviour
induced
enkephalinase
rats
phosphoramidon
inhibitor
in
naive
by
naloxone-precipitated withdrawal in the opiate-dependent rats.
METHODS
Adult, male Wistar rats (100-125 g) housed in transparent
in
a
constant
environment
plastic
cages
room with a light-dark cycle 14:10 (light phase
07.00 -21.00 h) were used.
Intracerebroventricular (i.c.v.) administration of drug solutions
For i.c.v.
administration of drugs a stainless steel guide
stereotaxically
mm above the lateral ventricle.
directed
(maximum volume 2
~1)
cannula
was
Drug solutions
were injected into the lateral ventricle with guage
needle, attached to a Hamilton microsyringe by polyethylene (PE) tubing.
length of the needle was made such that it protruded 1 mm
The
ventricle.
before
and
10
after
sec.
each
Correct
experiment
ventricular
using
technique previously described by Paakkari (24).
tubing
is
attached
to
the
injection
cerebrospinal fluid (CSF) or saline.
cannula
during
needle
In
The
lateral
and
a
cannulation
was
modification of the
this
procedure
a
PE
filled with artificial
To test the correct placement of i.c.v.
sugery, the tubing is raised above the head of the animal on
the stereotaxic apparatus, and a rapid inflow of
placement
the
injection was made over 10 sec and the needle maintained in
position for an additional
verified
into
30
of cannula.
saline
denotes
a
correct
The cannula was moved only in a downward direction to
avoid the possible false positive effect due to an
upward
movement
of
the
cannula after the first unsuccessful cannulation (14).
REM sle&p deprivation (REMSD)
REMSD
was
carried
out
according
to
the
conventional
-flower
pot-
technique
previously described (21). In this procedure, rats were placed on
platforms (14 cm2 /100 g rat) surrounded by water, such that the water level
was
on
0.5-1.0 em below the platform.
platform
and
deprived
of
REM
Rats made morphine-dependent were placed
sleep
from
day
7-11
(96
hr)
of
123
morphinization.
During
this
period
animals
received the normal doses of
morphine for these days.
Forty-five to 60 min after the discontinuation
injected
of
REMSD
the
with the enkephalinase inhibitor phosphoramidon.
time the animals were kept awake mannually.
The
animals
were
In this period of
behavioural
changes
were
-flower
pot-
scored during the following 30 min.
Control for the unspecific stress factors associated with
the
technique
In order to control for the known stress factors (dampness, isolation and
immobilisation)
associated
with the -flower pot- technique rats were placed
on platforms large enough (60 cm 2 /100 g rat) for them to curl up and have
normal
The large platform can simulate the chronic stress condition
sleep.
associated with REMSD without affecting REM sleep level after 96 hr (20,21).
Phosphoramidon-induced behaviour
Behavioural observation and scoring were carried out on
rat
each
individual
housed singly in Plexiglas cages (40 x 20 x 15 em) containing saw dust .
Wet-dog-shakes consisting of paraxysmal shudder of the whole body
spinal axis were registered and quantified.
along
the
The body scratching (BS) episode
is defined as head or body scratches followed immediately by the
licking
of
the paw used in scratching.
Induction of morphine dependence
Animals were
injection
made
procedure
dependent
previously
on
morphine
described
according
(32).
The
dosage
schedule
was
moles/kg/day; Days 3 and 4 ( 14 X
10- 5 moles/kg/day) and Days 7-11
follows:
as
, o- 5
(56
X
the
repeated
In this method rats were
given two intraperitoneal injections of morphine daily (at
hr).
to
Days
07.30
and
moles/kg/day); Days 5
10- 5 moles/kg/day).
and
2
and
(7
6
15.30
X 10- 5
(28
X
Precipitation of morphine withdrawal shaking behaviour
Abstinential behaviour in morphine dependent animals was provoked by
6
nalxone (3.1 x 10moles/kg i.p.) three hours after the last
injecting
injection of morphine.
allowed
a
Prior to receiving naloxone treatment
habituation
Naloxone-precipitated WDS
induced
period
in
of
30-60
min
morphine-dependent
in
the
rats
each
rat
observation
and
was
area.
phosphoramidon-
behavioural phenomena in naive animals were studied in the following
three groups of rats:- a) Control group:- these rats were
housed
singly
in
124
home
cages
throughout
amounts
o~
96
continous
hr
sleep;
the
experimental
procedure and allowed spontaneous
b) REM sleep deprived group:- these rats were submitted to
REM
sleep deprivation;
c) stressed group:- these animals
were chronically stressed for 96 hr.
Drugs
The following drugs were used:
hydrochloride
(Endo
Lab)
and
morphine hydrochloride (Merck).
naltrexone
administered dissolved in physiological
hydrochloride
saline.
(Endo
naloxone
Lab)
Phosphoramidon
were
(Peninsula
Lab) was dissolved in CSF prepared fresh and administered i.c.v.
Data Analysis
The results were analyzed using the Kruskal-Wallis
one-way
ANOVA.
The
statistical difference between two groups of treatment were carried out using
a two-tailed Mann Whitney U test. except vhen indicated in text.
RESULTS
Effects of REMSD on behavioural syndrome induced by enkephalinase inhibiton
The intracerebroventricular administration of the enkephalinase inhibitor
phosphoramidon
(1
x
10- 8
5.6 x 10- 7 moles i.c.v.) induced a behavioural
-
syndrome consisting of excessive grooming (as measured
and
stressed).
These
symptoms
appeared
vithin
5
min
administration and vere still observed after 240 min.
vay ANOVA shoved a significant difference in the
across
the
groups
(H=1 06.21,
NDF:8
body
scratching)
stressed
(p<0.02)
both
increasing
doses).
control
of
(p<0.05)
and
significantly
different
1,
p<0.02,
p<0.002,
p<0.002
The frequency of BS in the REM sleep
deprived animals vas significantly less intensive compared with
stressed animals (Fig 3, p<0.02).
WDS
frequency
REMSD significantly decreased the WDS
induced by three doses of phosphoramidon (Fig
for
The
Hovever, the frequency of the
phosphoramidon-induced WDS in stressed rats was not
respectively,
phosphoramdion
phosphoramdion-induced
in
groups of animals (Fig 1 ).
control animals (Fig 1. p>0.05).
after
The Kruskal-Wallis one
p<0.001).
phosphoramidon-induced WDS vas dose-related
from
by
vet-dog -shakes (WDS) in all three groups of animals (control, REMSD and
control
and
There vas no significant difference in the
mean BS between control and stressed animals (Fig 3, p>0.10).
6
Naltrexone (2.9 x 10moles/kg i.p .• 10 min prior)
significantly
125
(n=l2)
"
35
(n=9)
30
0
.E
(n,6)
~
~
STRESS
25
~
iii
:,
0
20
0
[ n · 5)
;;
( n '5)
~
'o
•'
( n o9)
15
D
E
0
z
( n =5)
10
REM SO
( n "5)
( n =9)
. 8. 5
7. 5
- 5. 5
log dose of phosphor;Jmidon (moles icv)
Figure 1:
The phosphoramidon (1 x 10-B- 5.5 x 10-7 moles
wet-dog-shakes
dose
of
(WDS).
phosphoramidon
phosphoramidon-induced
Each
is
WDS
point is mean ± S.E.M.
stated
in
i.c.v.)-
induced
The number of rats per
parentheses.
Note
that
the
was significantly lowered in REMSD rats compared
with control or stressed animals.
126
decreased
the
phosphoramdion-induced
stressed animals (p<0.05, Ducan
control
rats
Ne~
~S
in
Multiple
control (p<0.002), REMSD and
range
the phosphoramidon-induced BS were
after pretreatment with naltrexone (92.8±23.6.
test)(Fig
signi~icantly
n=11)
compared
2).
In
the
less frequent
with
saline
pretreated animals 201 .8±29, n=12, p<0.05).
'"
;;
So1!Jne (Sal) 2 vl lev
0
'
N<~ltrexol'le
"
(Nx) 2.9 x 1a"
6
mole5/kg lp
Phosphor.:.mldon (PM) 5.6 x 10" 7 mol.,,-. icv
~
"
&
0
8
"
"
~
0
~
"
§
z
;o
{n~14)
1"''1 (n;6}
L_j=:J
Sal
Nx
Ph11
REMSD
Figure 2:
(5.6
S.£./ff.
x
Effect oF naltrexone (2.9 x 10-6 moles(kg, i.p.) on phosphoramidon
7
10- moles i.c.v.)-induced wet-dog-shakes (WDS).
The
parentheses.
number
of
animals
per
treatment
group
Each bar is/mean±
is
indicated
in
Note that pretreatment ~ith naltrexone significantly decreased
phosphoramidon-induced WDS.
127
300
250
c
"E
.
Saline (Sal) 2 ul lev
Phosphoramidon (Pha) 5.6 x 10-
7
moles icv
200
1!v
~
Ji
,_
-g
150
m
'0
.....
•
1 00
.0
,
E
(n=9)
z
I]
50
(n=lS)
:c
(n=14)
0
Effect oF REMSD on
Figure 3:
induced
body
treatment group is
scotches
induced
stated
by
REMSD
phosphoramidon
in
parentheses.
phosphoramidon
in
Sal
__j
(5.6
Note
control
L_
10-7
x
Each bar is mean± S.E.M.
scratches.
LJ
Pha
Sal
L_
(n=S)
Pha
STRESS
moles,
__j
i.c.v.)
The number of rats per
that
the
intense
body
and stressed animals were
significantly decreased by REMSD.
Effect of REMSD on opiate withdrawal WDS
Naloxone (3.1 x 10- 6 mole/kg i.p.) precipitated WDS in morphine-dependent
rats
in
control.
Kruskal-Wallis
withdrawal
WDS
frequency of the
REM
one
way
across
sleep
deprived and stressed groups of animals.
ANOVA
the
showed
groups
precipitated
WDS
a
(Fig
was
significant
difference
in
4, H=16.12, NDF=2. p<0.001 ).
significantly
more
pronounced
The
the
The
in
128
8
{ n =1 0)
7
-
c
=
0
M
6
{n=1 0)
5
-
~
w
"'.crn
Vl
'
Ol
0
0
.:.
"'"
4
'•""
rn
"0
.c
{n =13)
3
"'
1
~
0
"w
.0
=
2
~
z
0
CONTROL
Figure
4:
-precipitated
£TTect
oT
withdrawal
REMSD
on
REMSD
naloxone
wet-dog-shakes
STRESS
(3 .1
X
70-S
moles/kg
(WDS).
i.p .)
Each bar is mean ± S.E.M.
The number oT rots per group is indicated in parentheses.
Note that the
total frequency of withdrawal WDS was significantly less in REMSD rats
compared with control (p<0.01) and stressed (p<0.002) animals.
129
animals
allowed
to sleep normally (control and stressed groups) than in the
REM sleep deprived rats (Fig 4, p<0.002).
induced
WDS
(Fig 4, p>0.2).
the
intensity
of
such
Body scratchings in naloxone-treated morphine dependent rats
irregular
few
were
However,
in control or stressed animals were not significantly different
and
therefore
omitted
from
further
detailed
quantitative evaluation.
DISCUSSION
The results of this
enkephalinase
study
showed
that
WDS
and
the
fact
that
(16,25).
induced
by
inhibitor, phosphoramidon, were inhibited by naltrexone, which
might indicate an involvement of opioid receptor(s).
blocking the
grooming
enkephalinase
inhibition
biotransformation
In
addition,
of
the
can
activate opioid receptors by
endogenously
WDS-induced
This is consistent with
released
by
i.c.v.
opioid
peptides
administration
of
enkephalins were attenuated by opiate antagonists (2,5,6,13)
Grooming behaviour has also been observed after
(29).
low
doses
of
morphine
Taken together, these data might suggest that WDS and grooming induced
by phosphoramidon or opioid substances share a common mechanism.
The biological significance of WDS
chemical
compounds
of arousal (10),
mechanism
is not clear.
whereas
(17).
and
grooming
behavioural
grooming
by
different
Some data suggest that WDS are indicative
grooming
might
be
a
-de-arousing-
homeostatic
In addition, it has been demonstrated that opioid peptides
facilitate arousal (37) and in higher doses induced
and
induced
phenomena
observed
in
similar
our
to
experiment
an
electrophysiological
epilepsy (7,8).
might
be
a
Thus the excessive
response
to
the
phosphoramidon-induced arousal, manifested as WDS.
However, the most important aspect of this report is the fact that
suppressed
rats.
Why REMSD decreased these behaviours in rats is not clear.
however,
be
suggested
that
REM
phosphoramidon
could
be
biochemical evidence for the
during
REMSD
this
less
pronounced.
insufficiency
possibility
It
could,
sleep deprived animals might have limited
availability of opioid peptides and hence the WDS and
by
REMSD
the WDS and grooming induced by enkephalinase inhibition in naive
could
of
grooming
precipitated
Although there is no direct
the
enkephalinergic
system
be considered since it is known that
130
REMSD is associated with the
concept
of
inhibition
of
protein
synthesis
(30).
system in REM sleep deprived animals receives further support from
that
The
a functional insufficiency in the enkephalinergic/endorphinergic
abolished
REMSD
antinociceptive
the
effects
of
the
fact
morphine
and
phosphoramidon (38).
This hypothesis of a functional insufficiency in this opioid system might
explain
the
increased
neuronal
excitability
during REMSD (4) since it is
known that oPioid peptides exhibit tonic inhibitory effects on the release of
excitatory
transmitters
(23).
However, additional experiments are required
to clarify whether this mechanism might
be
seizures
of REMSD in some types of endogenous
and
the
therapeutic
effect
involved
in
REMSD-precipitated
depression.
A second
important
abstinential
WDS.
finding
The
of
and probably involves alteration in
systems.
However,
this
study
is
that
REMSD
inhibited
mechanism of opiate addiction/withdrawal is complex
several
neurotransmitter/neuromodulator
the known changes in classical transmitters during REMSD
can not account for the decrease in naloxone-precipitated withdrawal
REM
sleep
deprived
animals.
For
WDS
in
example, REMSD increased the functional
activity of dopaminergic system (36), but did not alter the adrenergic system
(31 ).
However, substances which block these systems inhibited withdrawal WDS
(18,34) Furthermore, the known changes in brain serotonin
REMSD
metabolism
during
(33} probably play no role in the inhibiton of abstinential WDS in REM
sleep deprived rats, since the alteration of the serotoninergic system had no
clear
effect
on
WDS
induced by morphine withdrawal (1 ).
It is also known
that drugs which stimulate central muscarinic receptors inhibited the shaking
response
(39), whereas REMSD decrease the acetylcholine content of the brain
(3.35).
Although some high
morphine,
there
is
energy
phosphates
can
antagonize
the
effects
of
no evidence that the concentrations of AMP, ADP and ATP
are significantly altered by REMSD (11 ,22).
Therefore, an alternative explanation for the inhibitory effect of
on
morphine
withdrawal
during development of
synthesis
of
WDS should be considered.
morphine
secretory
dependence
proteins
in
the
REMSD
Namely, it is known that
there
is
brain
regions (pons-medulla and
stratum -septum)(27), which are particularly rich in
an
opioid
increase
of
receptors
the
(26)
131
and
functionally
involved
in
opiate
dependence
(15).
It has also been
demonstrated that REMSD decreased protein synthesis in the cerebral and brain
stem
fractions
phenomena (19).
(30)
and
that
synthesis
can
decrease
Thus the decrease of protein synthesis
opiate withdrawal
during
REMSD
might
explain the inhibitory effect of REMSD on withdrawal WDS.
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135
CHAPTER 9
ENKEPHALINASE INHIBITION ANTAGONIZES THE INCREASED SUSCEPTIBILITY TO
SEIZURE
INDUCED BY REM SLEEP DEPRIVATION
ABSTRACT
In order
to
enkephalinergic
and
elucidate
the
the
enkephalinase
handling-induced-convulsions
phosphoramidon (25-500
convulsions
relationship
between
REM
sleep
and
the
system, the effects of REM sleep deprivation (REMSD), stress
~g
inhibitor
were
studied
phosphoramidon
in
mice.
REMSD,
on
stress
and
i.c.v.) increased the frequency of handling-induced
(HIC) in normal mice.
However, only in the last two groups were
RIC antagonized by naloxone (1 mg/kg i.p.).
In
REMSD
mice,
phosphoramidon
decreased the frequency of HIC, this effect being abolished by naloxone.
increase of neuronal excitability during REMSD is suggested to be
The
associated
with an insufficiency of an enkephalinergic system.
INTRODUCTION
Rapid-eye-movement (REM) sleep is known to modulate neuronal excitability
in
man
and
animals
(Drucker-Colin et al., 1977;
Passouant et al., 1965).
However, in clinical reports and animal experiments, it has been demonstrated
that
REM
sleep
deprivation
(REMSD)
increases
facilitates seizure activity (Pratt et al .• 1969;
Evidently.
the
phasic
changes
in
neuronal
neuronal
Cohen and
activities
excitability and
Dement,
during
1965).
sleep can
influence the pathophysiology of seizures.
Recently, it has been suggested that the enkephalinergic system plays
important
1979).
REM
role
in
epileptogenesis
(Frenk
et
al .• 1978;
It is also known that ~-endorphin exerts an inhibitory
sleep
(King
et al., 1981 ).
influence
1983).
on
In addition, we have demonstrated that REM
sleep has an inhibitory effect on enkephalin-induced seizures (Ukponmwan
Dzoljic,
an
Dzoljic et al.,
and
These data might indicate an involvement of the endogenous
opioid system in mechanisms regulating REM sleep and neuronal excitability.
136
In order to eludicate further the
enkephalins,
we
studied
relationship
between
REM
sleep
and
the effects of REMSD, stress and the enkephalinase
inhibitor, phosphoramidon on handling-induced convulsions in mice.
MATERIALS AND METHODS
Animals:
Adult, male mice of B10 A strain (25-30 g) were used (Dlac Ltd, Bicester,
England).
Intracerebroventricular
(i.c.v.)
administration of drugs was by
means of a stainless steel cannula stereotaxically implanted in
ventricle.
the
lateral
A minimum of 3-5 days was allowed for recovery before experiments
were commenced.
Animals were then divided into three
housed
individually
sleep;
Group
B
in
mice
Group
groups:
A
(control)
were
subjected
to
72
h
REMSD
according
conventional
-flower pot- technique described by Fishbein {1970).
which
habituate
could
experimentation.
mice
transparent cages and allowed spontaneous amount of
to
the
platform
within
3
h
to
the
Only mice
were
for
used
Group C (stress) mice were placed on platforms large enough
(8.0 em diameter, for 72 h) for the animals to curl up and exhibit REM
sleep
without falling into the water.
Handling-induced convulsions (HIC):
HICs in mice were assessed using the criteria
alcohol
withdrawal
was picked up
the
by
characterised
(Goldstein and Pal, 1971 ).
by
tail
and/or
violent
jerking
tightening of facial muscle (grimace).
pick-up
were
tonic-clonic
picking
recorded.
convulsions
up-3,
grimace
pick
min
gently
or
twirling,
after
intervals
the
tonic
were
up-4,
for
180°.
HIC
assigned
as
tonic-clonic
was
convulsions
Only HICs occuring within 6-10
tonic
gentle
convulsions
spinning-0.5.
form 120 min.
after
possible
gentle
follows:
s
of
violent
convulsions
upon
spinning-1.
and
The scoring procedure is based
(1981 ).
RIC was determined
at
The intensity of convulsions is indexed by
total HIC during the first 30 min scoring period.
avoid
through
spun
upon the criteria described by Crabbe et al.
10
described
tonic convulsions upon picking up or tonic-clonic convulsions
after gently spinning-2,
facial
Scores
upon
previously
In this procedure each mouse
The period was
modifying influence of repeated handling.
chosen
to
The observer
137
was -blind- during the evaluation of dose-response curve
of
the
effect
of
phosphoramidon in all three groups.
Drugs:
Phosphoramidon
(Winthrop
(Peninsula
Laboratories)
were
naloxone
Laboratories)
and
dissolved
in
saline.
20
s
phosphoramidon administered i.c.v.
over
was
hydrochloride
The maximum volume of
Naloxone
1 .5~1.
was
administered intraperitoneally (i.p.).
Statistical analysis oF data:
The Kruskal-Wallis ANOVA was carried out on the
and
across
all
data
in
all the experiments.
mean
convulsions
score
Comparisons between any two
treatment groups were made with the Mann-Whitney U-test (Siegel, 1956).
RESULTS
The Kruskal-Wallis test
scores
across
the
showed
groups
significant
(H=174.47,
differences
p<0.001,
in
convulsion
The
NDF=15).
significance between two groups using the Mann-Whitney U test
levels
are
of
indicated
below.
Control.
In this study, 16% of control non-treated mice displayed mild signs
(1~1
i.c.v.) or
naloxone also induced signs of HIC consisting mainly of grimacing.
However,
of
HIC such as grimacing.
phosphoramidon
(25-500
In treated mice, saline injection
~g
i.c.v.) significantly increased the intensity and
degree of susceptibility to HIC in
a
dose-related
manner
(p<0.01 ).
This
effect was antagonized by naloxone (1 mg/kg. i.p.)(Fig 1A).
R£MSD group.
Mice
increase
the
in
subjected
incidence
to
and
72
h
REMSD
intensity
demonstrated
of HIC (p<0.001 ).
a
significant
This was not
affected by naloxone (p=o.1) but significantly inhibited by the enkephalinase
inhibitor
phosphoramidon
(25-500
termination (p<0.001, Fig 1B).
~g)
This effect of phosphoramidon
antagonised by naloxone (1 mg/kg i.p.
Stress
group.
Stress
also
administered within 10 min after REMSD
induced
was
partially
10 min prior, p<0.001 ).
an
increase
in
the
intensity
susceptibility to RIC (p<0.001) but less intensely than in REMSD group.
effect was further potentiated by phosphoramidon (100
(1
mg/kg,
i.p.)
(p<0.001 Fig 1C).
decreased
the
susceptibility
to
~g.
p<0.001 ).
HIC
and
This
Naloxone
in stress animals
138
"E"
c
B
A
"
10
0
~
0
•0
8
~
0
.,
0
·;;;
>
0
0
u
6
•
In·>)
~ ~.. I..
'""'.,
0
0
0
E
'""'"
ln•>J
2
<n-101
~ a@
(n•!OO)
0
ln•>l
9
l ""''-P
1
1a-c:
phosphoramidon
The
first 30 min.
erTect
(ph),
convulsions (HIC).
""
,f,'~j
control
Figure
'"'"·~
of
naloxone
'""
"'"
REMSD
REM
sleep
ln••l
"'
1""'"'G
I
'""''"c
'""''""
,:,""'lj
t______stress______j
(REI'ISD,
deprivation
72
h).
(nal) and stress (72 h) on handling-induced-
Each bar is mean intensity± S.E.M
of
HIC
during
The number of animals per group is stated in parentheses.
the
139
DISCUSSION
Mice subjected to REMSD, stress or
increase
in
the
susceptibility
phosphor amidon
to
HIC.
phosphoramidon-treated animals was the
naloxone.
This
might
treatment
HoYever,
convulsant
only
in
activity
agrees
with
antagonised
data -showing
that
various
stress
regimens
are
associated
opioid
with
endogenous opioid peptides (Christie and Chesher, 1982).
stress
and
system.
the
The
1981)
release
evidence
of
that
enkephalinase inhibition increase susceptibility to epileptiform
activity could be ascribed to the fact that opioid
induce
by
that concentrations of opioid peptides are
increased after treatment with enkephalinase inhibitors (Patey et al.,
and
an
indicate that the phosphoramidon- and stress-induced
proconvulsant activity is due to activation o£ an endogenous
This
showed
stressed or
seizure
phenomena.
The
epileptogenic
peptides
in
excess
potential of various opioid
peptides administered exogenously has been demonstrated frequently
al., 1977;
Dzoljic et al., 1979;
may
(Urea
et
Snead and Bearden, 1982).
However, the mechanism of epileptiform activity of opioid peptides is not
completely understood.
the
delta-subtype
(Dzoljic
1983).
may
the
epileptiform
and vd Poel-Heisterkamp, 1982;
The target area
(Henriksen
1980;
Recent data suggest that specific opioid receptors of
mediate
et
of
this
hippocampal
(Zieglgansberger et
increased
al.,
these
substances
Haffmans and Dzoljic, 1983;
action
susceptibility
population
1984).
seems
to
be
the
Frenk,
limbic
system
neurons
It has been shown that opioid peptides
by
1979).
Thus,
to
in
phosphoramidon is due to the
receptor
of
al., 1980), particularly the hippocampus (French and Siggins,
Haffmans et al., 1983;
excite
effect
HIC
inhibiting
it
might
animals,
a~tivation
of
a
adjacent
be
stressed
or
particular
in selective brain regions(s).
interneurons
suggested
that
treated
type
of
an
with
opioid
The potentiating effect
of phosphoramidon on stress-induced HIC is probably due to the protection
of
the released opioid peptides during the stress procedure.
A second significant aspect of
inhibitor
this
study
effect of enkephalinase inhibition might suggest
with
an
is
that
the
enkephalinase
phosphoramidon decreased convulsant behaviour in REMSD mice.
insufficiency
of
that
endogenous opioid peptides.
supported by the fact that REMSD decreases
peptide
REMSD
is
This
associated
This possibility is
synthesis
(Shapiro
and
140
Girdwood,
1981)
al., 1981 ).
and
In
secretion
of
affects the levels of some brain peptides (Mattiace et
addition,
humural
recent
data
indicate
that
nocturnal
episodic
endorphins occurs during REM sleep (Oksenberg et al.,
1980).
However,
antagonizes
the
exact
mechanism
REMSD-induced
by
neuronal
which
an
excitation
enkephalinase
is not clear.
inhibitor
One possible
explanation that should be considered is the fact that enkephalins block GABA
transport
across
plasma
membrane
(Cupello
and Hyden, 1981 ), leaving GABA
outside the neuronal membrane in contact with
time.
its
receptors
for
a
longer
Such mechanism might explain enkephalin-induced neuronal inhibition in
physiological circumstances.
decrease
o£ enkephalinergic
decrease
GABA
inhibitory
In the context of these
ac~ivi~y
activity
findings,
a
proposed
during REMSD would be associated with a
and
consquent
increase
in
seizure
susceptibil ty.
In summary, these results
have,
indicate
that
enkephalinase
animals) or anticonvulsant (in REMSD animals) action.
data
inhibition
may
depending on conditions. proconvulsant potential (in stress or control
concerning
the
proconvulsant
opioids have been reported.
these
potential
Both the pro-
and
o£
Similar
morphine
contradictory
and endogenous
anticonvulsant
activity
substances have been demonstrated (Gilbert and Martin, 1975;
and Marty, 1954:
Cowan et al.,
1979;
Tortella
et
al.,
1981;
o£
Verdeaux
Dzoljic,
1982).
An
by
insu£~iciency
this
study
of endogenous opioid peptides in REMSD animals suggested
might
be
of
importance
£or
the worsening o£ seizures or
improvement o£ depressive disorders during REMSD (Pratt et al., 1968;
et
al.,
1980).
It is of
interes~
Vogel
to note that in nacolepsy, the attacks of
REM sleep have been prevented by naloxone, suggesting a possible
involvement
o£ endogenous opioid systems (Pasi et al., 1983).
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physiologically
released
1173-1177
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phase, Science, 150:
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1318-1319
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JC,
Young
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v
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enkephalin in rat brain and the anatomical regions
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3 H-labelled
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enkephalin-
1021-1028
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King C, Masserano JM, Codd
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WL
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substance P and somatostatin levels in discrete areas of the
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Soroca-Lucas
E
Gras
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inhibiting
Opioid
adjacent
inhibitory
144
CHAPTER 10
REM SLEEP DEPRIVATION INHIBIT THE NITROUS OXIDE WITHDRAWAL CONVULSIONS
ABSTRACT
The e£fect of
withdrawal
REM
convulsions
sleep
in
deprivation
mice
(REMSD)
investigated
~as
using
following exposure to nitrous oxide (N D).
2
REMSD for 72 h significantly decreased the severity of withdrawal convulsions
following acute exposure to N 0.
2
The results were interpreted in the light of protein synthesis inhibition
during
REMSD
REMSD.
procedure
might
stimulate
a
new
approach
to the
treatment of withdrawal neuronal excitability.
INTRODUCTION
It is known that REM sleep deprivation (REMSD) results in a
protein
synthesis
(Drucker-Galin
associated with an increase
threshold
~or
in
and
Rojas-Ramirez,
neuronal
electroconvulsive
shocks
1976)
excitability.
(Cohen
and
decrease
and
For
example,
Dement,
in
is often
1965)
the
and
amygdaloid kindling (Calvo et al., 1982) is greatly reduced following REMSD.
Patients recovering from nitrous oxide anaesthesia are known
an
increase
in
excitability
(Eckenhoff
et
al.,
1961 ).
to
exhibit
Similarly, mice
exposed to nitrous oxide showed convulsions when picked up by the tail
removal
from the anaesthetic (Harper et al., 1980).
after
This seizure pattern is
known to occur after exposure to nitrous oxide, ethylene and cyclopropane and
it is considered to be a type of withdrawal convulsions (Smith et al., 1979).
It is of interest to note
associated
with
REM
that
some
types
of
epileptic
attacks
are
sleep dysfunction (Passouant, 1976) and also with drug
addicition (Herzlinger et al .• 1977;
Mendelson and Mello, 1978).
In this study we report on the inhibitory
oxide withdrawal convulsions.
effect
of
REMSD
on
nitrous
145
METHODS
Adult, male mice o£ the B10A strain weighing 32±0.2 g
experiment
under
were
constant
temperature
libitum.
was
used.
at
the
onset
of
Throughout the investigation the animals were housed
light/dark
cycle
maintained
at
(light
24±°C.
The number of animals used in
phase
Food
each
09.00-21 .00).
and
room
The
water were available ad
procedure
is
stated
in
the
results.
REMSD was carried out according to the
previously
described
(Fishbein
et
al.,
conventional
1971 ).
This
deprives mice of REM sleep and with slight effect on
this
study,
the water
least
each
le~el)
once
in
slow
tank
method
method selectively
wave
sleep.
In
mouse was placed on a platform (3 em diameter, 1 em above
surrounded by water (3 em deep).
24
water
h.
Only
mice
The water was replaced
at
which could adjust to this experimental
condition within 2 h were used in experiments.
Stress control group for REMSD consisted of mice placed on platform (6 em
in
diameter
) under the same experimental conditions as in REMSD.
A second
stress consisted of mice forced to swim in water 3 em deep at 18°C for two, 1
h
sessions
daily.
These two stress control procedures produced comparable
weight loss as in REMSD (10-15%).
Nitrous oxide withdrawal convulsion was induced and assessed according to
the
method of Goldstein and Pal (1971 ).
In this procedure mice were exposed
to a mixture of nitrous oxide and oxygen (80:20) at 1.6 atm for
mouse
was
picked up by the tail and spun gently through 180°.
Each
h.
Mice showing
violent jerking or twirling and/or grimace were quantified as positive.
Mice
were tested for the presence or absence of handling-induced convulsions (HIC)
every 10 min after the removal from N2 0, until there were no convulsions in
successive tests. Throughout this study, all testing of HIG was carried
two
out
between
10.00-14.00
h
to
avoid
circadian
alterations
in
seizure
threshold.
Statistical analysis:The number of animals convulsing after N2 D withdrawal ~n
control, stressed and REMSD groups were compared using the Kx2 method (de
Jong, 1963).
less.
Statistical signifcance was accepted at
P-values
of
0.05
or
146
RESULTS
(n~2D)
Nitrous oxide withdrawal convulsions
Mice exposed to N2 0 showed withdrawal convulsions lasting about 2 h with
incidence within 20 min after removal from N2 o. The mice convulsed
highest
when picked up by the tail following N 0 anaesthesia.
2
convuslions
components)
(HIC)
and
consisted
arching
of
of
the
violent
back
and
jerking
These handling-induced
and
twirling
contractions
of
head
(clonic
muscle
(-grimace-, tonic components).
REM sleep deprivation (n=20)
Mice subjected to REMSD for 24-120 h showed increased
HIC.
However.
mice
subjected
to
susceptibility
to
REMSD for 72 h showed a decrease in the
degree of susceptibility to N2 0 withdrawal convulsions (Fig A1 ).
Stress
did
inhibit N o withdrawal convuslions (n=10), but instead exacerbated it.
2
Combined REMSD and an enkephalinase inhibitor phosphoramidon (Hudgin et al.,
not
1981)
also
decreased the frequency of HIC but not to a degree significantly
different from REMSD or phosphoramidon given alone (Fig A.1 ).
DISCUSSION
Mice exposed to N2 o develop very quickly a drug dependence which is
characterized by withdrawal convulsions. This is demonstrated when mice are
picked up by the tail following N 0 anaesthesia. This type of convulsions
2
was conteracted by REM sleep deprivation (REMSD), in spite of the fact that
REMSD could induce neuronal excitation in the normal animals.
The
neurobiological
convulsions
is
still
basis
unclear,
withdrawal
N o tolerance
2
it is does not appear to involve changes in
of
acute
synaptic membrane fatty acid, phospholipid and cholesterol
1979).
(Koblin
et
al.,
However the possible involvement of endogenous opioid peptides in N o
2
Acute exposure to
tolerance and withdrawal excitation has been demonstrated.
N o induced an increase in the cerebrospinal fluid met-enkephalin levels
2
(Qock et al., 1985), whereas the administration of morphine or naloxone can
inhibit
and facilitate, respectively, the N o withdrawal convulsions (Manson
2
This suggests that N o stimulates an opioid-like dependence.
2
et al., 1983).
147
$ vs control
•
100
vs stress
\--Gil-·--·
.
~
0~ \/·~ ~/~.
)(\
80
0
\
\-STRESS
\-
\~EMSD
\
0
~
-CONTROL
s
~\A~-:
~\PHD~-~--\
20
\,..
0
-REMSD
~·
\
""'
~~~~
~~
0
PHOSPH.
OL-----N>----~--a-~,-~~~~~~----~----~~
20
40
NITROUS OXIDE
AFTER
Figure A.1:
The effect of
R£~
sleep deprivation
(REMSD)
on
withdrawal
convulsions.
treatment.
(*)denotes statistical significance at p < 0.05
to control or stress.
Control
consists
of
mice
alter
convuslions.
the
nitrous
oxide
subjected to any
level
compared
Note that 1) REMSD significantly reduced the frequency
and duration of nitrous oxide withdrawal convulsions;
not
not
inhibitory
action
of
REMSD
on
2) phosphoramidon
nitrous
did
oxide withdrawal
The effect of the enkephalinase inhibitor phosphramidon was not
considered in this chapter.
148
It
known
is
that
several drugs with the common ability to inhibit protein
synthesis reduced the development of
Cochin,
dependence
to
opiates
(Feinberg
and
Bowman and Rand, 1980) and decrease REM sleep (Drucker-Colin
1 972;
and Rojas-Ramirez, 1976).
In
addition
REMSD
has
also
been
reported
to
decrease protein synthesis in some nuclei of the rat brain stem (Bobillier et
al., 1974;
Panov, 1982).
convulsions,
might
be
The antagonistic effect of REMSD on N D withdrawal
2
to decrease in protein synthesis, which in turn
due
blocked the development of tolerance and physical dependence.
Stressed
convulsions
animals
showed
proving
that
enhanced
the
susceptibility
to
N2 o
withdra"W"al
inhibitory action of REMSD on N2 0 withdrawal
convulsions is not related to the unspecific effects (dampness, isolation and
restriction) of the experimental procedure.
Apart from the uncertainty of the
withdrawal
syndrome,
prove useful as
the
new
results
approach
to
between REMSD and N o
2
this study indicate that REMSD might
interactions
of
the
management
of
withdrawal
neuronal
hyperexcitability.
REFERENCES
Bobillier P, sakai T, Seguin S, Jouvet M (1974) The effect of sleep
upon
in
vivo
and
deprivation
in vitro incorporation of tritiated amino acids in brain
proteins in the rat at three different age levels, J Neurochem., 22:
Bowman
we, Rand MJ
(1980)
Drugs
used
to
relieve
pain,
23-31
Textbook
In:
of
Pharmacology, Blackwell Scientific Publications, pp 16.1-16.17
Calvo JM, Alvarado R, Briones R, Paz C, Fernandez-Guardiola A (1982)
kindling
29'
during
rapid eye movement (REMSD) sleep in cats, Neurosci.
Lett .•
255-259
Cohen HB, Dement WC (1965) Sleep:
shock
Amygdaloid
in
rats
after
changes
deprivation
of
in
threshold
to
-paradoxical
electroconvulsive
phase-,
Science, 150:
1318-1319
Drucker-Galin RR, Rojas-Ramirez JA (1976) New approaches to
neurochemical
basis
of
sleep
and
wakefulness.
the
In:
study
of
Advances
Psychobiology, Riesen AH, Thompson RF (eds), John Wiley, New York, pp.
Eckenhoff JE, Kneale DH, Dripps RD (1961) The incidence
and
etiology
anaesthetic excitement (a clinical survey), Anaesthiol., 22:
the
of
in
1-34
post
667-673
Feinberg MP, Cochin J (1972) Inhibition of development of tolerance to
morphine
149
by cycloheximide, Biochem.
Pharmacal., 21:
3083-3085
Fishbein W, McGraugh JL, Swarz JR (1971) Retrograde amnesia:
shock
effect
after
Science. 172:
80-82
termination
of
rapid
Electroconvulsive
eye movement sleep deprivation,
Goldstein DB, Pal N (1971) Alcohol dependence produced in mice by inhalation
ethanol grading the withdrawal reaction, Science, 172:
Harper MH,
Winter
PM.
Johnson
BH,
Koblin
DD,
Eger
withdrawal, J.
Pediatr., 91:
Neonatal
selective peptide inhibitors,
Pharmacal.
Jonge
H
de
Exp.
Ther., 211:
Inleiding
Withdrawal
Analg., 59:
19-21
with
narcotic
638-641
Li~e
(1979)
(1963)
(1980)
seizures
Hudgin RL, Charleson SE, Zimmerman M, Mumford R, Wood PL
Koblin DD, Dong DE, Eger EI
288-290
EJ
convulsions in mice following nitrous oxide, Anaesth.
Herzlinger RA, Kandall SA, Vaughan HG (1977)
of
Sci., 29:
Tolerance
(1981)
Enkephalinase:
2593-2601
of
mice
to
nitrous
oxide,
J
317-325
tot
medische
statistiek.
Wolters-Noordhoff,
Groningen
Manson HJ, Dyke G, Melling J, Gough M (1983) the
on
convulsions
Anaesth.
Soc.
in
mice
J., 30:
following
ef~ect
withdrawal
from
alcohol, Ann.
In:
NY.
nitrous
oxide,
Can.
28-31
Mendelson JH, Mello NK (1978) Recent developments in
addiction.
of naloxone and morphine
Mechanisms
Acad.
Sci., New York, Kissin B (ed) 311:69-79
de
vigilance
physical
of
Basic
Passouant P (1977) Influence des etats
underlying
chemotherapy
sur
Sleep 1976, WP Koella, P Levin (eds), Karger, Basel, pp.
narcotic
dependence
les
epilepsies
In:
57-65
Panov A (1982) RNA and protein synthesis and contents of brain stem cells
sleep deprivation, Rev biol., 75:
upon
after
95-99
Qock RM, Kouchich FJ, Tseng L-F (1985) Does
release
of
Smiths RA, Winter PN, Smith M, Eger EI II (1979) Tolerance to and dependence
on
brain opioid peptides ?, Pharmacal., 30:
nitrous
oxide
induce
95-99
inhalational anaesthetics, Anaesthesiol., 50:
505-509
Snead OE, Bearden LJ (1980) Anticonvulsants specific for petit
epileptogenic effect of leucine enkephalin, Science, 210:
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antagonize
1031-1033
150
CONCLUDING REMARKS AND S1JMMARY
In
chapter
mechanisms,
a
general
review
of
the
literature
on
sleep-waking
functions of sleep, and the biological and therapeutical effects
of sleep deprivation was provided.
Chapter 2 gave a brief review of the role
of the endogenous opioid system in physiological regulation.
In this thesis the role of endogenous opioid peptides in
of
sleep-waking
endogenously
opiates,
states
released
are
and
and
reported.
the
effects
exogenously
We
of
REM
sleep
administered
explored
the
the
on reactivity to
opioid
possibility
regulation
peptides.
that
REM sleep is
involved in regulating the functioning of opioid peptidergic neurons
central
nervous
system
by
studying
the
or
in
the
effects of REM sleep deprivation
(REMSD) on behavioural responses to endogenously released opioid peptides and
peptides.
The
opioid/opiate-induced behavioural responses were examined in
exogenously
administered
this
analgesia,
opiates
akinesia-catalepsy,
and
opioid
spontaneous
following
thesis
vertical motor activity (SVMA),
convulsions, grooming and nitrous oxide and morphine withdrawal symptoms.
Endogenous opioid peptides and waking mechanism (s)
The possible involvement of
waking
and
mechanism
~-endorphin
regulation
released
opioid
concentrations in brain nuclei, involved in
(chapter
~-endorphin,
dark,
endogenously
1,
sections
1.2,
1.3.2i).
Thus,
in
vigilance
state
concentrations of
enkephalins and dynorphin in the rat brain are
during
peptides
(s) is suggested by the diurnal oscillations in enkephalin
highest
in
the
which wakefulness is high, and lowest in the light phase, when
propensity to sleep is at its highest.
In our study we demonstrated that the inhibition of
phosphoramidon,
induced an increase in wakefulness.
stages were suppressed.
inhibition
was
enkephalinase,
Both NREM and REM sleep
The increased wakefulness induced
accompanied
by
excitatory
symptoms,
by
rats
(chapters
2,
section
1 .3.2i
and
phosphoramidon was decreased by naltrexone.
receptors
and
8).
The
enkephalinase
such as head shakes,
scratching (chapter 3) and excessive grooming, which normally
in
with
precede
insomnic
These data suggest
sleep
action of
that
opioid
endogenous enkephalins play an important role in sleep-waking
151
mechanism (chapter 3).
For
situations.
o~
This idea might be
example,
an
increase
in
relevance
in
some
clinical
opioid peptide levels has been
demonstrated during stress, anxiety and psychic disturbances associated
insomnia.
is
It
also
conceivable
that
this
new
class
of
with
drugs, the
enkephalinase inhibitors, might be of potential use in the treatment of sleep
disorders characterised by excessive somnolence.
Sleep-waking states and enkephalin induced neuronal excitation
The intracerebroventricular (icv) administration of enkephalin was
to
induce
epileptiform
found
activity in the hippocampal and cortical EEG and in
the EMG derived from submandibular muscle in freely moving rats (chapters 4).
Sleep, in particular REM sleep, decreased the enkephalin-induced epileptiform
discharges.
These observations
states
can
indicate
that
phasic
changes
during
sleep-waking
modulate the neuronal excitability regulated by opioid peptides.
This might be of importance for some forms of human
epilepsies
affected
by
the sleep-waking cycle.
RE~
sleep deprivation and nociception
REMSD decreased the pain threshold and abolished the analgesic effects of
morphine.
6).
enkephalinase inhibition and cold-water-swim (CWS) (chapters 5 and
The pain threshold to noxious electric shock was
REMSD
(chapter
1,
section
1 .5.2d,
vii).
similarly
reduced
sleep is an important factor in the physiological regulation of
The finding that
relevance
for
REMSD
those
can
individuals
reduce
the
who
suffer
insomnia or people working in shifts.
pathological
modify
the
by
All these data suggest that REM
pain
threshold
from
nociception.
might
disturbed
be
of
sleep
e.g.
In general it should be expected
that
conditions. and/or drug treatment accompanied with REMSD, could
therapeutic
effectiveness
of
opiates
and
other
analgesic
procedures.
REM sleep deprivation and monoamine-opioid interactions
There are abundant data showing that opioid peptides interact
other
physiologically
attention to
active
~-phenylethylamine
substances.
(PEA), a
with
many
In our study we paid particular
substrate
for
the
MAO-B
enzyme,
152
since
it
is known that inhibition of MAO-B modulates some effects of opioid
peptides (chapter 6, introduction).
(the
enzyme
which
biodegradates
MAO-B) had a stimulatory effect
released
In our study, the
PEA)
on
the
and
inhibition
of
MAO-B
excess of PEA (a substrate for
analgesic
action
of
endogenously
opioid peptides in rats allowed undisturbed sleep, but not in REMSD
animals (chapter 6).
The described facilitatory action of MAO-B inhibition on
enkephalinergic
transmission might be of relevance not only in the physiology of nociception,
but also in conditions associated with the alterations of MAO-B enzyme and/or
REM sleep (endogenous depression, ageing etc).
REM sleep deprivation induces a Functional deFiciency oT
~
opioid
receptor
system
The finding that opiate-induced analgesia, which is due to a preferential
activation
~-opioid
of
receptors,
~-receptor
functional insufficiency of a
test
this
concept.
inhibition.
In
mainly by the
motor
be
blocked
by
REMSD suggested a
system (chapters 5, 6).
In order to
we studied the effects of REMSD on opiate induced motor
this
akinesia/catalepsy
can
study
we
demonstrated
that
the
morphine
induced
syndrome, which is characterised by rigidity and mediated
~-opioid
receptors. was abolished
by
REMSD
and
replaced
by
excitation, seen as an increase in spontaneous vertical motor activity
(SVMA).
Naltrexone blocked the morphine induced akinesia/catalepsy
allowed
undisturbed
sleep,
REMSD animals (chapter 7).
opioid/opiate-induced
but
In
in
rats
not the opiate induced increase in SVMA in
addition
analgesia
to
the
fact
that
akinetic-cataleptic
REMSD
blocked
syndrome,
the
naltrexone sensitive wet-dog-shakes and grooming behaviours stimulated by
an
enkephalinase inhibitor were attenuated in REMSD animals (chapter 8).
These data collectively indicate that the blockade of
akinesia/catalepsy
syndrome and the reduced effects of enkephalinase inhibition in REMSD animals
are due to a
allowing
functional
for
insufficiency
of
the
~-opioid
blockade
morphine
functional
system,
an increased expression of the naltrexone resistant excitatory
opioid receptors responsible for the increased SVMA.
that
receptor
of
(chapter
~-opioid
7,
It is known for example
receptors facilitates the excitatory effects of
discussion).
The
idea
that
REMSD
can
induce
a
deficit of an opioid system derives further support from the fact
153
that enkephalinase inhibition
(chapter
9).
This
blocked
proposition
the
can
be
proconvulsant
~-opioid
(chapter 7. discussion).
REMSD
o~
supported by the known inhibitory
influence of opioid peptides on the release of
that blockade of
action
excitatory
transmitters
and
receptors vith naloxone facilitates epileptogenesis
The suggested functional deficiency
of
an
opioid
system might explain the increase in neuronal excitability in REMSD animals.
Some recent biochemical studies. provide
derangement
of
endogenous
opioid
decreased the concentrations of
in
the
(Haf~mans,
~-endorphin
during REMSD.
the
insuf~iciency o~
therapeutic
o~
o~
the
concept
of
For example REMSD
in the pituitary but increased it
We have also observed
leu-enkephalin in some
brain
areas
Ukponmwan, Dzoljic in preparation).
The proposed
cases
system
for
hypothalamus (chapter 1, section 1 .5.2b, vi).
that REMSD reduced the concentrations
~or
evidence
and
an opioid system during REMSD could account
o~
diagnostic value
REM sleep curtailment in some
depression and epilepsy respectively (chapter 7, discussion).
REM sleep deprivation and opiate dependence
Acute expose to nitrous oxide can lead to the release
in
the
iv).
CSF
o~
and opiate-like dependence phenomena (chapter 2, section 2.5.2,
The withdrawal convulsions in mice following exposure to nitrous
were
reduced
by
REMSD
(chapter
10).
Similarly,
8).
It
also
has
been
demonstrated
naloxone-precipitated jumping and myoclonic contractions
dependent
rats (Dzoljic et al.
in preparation).
basis of drug dependence is not fully understood,
that
several
has
also
been
attenuate
in
acute
morphine
Although the neurochemical
it
has
been
established
(chapter
2,
section
2.4.8).
demonstrated to decrease protein synthesis in the rat
brain (chapter 1, section 1 .5.2b, vii).
antagonistic
(chapter
drugs which can inhibit protein synthesis reduced the severity
opiate dependence and withdrawal phenomena
REMSD
REMSD
REMSD
that
oxide
naloxone-precipitated
wet-dog-shakes in morphine-dependent rats were attenuated by
o~
met-enkephalin
It is therefore suggested
that
the
of REMSD on opiate or nitrous oxide withdrawal phenomena
e~~ect
might be due to a decrease in protein synthesis in animals
deprived
of
REM
sleep.
The
the
~inding
existence
that REMSD can attenuate abstinential symptoms might
of
a
common
link
between
REM
sleep
de~iciency
suggest
and drug
154
dependence.
syndromes
However, the mechanism
and
whether
it
by
which
REMSD
inhibits
abstinential
modulates drug dependence in humans remain to be
clarified.
The following general conclusions can be made from this thesis:7o) Activation of endogenous opioid peptides increased wakeFulness
1b) Sleep, particularly the REM sleep stage, decreased the epileptic effects
enkephalin
when
compared
with
the
waking
of
Statements 1a and 1b
state.
indicate that there is an interaction between vigilance and
the
endogenous
opioid system.
2)
Inhibition of
facilitated
but not in
clinical
monoamine
oxidase
enkephalinergic
RE~SD
animals.
situations
B
and/or
excess
of
~-phenylethylamine
transmission in rats allowed undisturbed sleep
This
associated
finding
with
might
the
be
of
relevance
in
some
alterations of MAO-B and/or REM
sleep (endogenous depression, ageing, etc).
3)
Stimulation of the endogenous opioid system, with enkephalinase
attenuated
the
inhibition,
proconvulsant action of REMSD, suggesting an involvement of
opioid peptides in the regulation of neuronal excitability.
4)
The analgesic effects of endogenously released enkephalins
administered
opiates,
abolished by REMSD.
and
exogenously
which are mediated mainly by #-opioid receptor, were
This suggests that normal REM
sleep
is
an
important
factor for proper regulation of the pain threshold.
5)
The akinetic/cataleptic
effect
of
morphine,
also
mediated
by
#-opioid
receptors, was replaced by excitatory motor activity in REMSD rats.
6)
Conclusions 4 and 5 suggest that REM sleep
7)
REM sleep deprivation
deprivation
may
be
associated
with a functional insufficiency of #-opioid system.
sleep
deprivation
dependence.
decreased
abstinential
phenomena,
suggesting
that
could be an attractive tool in the investigation of drug
155
SAMENVATTING EN CONCLUSIES
In het eerste hoofdstuk werd een algemeen literatuuroverzicht gegeven
slaap-waak
mechanismen,
therapeutische
effecten
fysiologische
systeem
in
de
van
regulaties
de
proefschrift
vorm
functies
beschrijft
slaap
slaapdeprivatie.
korte
een
In
en
de
hoofdstuk
over
biologische
en
2
de
werden
toegespitst op de rol van het endogene opioid
hierin
van
van
samenvatting
van
de
literatuur.
Dit
een onderzoek naar de rol van endogene opioid peptiden
in de regulatie van slaap-waak stadia en de invloed van REM slaap op de effecten
van
endogeen
gesecreteerde- en exogeen toegediende opioid peptiden en opiaten.
Met name is de mogelijke betrokkenheid van
de
REM
slaap
bij
de
functionele
regulatie van opioid peptiderge neuronen in het centrale zenuwstelsel van de rat
onderzocht, via het bestuderen van de
e~~ecten
bij
endogeen
bepaalde
op
ge~ragsresponsies
van REM slaap deprivatie
gesecreteerde opioid peptiden en
exogeen toegediende opiaten en opioid peptiden.
volgende
door
opiaat/opioid
(REMSD)
In
geinduceerde
dit
onderzoek
gedragsresponsies
werden
de
bestudeerd:
analgesie, akinesie-catalepsie, spontane verticale motorische activiteit (SVMA),
convulsies,
poetsgedrag en opiaat onthoudingssymptomen opgewekt door lachgas en
morfine.
Endogene opioid peptiden en waak mechanisme(n)
De
mogelijke
mechanisme(n)
betrokkenheid
van
endogene
zou afgeleid kunnen worden uit de
E-endorfine concentraties in
bepaalde
opioid
peptiden
dag~luctuaties
hersenkernen
die
alertheidsregulering (hoofdstuk 1, sectie 1 .2, 1 .3.2i).
een
hoogste
in
het
grootst
van het farmacon
induceren.
toegenomen
vergezeld
is.
spelen
in
rattehersenen
Zowel
de
NREM
symptomen
licht,
wanneer
de
neiging
als
bleek
een
verhoging
van
de
vooraf
tot
waakzaamheid
als de REM slaap periodes werden onderdrukt.
gevolg
van
de
remming
van
enkefalinase
te
Deze
werd
van geprikkeldheid zeals schokbewegingen van de kop,
krabben (hoofdstuk 3) en overmatig poetsgedrag, dat normaal bij ratten
slapen
bij
De remming van het enzym enkefalinase door toediening
fosforamidon
waakzaamheid
door
rol
de nacht of in het danker, wanneer de waakzaamheid hoog is, en
zijn ze het laagst gedurende de dag of in het
slapen
waak
Dienovereenkomstig zijn
de concentraties van E-endorfine, de enkefalines en dynorfine
het
bij
in enkefaline en
gaat (hoofdstuk 2, sectie 1 .3.21 en hoofdstuk 8).
aan
het
Het bleek dat
156
de opwekkende Werking van fosforamidon werd verminderd door de opiaat antagonist
naltrexon.
Deze
gegevens
suggereren
dat
opioid
receptoren
enkefalines een belangrijke rol spelen in het slaap-waak
3).
Dit
mogelijke
verband
zou
in
mechanisme
Er is bij voorbeeld bij de mens een toename in
concentraties
aangetoond
gedurende
stress.
storingen die samengaan met slapeloosheid.
type
endogene
(hoofdstuk
bepaalde situaties klinische implicaties
kunnen hebben.
nieuw
en
endogene
angsttoestanden
Het
en
voorstelbaar
ook
is
opioid
psychische
dat
een
van farmaca, de enkefalinase remmers, potentieel van nut zou kunnen
zijn bij de behandeling van patienten die lijden aan excessieve slaperigheid.
Slaap-waak stadia en inductie van neuronal& excitatie door enkefaline
De intracerebroventriculaire (i.c.v.) toediening van
epilepti~orme
activiteit
ook zichtbaar aan het EMG van de
submandibulaire
ratten
en
(hoofdstuk
4).
Slaap,
enkefaline geinduceerde
enke~aline
induceerde
in de hippocampus en de cortex volgens het EEG en was
epilepti~orme
spieren
bij
vrij
bewegende
in het bijzonder de REM slaap, kon de door
ontladingen doen verminderen.
Deze observaties geven aan dat fasische verschuivingen in slaap-waak stadia
het
van
ef~ect
moduleren.
opioid
peptiden
op
de
neuronale
prikkelbaarheid,
kunnen
Dit zou bij de mens betekenis kunnen hebben voor bepaalde vormen van
epilepsie, waarop de slaap-waak cyclus een invloed heeft.
REM slaap deprivoti& en pijnprikkeling
REMSD verminderde de pijndrempel en antagoniseerde het analgetische
resp.
enkefalinase
mor~ine,
remming,
e~~ect
in-koud-water-zwemmen
en
(CWS)(hoofdstukken 5 en 6).
De pijndrempel veer een electrische schok kan eveneens
REMSD
(hoofdstuk
slaap
een
1,
sectie 1.5.2d.vii).
belangrijke
~actor
is
in
verminderd
worden
door
Al deze gegevens duiden erop dat REM
de
regulatie
~ysiologische
van
pijnprikkeling.
De waarneming dat REMSD de pijndrempel kan verlagen, zou
hebben
voor
verwachten
behandeling
effektiviteit
kunnen
die personen die aan een gestoorde slaap lijden zeals bijvoorbeeld
slapeloosheid bij mensen die in ploegendienst werken.
kunnen
betekenis
met
dat
farmaca
pathologische
die
opiaten
vergezeld
en
andere
In het algemeen
en/o~
omstandigheden
gaan
met
REMSD,
analgetische
de
een
zou
men
bepaalde
therapeutische
behandelingen
kunnen
157
beinvloeden.
REM sloop deprivatie en monoamine-opioid interakties
Er is een veelheid aan
effecten
van
actieve stoffen.
besteed
aan
laat
dat
zien
de
In dit proefschrift is
in
dit
verband
bijzondere
aandacht
t-fenylethylamine (PEA), een substraat voor het MAO-B enzym, omdat
bekend is dat remming van
beinvloeden
in de literatuur die
in~ormatie
opioid peptiden interfereren met die van vele andere fysiologisch
(hoofdstuk
MAO-B
6:
bepaalde
effecten
-introduction-).
PEA afbreekt) en een overmaat aan PEA (het
opioid
peptiden
kan
Remming van MAO-B (het enzym dat
substraat
~erking
stimulerend effect op de pijnstillende
van
voor
MAO-B)
hadden
een
van endogeen gesecreteerde opioid
peptiden bij ratten die ongestoord konden slapen, maar niet bij
ratten
waarbij
de REM slaap werd onthouden (REMSD)(hoofdstuk 6).
Het effect van
beschreven
MAO-B
remming
neurotransmissie
op
enkefalinerge
transmissie,
die
zoals
bevorderend is, kan niet alleen van betekenis zijn
voor het begrip van de fysiologie van pijnprikkeling bij de mens, maar oak
voor
die van bepaalde pathologische condities die verband houden met veranderingen in
de activiteit van het MAO-B enzym of van de
hoeveelheid
REM
slaap
zoals
bij
endogene depressies of bij het verouderen.
REM slaap deprivatie en de inductie
het
van
een
in
het
De bevinding dat de analgesie die door een opiaat wordt geinduceerd en
die
~-opioid
gevolg is van een stimulering van voornamelijk de
geblokkeerd kan worden door REMSD,
insufficientie
in
het
~-type
opiaten
veroorzaakte
motorische
studie kwam naar voren dat het
syndroom,
deficientie
werd
dat
geinterpreteerd
vervangen
~-receptor
door
opioid receptoren.
als
een
functionele
effect
van
REMSD
op
activiteitsvermindering bestudeerd.
door
gekarakteriseerd
een preferentiele
~-type
receptor systeem (hoofdstukken 5 en 6).
opvatting te kunnen toetsen, is vervolgens het
werd
funktionele
receptor system
morfine
geinduceerde
de
door
Uit deze
akinesiejkatalepsie
wordt door een lichaamsstarheid als gevolg van
stimulering, werd tegengegaan door REMSD
motorische
Om deze
activiteitsvermeerdering
toegenomen spontane vertikale motorische activiteit (SVMA).
in
en
zelfs
de vorm van een
De opioid
receptor
antagonist naltrexon blokkeerde de door morfine geinduceerde akinesiejkatalepsie
in ratten die ongestoord konden slapen, maar niet de door
morfine
geinduceerde
158
toename
in
in
SVMA
dieren
met
REMSD
beschreven waarnemingen dat REMSD
de
(hoofdstuk
door
opioiden
7).
en
Naast
de hierboven
opiaten
geinduceerde
analgesie en het akinesie/katalepsie syndroom kan blokkeren, bleek dat in dieren
met
het
REMSD
opioid
-wet-dog-shakes-
en
geinduceerde
gedrag,
zeals
de
naltrexon-sensitieve
het door een enkefalinase remmer geinduceerde poetsgedrag,
eveneens verzwakt werd (hoofdstuk 8).
Uit het geheel van deze waarnemingen zou
blokkering
van
men
kunnen
concluderen
van
het
receptor
~-opioid
hierbij toegeschreven aan een zichtbaar
opwekkende
receptor
de
het akinesie/katalepsie syndroom en de verminderde effecten van
de enkefalinase remming in REMSD dieren worden veroorzaakt door een
insufficientie
dat
opiaat-effecten,
type.
Het
is
middels
systeem.
geworden
een
bijvoorbeeld
De toegenomen SVMA wordt
expressie
ander
bekend
functionele
(demaskering)
van
en naltrexon-resistent opioid
dat
blokkering
van
~-opioid
receptoren de expressie van opwekkende effecten van morfine kan vergemakkelijken
(hoofdstuk 7:
een
opioid
discussie).
systeem
kan
Het denkbeeld dat REMSD een
(hoofdstuk 9).
het
waarbij
in
proconvulsieve
effect
van
REMSD
kan
Deze bewering wordt ondersteund door het bekende feit
van de remmende werking van opioid
neurotransmitters,
tekort
induceren, wordt verder ondersteund door het feit dat
remming van het enzym enkefalinase
blokkeren
functioneel
een
het ontstaan
van
epileptische
discussie).
Het
veronderstelde
peptiden
op
blokkering van
verschijnselen
functionele
de
afgifte
~-opioid
van
stimulerende
receptoren met naloxon
vergemakkelijkt
(hoofdstuk
7:
tekort in een opioid systeem zou
aldus de toename in normale prikkelbaarheid in REMSD dieren kunnen verklaren.
Enkele recente
biochemische
studies
ondersteunen
het
concept
van
een
verstoring van endogene opioid systemen als gevolg van deprivatie van REM slaap.
REMSD verminderde bijvoorbeeld de concentratie van E-endorfine in
maar
in
vi).
In overeenstemming hiermee
de
hypofyse.
de hypothalamus nam deze concentratie toe (hoofdstuk 1, sectie 1 .5.2b.
verminderde
concentraties
Ukponmwan, Dzoljic;
van
vinden
wij
leu-enkefaline
in
bepaalde
REMSD
zou
verantwoordelijk
kunnen
een
zijn
opioid
voor
systeem
het
respectivelijk
discussie).
bepaalde
types
depressie
en
epilepsie
als
gewenste
therapeutische effect en de diagnostische waarde van het beperken van REM
bij
ook
in voorbereiding).
De veronderstelde functionele insufficientie van
gevolg
hersengebieden
als gevolg van REMSD (Haffmans,
slaap
(hoofdstuk
7:
159
REM slaap deprivatie en lichamelijke afhankelijkheid van opiaten
Een acute inhalatie van lachgas kan de afgifte
hersenvloeistof
verhogen
fenomenen oproepen.
van
Met-enkefaline
in
de
en aldus opiaat-achtige lichamelijke afhankelijkheids
De convulsies als element van de
onthoudingsverschijnselen
bij muizen na lachgas inhalatie waren verminderd in REMSD dieren (hoofdstuk 10).
Hiermee vergelijkbaar was het door naloxon opgeroepen -wet-dog-shakes- gedrag in
ratten
die
tevoren
REMSD dieren.
gedrag
en
Oak
i~
morfine
afhankelijk gemaakt waren, minder uitgesproken in
aangetoond dat REMSD het door
naloxon
opgeroepen
spring
de myoclonische contracties bij ratten die acuut morfine-afhankelijk
gemaakt zijn, kan verzwakken (Dzoljic et
neurochemische
basis
van
al.,
afhankelijkheid
in
van
voorbereiding).
verdovende
Hoewel
substanties niet bekend is, is wel vastgesteld dat verschillende stoffen die
eiwitsynthese
remmen
de
mate
van
opiaat
afhankelijheid
onthoudingsverschijnselen doen verminderen (hoofdstuk 2.
aangetoond,
1.
sectie
dat
de
middelen en andere
en
sectie 2.4.8).
de
van
Het is
REMSD de eiwitsynthese in ratte hersenen vermindert (hoofdstuk
1 .5.2b,
antagonistische
vii).
Om
deze
reden
wordt
voorgesteld,
dat
het
effect van REMSD op opiaat en lachgas onthoudingsverschijnselen
veroorzaakt zou kunnen worden door een afname van de
eiwitsynthese
bij
dieren
met REM slaap onthouding.
De waarneming dat REMSD opiaat
veronderstelt
lichamelijke
het
bestaan
afhankelijkheid
onthoudingsverschijnselen
lichamelijke
kan
afhankelijkheid
onthoudingsverschijnselen
een
verband
opiaten.
tussen
Het
REM
mechanisme
kan
slaap
verzwakken,
tekort
waardoor
en
REMSD
verzwakken en of REMSD invloed kan uitoefenen op
van
opiaten
bij
de
mens
meet
echter
nader
onderzocht worden.
Uit de resultaten van dit proefschrift kunnen de
volgende
algemene
conclusies
getrokken worden:
1a)
Activering van het endogene opioid systeem doet de waakzaamheid toenemen.
1b)
Slaap, in het bijzonder het REM slaap stadium, vermindert
de
effecten van enkefaline. vergeleken met het waak-stadium.
De stellingen 1a
epileptische
en 1b geven aan dot er een interactie is tussen de mate van waakzaamheid en
het endogene opioid systeem.
2)
Remming van monoamine oxidase 8 en/of een
vergemakkelijkt
enkefalinerge
overmaat
aan
~-fenylethylamine
transmissie in rotten die ongestoord kunnen
160
slapen, maar niet in rotten met REM sloop deprivatie.
voor
de
mens
betekenis
kunnen
hebben
Daze
conclusie
zou
in bepaalde klinische situaties,
waarin veranderingen in MA0-8 enzym activiteit
en/of
REM
sloop
een
rol
spelen (endogene depressie, veroudering, enz.).
3)
Stimulering van het endogene opioid
enkefalinase
betrokkenheid
verzwakt
van
de
systeem
proconvulsieve
opioid
peptiden
in
via
ramming
werking
de
van
het
enzym
van REM$0, hetgeen een
regulatie
van
neuronale
prikkelbaorheid gesuggereert.
4)
De analgetische werking van endogeen gesecreteerde enkefalines
exogeen
toegediende
opiaten,
die voornamelijk het
stimuleren, vordt tegengegaan door REMSD.
een
normale
REM
slaap
een
~-opioid
en
van
Hieruit kan men concluderen
belangrijke
Taktor
is
voor
de
receptor type
een
dat
adequate
installing van de pijndrempel.
5)
Het akinetische/kataleptische effect van morfine, dat ook via de stimulatie
van
~-opioid
receptors
werkt,
wordt
in REMSD rotten vervangen door een
verhoogde motorische activiteit.
6)
Uit de conclusies 4 en 5 zou men kunnen afleiden dat REM
geassocieerd
is
met
een
functionele
insufficientie
slaap
van
deprivatie
het
~-opioid
systeem.
7)
REM slaap deprivatie vermindert opiaat
onthoudingsverschijnselen,
hetgeen
doet vermoeden dot slaap deprivatie een aantrekkelijk hulpmiddel zou kunnen
zijn bij verslavingsonderzoek.
161
List of Publications
*Dzoljic
MR.
Ukponmwan
OE,
Baas
AAG,
vd
Berg
DT,
Rupreht
(1984)
J.
Enkephalinase inhibition and REM sleep deprivation inhibit the nitrous oxide
withdrawal convulsions, In:Epilepsy, Sleep and Sleep Deprivation •
Degen
R
and E Neidermeyer (eds) Elsevier, pp 305-310
*Dzoljic
MR,
Ukponmwan
enkephalinergic
OE,
system
Rupreht
Haffmans
J,
(1985)
J.
The
role
of
in sleep studied by an enkephalinase inhibitor, In:
Sleep, Neurotransmitter and Neuromodulators, Mauquier et al (eds), New York,
pp 251-263
Dzoljic MR, Haffmans J, Ukponmwan OE.
Endogenous opioid
peptides
(endorphins)
and neuronal excitation, Acta Yugoslav Pharmacal (in press).
Dzoljic MR, Haffmans J, Rupreht J, Ukponmwan OE (1985) Endogene opioid
en pijn, IKR-Bulletin, 9:
Dzoljic MR, Ukponmwan OE (1985) The inhibitory effect of REM
(REMSD)
In:
peptiden
12-14
sleep
deprivation
on opiate withdrawal and quasi-morphine withdrawal syndrome (QMWS),
Sleep 1984, Koella
WP,
Ruther
E,
Schultz
H
(eds),
Gustav
Fisher
Verglag, Stuggart, New York, pp 397-399
Dzoljic MR, Radermaker B,
Enkephalinase
Poel-Heisterkamp
inhibition
supresses
ALvd,
Ukponmwan
naloxone-induced
DE,
Haffmans
jumping
J,
in morphine
dependent mice, Arch Int Pharmacodyn (in press)
Grillo SA, Ukponmwan DE (1981) The effect of amphetamine and
the
electrolyte
Pharmac 12:
contents
chlorpromazine
on
of the cerebrospinal fluid of the cat, Nig Journ
300-303
Haffmans J, Blankwater YJ, Ukponmwan DE, Zijlstra FJ, Vincent JE,
Dzoljic
MR.
(1983)
enkephalin in rat brain
Hespe W and
between the distribution of 3 -H-labelled
Correlation
and
anatomical
induced seizures, Neuropharmacol 22:
Rupreht J,
Ukponmwan
phosphoramidon-
DE,
a
Admiraal
selective
behaviour, Neurosci Lett 41:
Rupreht
J,
Ukponmwan
Enkephalinase
DE,
inhibition
Dzoljic
MR.
involved
in
enkephalin-
1021-1028
and
Dzoljic
enkephalinase
MR.
inhibitor
(1983)
on
Effect
of
nociception and
331-335
Dworacek
B,
Admiraal
PV,
Dzoljic
MR.
(1984)
prevented tolerance to nitrous oxide analgesia in
rats, Acta Anaesthesiol Scand 28:
Ukponmwan DE,
PV
regions
(1981)
617-620
The
modulatory
effect
of
morphine
and
162
monoamine
B
oxidase
system
(MAO-B)
on
enkephalin-induced
seizures,
In:Advances in Endogenous and Exogenous Opioids, Takagi H and EJ Simon (eds)
Kodanska LtD, Tokyo, pp 226-228
Ukponmwan OE, Poel-Heisterkamp ALvd,
deprenyl
inhibitor
Haffmans
J,
Dzoljic
B-phenylethylamine
Met-enkephalinamide-induced seizures,
MR.
(1983)
MAO-B
potentiate
Naunyn-Schmied
Arch
Pharmacal
322:
38-41
**Ukponmwan OE, Dzoljic MR.(1983) Stages
of
vigilance
and
enkephalin-induced
seizures, In:Sleep 1982, Koella WP (ed), Karger, Basel pp 250-252
**Ukponmwan OE, Rupreht J, Dzoljic MR (1984) REM sleep deprivation decreases the
antinociceptive
property
enkephalinase
of
cold-water-swim, Gen Pharmacal 15:
**Ukponmwan OE, Dzoljic
increased
MR
(1984)
susceptibility
Psychopharmacol 83:
to
inhibition,
Enkephalinase
seizure
inhibition
induced
by
REM
the
OE.
enkephalinase
grooming
Rupreht
antagonizes
the
sleep deprivation,
and
shaking
behaviour
sleep
induced
J,
inhibition
Dzoljic
MR
(1986)
An
deprivation
by enkephalinase
inhibitor or opiate withdrawal, Pharmacal Biochem Behav 23:
**Ukponmwan
and
229-232
**Ukponmwan OE, Poel-Heisterkamp ALvd, Dzoljic MR (1985) REM
decreases
morphine
255-258
385-389
analgesic
effect
of
is modulated by monoamine oxidase B and REM sleep
deprivation. Naunyn-Schmied Arch Pharmacal (in press)
**Ukponmwan OE, Poel-Heisterkamp ALvd, Dzoljic MR (1986) REM
sleep
deprivation
antagonized the morphine-induced akinesia and catalepsy, Sleep (in press)
(*) Articles partly incorporated in this thesis
(**) Articles fully incorporated in this thesis
163
Curriculum Vitae
Name ....... Otasowie Eghe Ukponmwan
Date and place of birth ..... July 31st 1951, Benin City, Nigeria.
Education:Secondary:
Edo (Govt) College, Benin City, Nigeria
West ATrican School Certificate (1969)
General Certificate of Education (GCE 0/L Lond, 1970)
University:
School o£ Pharmacy, University of Ife, Nigeria (1970/75):
B.Pharm (Hons, 1975)
Professional training:
Specialist Hospital, Benin City (1975/76):
Internship (MPSN)
Post~aduate
studies and research training:
1) Faculty of Health Sciences, University of Ife, Nigeria
Courses: Neurobiology, Individual Behaviour and Psychophysiology,
Neuromuscular Pharmacology.
M.Sc (Human Bioi, 1978).
2) Division of Biocybernetics. Institute £or Medical and Dental
Engineering, Tokyo Medical and Dental University.
Tokyo
(1979/1980)(Sponsored by Japan International Co-operation
Agency JICA):
Sleep studies (supervision by Pro£ S.
Inoue).
Certificate in Neurophysiology
3) Dept o£ Pharmacology, Erasmus University Rotterdam, (1981/85).
Free doctoraal examinations:
Courses:
Biostatistics (Prof R.
van Strik), Neurophysiology
(Prof M.W van Hof and Dr JF Hobbelen), Chemical Pathology (Prof
H.G van Eijk), Pharmacology (Prof I.L.
Bonta, Prof J Bruinvels
and Prof P.R Saxena), Clinical Pharmacology (A.J Man in'
t
Veld,
Internist).
Research project
Sleep-vaking states and endogenous opioid
system (supervision by Dr MR Dzoljic).
164
ACKNOWLEDGEMENT
My gratitude to all the people
completion of this thesis cannot
~ho
have contributed
to
be adequately expressed.
the
successful
Therefore, I wish
to express my thanks to the following people:-Dr. MR Dzolj ic f'or his good supervision and
critical
remarks
throughout
the duration of this project.
-Prof. IL Bonta and other members
of
the
-promotiecommissie-
for
their
constructive criticisms.
-Special thanks go to Loeky van der
Poel-Heisterkamp
for
her
efficient
assistance in carrying out several parts of this study.
-I would like to thank Graham Elliott for his useful suggestions
and
the
linguistic revisions of several portions of this manuscript.
-My thanks also go to Fred Opmeer for the lively
the Dutch version
-The
collegial
o~
discussions
and
making
the summary.
and
collaborative
attitude
o~
JoZe
Rupreht is deeply
appreciated.
-I also wish to thank Judith Haffmans
~or
her contributions and help as
a
contact person during the -promotie- arrangement.
-The
excellent
administrative
help
rendered
by
Magda
Busscher-Lauw
throughout my stay in the department is highly appreciated.
-My thanks also go to
personnel
o~
Dh~
WJ van Alphen, Dhr. F Schumacher(G-CRW)
and
the
the-Automatische Signaal Ver'Werking- and ""Audiovisueel Centrum··
for all their technical assistance.
-To all other members of the department -a big thank you- for the
cordial
atmosphere which made my stay pleasant.
-Special thanks go to my parents and the Bendel State Government (Nigeria)
for their moral and financial support.
-Finally special thanks also go to my wife Catherine
~or
putting
up
me during the long period necessary for the preparation of this thesis.
'With