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12-1993
Co-sensitization of Dopamine and Serotonin
Receptors Occurs in the Absence of a Change in
the Dopamine D1 Receptor Complex After a
Neonatal 6-ohda Lesion
Li Gong
East Tennessee State University
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Gong, Li, "Co-sensitization of Dopamine and Serotonin Receptors Occurs in the Absence of a Change in the Dopamine D1 Receptor
Complex After a Neonatal 6-ohda Lesion" (1993). Electronic Theses and Dissertations. Paper 2686. http://dc.etsu.edu/etd/2686
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Co-sensitization of dopamine and serotonin receptors occurs in
the absence of a change in the dopamine D1 receptor complex
after a neonatal 6-OHDA lesion
Gong, Li, Ph.D.
East Tennessee State University, 1993
UMI
300 N. Zeeb Rd.
Ann Aibor, MI 48106
CO-SENSITIZATION OF DOPAMINE AND SEROTONIN RECEPTORS OCCURS
IN THE ABSENCE OF A CHANGE IN THE DOPAMINE D1 RECEPTOR
COMPLEX AFTER A NEONATAL 6-OHDA LESION
A Dissertation Presented to
The Faculty of the Department of Pharmacology
James H. Quillen College of Medicine
East Tennessee State University
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy in Biomedical Science
by
Li Gong
December 1993
APPROVAL
This is to certify that the Graduate Committee of
LI GONG
met on the
20^-
_day of^
The committee read and examined his dissertation,
supervised his defense of it in an oral examination, and
decided to recommend that his study be submitted to the
Graduate Council and the Associate Vice-President for
Research and Dean of the Graduate School, in partial
fulfillment of the requirements for the degree Doctor of
Philosophy in Biomedical Science.
Chairman, Graduate Committee
V Iug*y C, CjlMuidt
Signed on behalf of
the Graduate Council
7
Associate Vice-President for
Research and Dean of the
Graduate School
ii
ABSTRACT
CO-SENSITIZATION OF DOPAMINE AND SEROTONIN RECEPTORS OCCURS
IN THE ABSENCE OF A CHANGE IN THE DOPAMINE D, RECEPTOR
COMPLEX AFTER A NEONATAL 6-OHDA LESION
by
Li Gong
To test whether SKF 38393 could ontogenetically
sensitize dopamine (DA) D, receptors and whether this
sensitization would be associated with biochemical changes,
intact and neonatal 6-hydroxydopamine (6-OHDA)-lesioned rats
(200 pq i.c.v.) were treated daily from birth with SKF 38393
(3.0 mg/kg i.p. x 28 days) or its vehicle. In DA D,
neonatally sensitized 6-OHDA rats, enhanced locomotor
responses were observed with the first SKF 38393 challenge
dose (3.0 mg/kg i.p.) at 6 weeks. This response increased
further with weekly SKF 38393 treatments. Enhanced
stereotyped behaviors were seen in both lesioned and
sensitized rats at 8 weeks. There was no change in the
percentage of high affinity D, sites in these groups of
rats. Striatal mRNA levels for D, receptors were reduced in
the lesioned rats, but restored to control level after
treatments with SKF 38393 in adulthood. Basal, DA-, NaFand forskolin-stimulated adenylate cyclase activities were
similar among treatment groups. Striatal DA content was
reduced (>99%), whereas serotonin (5-HT) content was
elevated (>50%) in the 6-OHDA groups. To study possible
interaction between DA and 5-HT systems, the effects of a
series of 5-HT agents on the induction of oral activity were
determined. The 5-HTlc receptor agonist, mchlorophenylpiperazine (m-CPP), produced a marked increase
in oral activity in 6-OHDA-lesioned rats. The respective 5HT,A and 5-HT,B agonists, 8-OH-DPAT and CGS-12066B did not
increase oral activity. The m-CPP-induced oral response in
the lesioned rats was attenuated by mianserin, a 5-HT,c
antagonist, but not by ketanserin or MDL-72222, 5-HT2 and 5HTj antagonists, respectively. Although the supersensitized
oral response of lesioned rats to m-CPP was not attenuated
by SCH 23390, the enhanced response of SKF 38393 was
attenuated by mianserin. Additionally, mRNA levels for 5HT1C receptor were not altered in both intact and lesioned
rats. These findings demonstrate that ontogenetic
treatments of neonatal 6-OHDA-lesioned rats with a D,
agonist produce partial sensitization of DA D, receptors in
adulthood without altered biochemical markers, and that this
neonatal lesion is associated with both supersensitized DA
D, and 5-HTlc receptors. Moreover, induction of oral
activity by DA agonists is mediated via a serotonergic
system.
iii
DEDICATION
To my parents for their love and encouragement.
iv
ACKNOWLEDGMENTS
I would like to thank my major advisor, Dr. Richard
Kostrzewa, for his continued guidance, constant advice and
efforts throughout this thesis project.
I also wish to
thank Dr. Ernest Daigneault, Department Chairman, who took
precious time from his own busy work to provide advice and
support.
My thanks go to other members of my graduate
advisory committee: Drs. Peter Rice, Scott Champney, George
Youngberg and Hugh Criswell, for their assistance and
support.
I am indebted to Dr. Chuanfu Li for his generous
support and constant patience to provide technical
assistance in mRNA assays.
Drs. Ray Fuller and Kenneth
Ferry are acknowledged for their generously providing
monoamine assays.
I also wish to thank Dr. Ryszard Brus for
his help in our arduous but enjoyable behavioral
observations.
v
CONTENTS
Page
APPROVAL............................................. ii
ABSTRACT........................................... iii
DEDICATION........................................... iv
ACKNOWLEDGMENTS
...................................
V
LIST OF F I G U R E S ...................................
X
LIST OF TABLES
................................... xii
LIST OF PHARMACOLOGICAL AGENTS .....................
xiii
Chapter
1. INTRODUCTION
A. Anatomical Review of Dopamine System . . . .
l
B. Ontogny of Dopamine System . . . . . . . . .
2
C. Proposed Functions of Dopamine System
...
4
D. Dopamine Receptors .......................
5
E. Characterization and Localization of
Dopamine D, Receptors .....................
6
F. Signal Transduction systems of Dopamine
D, R e c e p t o r s ............................... B
G. Dynamics of Dopamine Dt Receptors .......... 9
H. Ontogeny of Dopamine D, Receptors .......... 9
I. Molecular Biology of Dopamine Receptors. . . 10
J. Innervation of Serotonin Fibers
to the S t r i a t u m ........................... 12
K. Serotonin Receptors in Striatum
L. 6-Hydroxydopamine and Dopamine
vi
.......... 13
Chapter
Page
Receptor Supersensitization
.............. 17
H. 6-Hydroxydopamine and Serotonin System . . .
19
N. Dopamine System and Animal
Behavioral Models
........................ 20
0. Brain Dopamine System and
Clinical Diseases
.......................
23
P. 5-HTlc Receptor-Related Animal
Behaviors and Clinical Implications
....
25
Q. R a t i o n a l e ................................ 28
2. MATERIALS AND METHODS
A.
Animals and T r e a t m e n t .................... 33
B. Behavioral Observations
.................
34
C. Determination of DA, 5-HT and Their
Metabolites in the S t r i a t u m ............... 37
D. Determination of DA Competition
for [3H]SCH 23390 Binding to
Striatal Homogenates
..................... 38
E. Determination of mRNA Levels for D,
and 5-HTlc Receptors in the Striatum......... 40
F. Determination of DA-Stimulated Adenylate
Cyclase Activity in the Striatum .......... 42
6 . Data A n a l y s i s ............................ 44
3. RESULTS
A.
Locomotor Activity of R a t s ................ 47
B.
Stereotyped Behaviors of R a t s ............ 49
vii
Chapter
Page
C. Striatal Levels of DA,DOPAC, HVA,
5-HT and 5 - H I A A ........................... 50
D. Agonist Competition for DA D, Receptor
Binding in the Striatal Homogenates
....
E. Striatal Levels of DA D, Receptor mRNAs
51
. . 51
F. striatal DA-Stimulated Adenylate
Cyclase Activity ..........................
52
G. Dose-Response Study of SKF 38393Induced Oral A c t i v i t y ..................... 53
H. Effect of Combined D( Agonist and Dt
Antagonist Treatments
.................... 53
I. Dose-Response Study of m-CPPInduced Oral A c t i v i t y ..................... 54
J. Effects of 5-HT,a and 5-HTlB Receptor
Agonists on Oral A c t i v i t y ................. 54
K. Effects of 5-HT Receptor Antagonists
on m-CPP-Induced Oral Activity ............ 55
L. Effect of a DA D, Receptor Antagonist
on m-cpp-lnduced Oral Activity ............ 55
N. Effect of a 5-HT)C Receptor Antagonist on
DA Dt Agonist-Induced Oral Activity
....
56
N. Effect of Neonatal 6-OHDA Treatment on
Concentration of DA, 5-HT an Their
Metabolites in the s t r i a t u m ............... 56
0. Effects of Neonatal 6-OHDA and Ontogenetic
viii
Chapter
Page
SKF 38393 Treatments on mRNA Levels for
5-HTjC Receptors of the S t r i a t u m ......... . 5 7
4. DISCUSSION
A. DA D,ReceptorSupersensitization
........... 83
B. Mediation of DA Agonist-Induced Oral
Behavior by the
5-HTs y s t e m ................. 93
5. S u m m a r y ...................................... 99
BIBLIOGRAPHY
.....................................
100
V I T A ................................................. 123
ix
LIST OF FIGURES
Figure
Page
1. Effect of SKF 38393 on Locomotor Activity
in Rats (First Injection)
................... 63
2. Effect of SKF 38393 on Locomotor Activity
in Rats (Second Injection) ........
.....
64
3. Effect of SKF 38393 on Locomotor Activity
in Rats (Fourth Injection)...................65
4. Dopamine Inhibition of [3H]SCH 23390
Binding in Rat Striatum (Saline) ............. 66
5. Dopamine Inhibition of [3H]SCH 23390
Binding in Rat Striatum(6-O H D A ) .............. 67
6 . Dopamine Inhibition of [3H]SCH 23390
Binding in Rat striatum
(SKF38393)
7. Dopamine Inhibition of [JH]SCH 23390
Binding in Rat striatum (SKF 38393+6-ohda) . . 69
8A.Striatal Dopamine D, Receptor mRNA
Levels Before Treatments with SKF 38393
in Adult R a t s ........................... . . 70
8B.Striatal Dopamine D, Receptor mRNA
Levels After Treatments with SKF 38393
in Adult R a t s ............................... 71
9. Dopamine-Stimulated Striatal Adenylate
Cyclase Activity in R a t s ..................... 72
10, Striatal Basal, NaF- or Forskolinx
68
Figure
Page
Stimulated Adenylate Cyclase Activity
in Adult R a t s ................................. 73
11.
SKF 38393-Induced Oral Activity...............74
12. Attenuation of SKF 38393-Induced Oral
Activity in Rats by SCH 23390 ................ 75
13.
M-CPP-Induced Oral Activity in R a t s ...........76
14.
Effects of Serotonin 1A and IB Receptor
Agonists on Oral Activity in R a t s ............. 77
15. Effects of Serotonin Receptor Antagonists
on m-CPP-Induced Oral Activity in Rats
....
78
16. Lack of Effect of SCH 23390 on
m-CPP-Induced Oral Activity in R a t s ........... 79
17. Attenuation of SKF 38393-Induced Oral
Activity in Rats by Mianserin................. 80
18A.striatal Serotonin 1C Receptor mRNA
Levels After Treatments with SKF 38393
in Adult R a t s .................................81
18B.Striatal Serotonin 1C Receptor mRNA
Levels After Treatments with SKF 38393
in Adult R a t s .................................82
xi
LIST OF TABLES
Table
Page
1. Behavioral Effects of Acute Saline
Challenge in R a t s ............................. 58
2. Behavioral Effects of Acute SKF 38393
Challenge in R a t s ............................. 59
3. Effects of Neonatal 6-OHDA and Ontogentic
SKF 38393 Treatments on Striatal Concentrations
of DA, 5-HT and Their Metabolites............. 60
4. Computer-modeled Parameters for Dopamine
Inhibition of [3H]SCH 23390 Binding
in Striatal Homogenates ......................
61
5. Effect of Neonatal 6-OHDA Treatment on
Concentrations of DA, 5-HT and Their
Metabolites in the striatum of Rats . . . . . .
xii
62
LIST OF PHARMACOLOGICAL AGENTS
SKF 38393 ------ DA D, agonist
SCH 23390 ------ DA D, antagonist
m - C P P ---------- 5-HTlc>2 agonist
8-OH-DPAT------ 5-HTia agonist
CGS 12066B----- 5-HT,b agonist
Mianserin------ 5-HTlcl antagonist
Ketanserin -----
5-HTj antagonist
MDL 72222 ------ 5-HT3 antagonist
6-OHDA
catecholamine neurotoxin
Desipramine ---- noradrenergic reuptake blocker
xiii
Chapter 1
Introduction
Anatomical Review of Dopamine system
Dopamine (DA), a veritable newcomer to the field of
monoamine transmitters in mammalian central nervous system
(CNS), belongs to a class of compounds termed catecholamines
(Montagu, 1957).
Until the mid-1950s DA was simply
considered to represent an intermediate precursor in the
biosynthesis of norepinephrine and epinephrine.
Because the
concentration of DA is almost equal to those of
norepinephrine in the brain, and because the regional
distribution of these two catecholamines is markedly
different, it was proposed that the biological role for DA
in CNS is independent of its function as a precursor in
norepinephrine synthesis (Carlsson et al., 1958? 1959).
With the development of histochemical and immunocytochemical
methods for visualizing catecholamines in brain tissue
sections (Dahlstrom and Fuxe, 1964? Fuxe et al., 1978), it
is now convenient to localize dopaminergic pathways under
several major categories as follows:
l. Nigrostriatal DA system,
A group of cell bodies,
designated the A9 group, are located primarily in
the substantia nigra pars compacta.
These neurons
project mainly into neostriatum (principally the
caudate and putamen), globus pallidus and
1
subthalamic nucleus.
2. Hesollmbic DA system.
One group of DA-containing
neurons, known as the A10 group, originates in the
ventral tegmental area and terminates in the
nucleus accumbens, olfactory tubercle and nucleus
interstitialis stria terminalis.
3. Mesocortical DA system.
Neuronal cell bodies are
also located in the ventral tegmental area (A10
group), but their projections end in the septum,
prefrental cortex, cingulate, hippocampus and
amygdala.
4. Mesothalamic DA system. Another group of cell
bodies originates in the ventral tegmental area (A10
group) and terminates in the medial and lateral
habenula.
5. Tuberoinfundibular and tuberohypophyseal DA system.
A system consisting of both short and intermediatelength neurons, designated the A12 group, arises
from the arcuate and periarcuate nucleus and the
ventral periventricular hypothalamic nucleus, and
terminates in the median eminence, infundibular
stalk, intermediate and neural lobes of the
pituitary.
Ontogeny of Dopamine System
Neuron production in the rat is largely prenatal
(Brasel et al., 1970).
The features of the postnatal
developmental process are mainly related to events involving
neuronal growth, axonal outgrowth and the synaptic
formation.
The development of dopaminergic fiber systems in
rats takes place in the first few postnatal weeks.
Several
striatal components including DA-containing nerve endings,
the neurotransmitter DA and DA receptors show transformed
patterns during development.
At birth, dopaminergic fibers can be visualized in
distinct patches' revealed by histofluorescence and
immunocytochemistry (Moon Edley and Herkenham, 1984; Voorn
et al., 1988).
Soon after birth, denser appearance of DA
terminals in a *matrix' pattern in the striatum can be
discerned (Gerfen et al., 1987).
By 3-4 weeks after birth,
the striatal pattern of DA fiber innervation continues to
mature and appears to reach an adult-like alignment (Moon
Edley and Herkenham, 1984; Voorn et al., 1988).
similarly, DA content in the developing brain exhibits
the same picture (Loizou and Salt, 1970).
At the first day
after birth, the striatum contains only about 12% of the
adult level of DA (Giorgi et al., 1987).
The striatal DA
level increases slowly thereafter, reaching adult levels 7-8
weeks after birth and remaining constant throughout
adulthood (Breese and Traylor, 1972; Giorgi et at., 1987;
Broaddus and Bennett, 1990).
Ontogenetic studies with neonatal rats demonstrated
that a first-week postnatal striatum lacks spontaneous
neuronal activity (Cheronis et al., 1979; Napier et al.,
1985).
Striatal neuronal activity can be recorded from 17
day old pups.
As the variety of striatal cell firing
patterns increased with age, 28 day old rats have been
observed to have similar patterns often seen in adult
striatum (Napier et al., 1985).
The above mentioned findings indicate that
developmental changes occur in nigrostriatal dopaminergic
projections and their connections especially during the
first 8 weeks after birth.
proposed Functions of Dopamine System
Based on anatomical, biochemical, behavioral and
pharmacological characteristics, there is compelling
evidence that DA serves as both a neurotransmitter and a
modulator in the brain.
The physiological role of the
central dopaminergic system has been proposed as follows:
1. Behavioral modulation.
A variety of behaviors are
found to be regulated by the dopaminergic system.
In general, the nigrostriatal pathway may largely
modulate motor behavior (Ungerstedt and Arbuthnott,
1970; Ungerstedt, 1971a,b), as evidenced by
characteristic motor disorder associated with its
degeneration in Parkinson's disease (Hornykiewicz,
1966).
Numerous studies have suggested that
mesolimbic and mesocortical pathways regulate not
only motor function, but mainly emotional and reward
behavior, as well as cortical and subcortical
functions, i.e. stereotyped behaviors (Creese and
Iversen, 1973), self-stimulation behavior (Phillips
and Fibiger, 1973), stimulus control behavior (Ho
and Huang, 1975) and locomotion (Pijnenburg et al.,
1976).
Dysfunction of mesolimbic dopaminergic
pathway has been hypothesized to underlie the
etiology of schizophrenia and depression (Stevens,
1973).
2. Neuroendocrine function.
The tuberinfundibular DA
system regulates secretion of prolactin and
gonadotropin releasing hormone, and inhibits release
of somatostatin and thyrotropin releasing hormone.
Dopamine Receptors
Ample evidence has been provided that there are
multiple receptors for DA in the CNS, on the basis of
pharmacological, biochemical, anatomical and physiological
features (Kebabian and Caine, 1979; Creese and Leff, 19S2;
Creese et al., 1983; Stoof and Kebabian, 1984).
Classically, upon agonist stimulation, DA D, receptors are
associated with activation of adenylate cyclase (AC)
(Kebabian and Caine, 1979; Seeman, 1980).
DA Dj receptors,
on the other hand, are either unlinked to stimulation of AC,
or have an inhibitory effect on the enzyme (Memo et al.,
1986; Onali et al., 1984; 1985).
characterization and Localization of Dopamine p. Receptors
Pharmacological studies, performed with radioligand
binding to discriminate between these two receptor
categories, have aided substantially in characterizing DA
receptor populations.
Dt receptors have been characterized
by using (JH]-cis**(Z)-flupenthixol (Hyttel, 1978; Murrin,
1983) and [3H]-cis-(Z)-piflutixol (Hyttel, 1981).
However,
both radioligands produce similar affinities for D( and Dj
receptors and cause a high degree of nonspecific binding.
SCH 23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl5-phenyl-lH-3-benzazepine-7-ol] was later introduced as the
first highly selective D, receptor antagonist and designated
as the most suitable ligand for D, receptors (Hyttel, 1983;
Iorio et al., 1983; Billard et al., 1984; Schulz et al.,
1984; Andersen et al., 1985).
In vitro, SCH 23390 potently
blocks DA-stimulated adenylate cyclase (Iorio et al., 1983)
and selectively displaces [3H]-piflutixol binding from
striatal slices (Hyttel, 1983).
However, in the [JH]-
spiperone binding assay (a ligand for D2 receptor), SCH
23390 displays a relatively low affinity at the micromolar
range and is about 460 times weaker than other neuroleptics
such as haloperidol.
in vivo, this compound is a potent and
selective antagonist of the D, receptor, with little
affinity toward D2 receptors in the CNS (Andersen and
Groenvald, 1986; Andersen and Nielsen* 1986).
Regional distribution and density of DA Dt receptors in
rat brain have been described by using autoradiographic
studies with [3H] SCH23390.
The highest density of D,
receptors was found in the caudate-putamen, nucleus
accumbens, olfactory tubercle and the substantia nigra par
compacta or pars reticulata (Dawson et al., 1986; Savasta et
al., 1986).
High densities was also observed in amygdala,
nucleus interstitialis stria terminalis, entopeduncular
nucleus and island of Calleja.
Moderate to low numbers of
Dj sites was found in cerebral cortex, several thalamic,
hypothalamic and hippocampal areas, the habenula, the
ventral tegmental area, the cingulate and the cerebellum.
The topographical distribution of D, receptors in the
striatum was much greater in the ventrolateral and medial
margin subregion than in the ventromedial and dorsolateral
sectors, and a rostrocaudal decrease in the densities of D,
receptors was also noticed (Savasta et al., 1986).
The regional localization of mRNA for Dt receptors has
been determined by northern blot and by in situ
hybridization histochemistry.
In general, the areas of
highest expression of D, receptors are the caudate-putamen,
nucleus accumbens and olfactory tubercle.
D, receptor mRNA
is also observed in the cerebral cortex, limbic system,
hypothalamus and thalamus (Fremeau et al., 1991; Sibley and
8
Monsma, 1992).
Signal Transduction Systems of Dopamine D, Receptors
Striatal DA D, receptors have been shown to mediate the
DA-stimulated increase of adenylate cyclase (AC) activity
(Kebabian and Caine, 1979).
In membrane preparations, the
stimulation of AC activity can also be elicited by GTP and
forskolin, which specifically act on stimulatory guanine
nucleotide regulatory protein (N.) or catalytic subunit of
the enzyme, respectively, rather than the receptor site
(Rodbell, 1980; Seamon et al., 1981).
DA, GTP and forskolin
thus have been widely used as biochemical markers in
adenylate cyclase assays to determine the functional
integrity of each subunit in DA Dt receptor complex system,
and to investigate Dt receptor/effector mechanisms.
There are also growing lines of evidence that
stimulation of DA receptors leads to changes in
phosphoinositide metabolism.
An early study by Hokin (1970)
showed variable effects of DA on incorporation of nP into
phosphatidate and phosphatidylinositol in slices of the
guinea pig brain.
Later, both stimulatory and inhibitory
effects of DA on phosphoinositide labeling were found
(Abdel-Latif et al., 1974; Odarjuk et al., 1986).
Undie and
Friedman (1990) demonstrated that the stimulation of DA D(
receptors enhances inositol phosphate formation in rat
brain.
By injecting mRNA from rat striatum into oocytes of
9
Xenopus laevis, Mahan et al. (1990) also showed the
expression of striatal D, receptors coupled to inositol
phosphate production.
Dynamics of Dopamine D, Receptors
The binding of antagonists to DA D, receptors show mass
action characteristics, whereas agonist binding displays two
separate states of high and low affinity (Hamblin et al.,
1984; Leff et al., 1985).
This heterogeneity was
demonstrated by shallow two-component curves of agonist
competition for radiolabeled antagonist binding.
In the
presence of guanine nucleotides, the high affinity agonistreceptor complex is converted to low affinity agonist
binding states of the receptors (Hamblin et al., 1984; Leff
et al., 1985; Hess et al., 1986).
These studies have
proposed a generalized ternary complex model to explain
these phenomena, which has been modified from the original
models of Boeynaems and Dumont (1975) and Jacobs and
Cuatrecasas (1976), and proposed for several other
monoaminergic receptors (Limbird, 1981).
Qntoaenv of Dopamine D, Receptors
Dopaminergic innervation of the striatum plays an
important role in regulating the development of neurons
intrinsic to the striatum (Rosengarten and Friedhoff, 1979).
There is increasing evidence that the ontogeny of DA
receptors and their mediated responses are dependent upon
the maturation of their corresponding presynaptic nerve
terminals.
DA Dt receptors are at low levels at birth (9%
of the adult value) and increase in number thereafter
(Giorgi et al., 1987; Zeng et al., 1988), attaining a peak
value at postnatal day 35 (Giorgi et al., 1987).
There were
no alterations in affinity (Kd) of [JH]piflutixol and[3H]SCH
23390 for D, receptors during development (Zeng et al.,
1988; Gelbard et al., 1989; Broaddus and Bennett, 1990).
Giorgi et al. (1987) showed that maximum DA-stimulated
adenylate cyclase (AC) activity in striatum has a
developmental profile similar to DA Dt receptors, suggesting
that the postnatal increase in AC activity is attributable
to the increase in stimulatory receptor density.
The
ontogeny of D, receptors parallels those seen with several
other markers associated with presynaptic dopaminergic
terminals in striatum, including DA content, DA uptake
(Coyle and Campochiaro, 1976), and tyrosine hydroxylase
activity (Coyle and Axelrod, 1972).
Molecular Biology of Dopamine Receptors
With the molecular characterization of DA receptors
being facilitated by the cloning of cDNAs and/or genes, two
subtypes of DA D, receptors and four subtypes of D2 receptors
have already been identified.
The D, receptor of human and
rats in CNS has been cloned, expressed and characterized
11
(Zhou et al., 1990; Dearry et al., 1990), while its subtype,
termed Djr has recently been identified and cloned as well.
The Dj receptor bears resemblance to the cloned D, receptor.
But pharmacologically, the D5 receptor has a 10-fold higher
affinity for the endogenous agonist, DA (Sunahara et al.,
1991.).
Two forms of the DA D* receptor, D2 short and D2 long,
were identified by gene cloning and found to be derived from
alternative splicing of a common gene (Dal Toso et al.,
1989; Giros et al., 1989).
A third subtype of the D2
receptor, termed Dj receptor and characterized by its
different anatomical distribution and pharmacological
profile, was isolated by the screening of rat brain cDNA and
genomic libraries (Sokoloff et al., 1990; Bouthenet et al.,
1991).
The fourth subtype of the D2 receptor, the D4
receptor, has been recently cloned.
Its pharmacological
profile is similar to that of the D2 and Dj receptors.
The
affinity of the D4 receptor for the atypical antipsychotic
drug, clozapine, however, is an order of magnitude higher
than for D2 and D3 receptors (Van Tol et al., 1991).
A better understanding of which DA receptor subtype
mediates particular physiological effects would greatly
facilitate the development of more specific dopaminergic
agents and provide insight into the mechanisms of disorders
thought to be related to the malfunctioning of the DA
system.
12
Innervation of Serotonin Flbera to the Striatum
The existence of serotonin (5-HT) innervation in
mammalian neostriatum was first shown by regional
biochemical examination of this monoamine, and by activity
of its biosynthetic enzyme, tryptophan hydroxylase in the
dog and cat brain (Bogdanski et al., 1957; Peters et al.,
I960).
Fluorescent histochemical and autoradiographic
investigations further provided evidence of this 5-HT
innervation in rat striatum (Arluison and Delamanche, 1980;
van der Kooy and Hattori, 1980; Parent et al., 1981).
The
distributional characteristic of the 5-HT innervation in
adult rat striatum demonstrated a marked heterogeneous
pattern (Ternaux et al., 1977; Steinbusch, 1981).
The
neostrlatal 5-HT fiber network has a higher density in
ventral vs. dorsal parts, and a caudo-rostral decreasing
density gradient was also found (Steinbusch, 1981;
Soghomonian et al., 1987).
This topographic distribution
was consistant with regional biochemical measurements,
demonstrating that higher levels of intrastriatal 5-HT
content or [JH]5-HT uptake is noted in the ventral vs.
dorsal, and the caudal vs. rostral parts of adult striatum
(Beal and Martin, 1985; Widraann and Sperk, 1986).
Although
the heterogeneous distribution of the striatal 5-HT
innervation remains of obscure significance, a complementary
striatal pattern of the distribution of DA and 5-HT
terminals has been found in the rostral to caudal, and
13
dorsal to ventral axes (Tassin et al., 1976; Doucet et al.,
1986).
On the other hand, the striatal 5-HT innervation
density is ouch less than that of DA fiber innervation
(Doucet et al., 1986).
A critical balance between
dopaminergic and serotonergic neurons has been postulated to
underlie normal motor activity, based on biochemical and
lesion studies (Sourkes and Poirier, 1968).
Lidov and Molliver (1982) reported that 5-HT
innervation in neonatal rats does not show a mosaic pattern
of patches and matrix organization, and the major
development of this innervation takes place 12-15 days after
birth.
This is in contrast with the DA innervation in
striatum, where at the earlier postnatal stage, the
prominence of DA islands is found (Olson et al., 1972; Voorn
et al., 1988).
Serotonin Receptors in Striatum
Based on biochemical, pharmacological and behavioral
studies, at least seven distinct subtypes of 5-HT receptors
are currently identified in mammalian central nervous system
(Frazer et al., 1990; Peroutka et al., 1990; Glennon and
Dukat, 1991).
Many subtypes of this receptor family have
now been cloned (Julius et al,, 1988; Pritchett et al.,
1988; Albert et al., 1990; Hamblin and Metcakf, 1991; Maricq
et al., 1991; Voight et al., 1991; Weinshank et al., 1992).
5-HT, group can be divided into four classes (labeled 5-HT,A
14
through 5-HT1D), while the three other groups are designated
5-HT2, 5-HTj and 5-HT4.
There is a relative lack of 5-HT,A receptor in rat
striatum (Marcinkiewicz et al., 1984; Verge et al., 1986),
recently confirmed by immunocytochemistry and in situ
hybridization of the mRNA (El Mestikawy et al., 1991; Riad
et al., 1991; Chalmers and Hatson, 1991; Miquel et al.,
1991).
Autoradiographic studies show that the 5-HT10
receptor subtype occurs in rat striatum (Pazos and Palacios,
1985).
Destruction of 5-HT innervation to striatum with the
5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), results
in increased striatal [m I]cyanopindolol binding, suggesting
postsynaptic 5-HTtB sites in the striatum (Offord et al.,
1988; Palacios and Dietl, 1988).
5-HTlD receptors have been
identified in the brain of rat, guinea pig and other species
(Herrick-Davis and Titeler, 1988; Herrick-Davis et al.,
1989; Waeber et al., 1990).
Quinalinic acid lesion of
striatal neuronal bodies induces a decrease of 5-HTID
receptors in substantia nigra, globus pallidus and striatum,
suggesting that their possible location either on cell
bodies, dendrites or terminals of interneurons (tfaeber et
al., 1990).
in the striatum, 5-HTj receptors have an
intermediate density, as determined by autoradiographic
studies (Pazos et al., 1985a; Palacios and Dietl, 1988).
Lesion of 5-HT system by 5,7-DHT did not reduce the number
of 5-HTj sites in rats (Fischette et al., 1987).
Therefore,
IS
it is likely that the 5-HTj receptors are located on non-5HT neurons.
The 5-HTlc receptors were initially found to be present
at high density in the choroid plexus (Pazos et al., 1985b).
Since the pharmacological characteristics of this receptor
subtype are similar to those of 5-HT2 receptors, it was
suggested that they might be in the same receptor family
(Hoyer et al., 1988).
Autoradiographic studies demonstrated
that 5-HTic receptors are present at low levels in many
areas of the brain.
But studies of the distribution of the
mRNA encoding for 5-HT]C receptors confirmed and also
extended these results (Hoffman and Mezey 1989; Molineaux et
al., 1989; Mengod et al., 1990), indicating that mRNA and
binding sites for this particular 5-HT receptor subtype are
heterogeneously distributed in the rodent brain including
striatum.
It was proposed that 5-HT,c receptors could be
involved in the regulation of many different brain functions
(Julius et al., 1988; Hoffman and Mezey, 1989; Mengod et
al., 1990).
The 5-HT,c receptors have been studied more
extensively in choroid plexus than in any other brain area.
They appear to be located on epithelial cells where they
regulate the production and composition of cerebrospinal
fluid (Pazos and Palacios, 1985).
Since lesion of the
dopaminergic system did not alter 5-HTlc binding in this
area, it was suggested that this receptor subtype is not
located on dopaminergic neurons (Palacios and Dietl, 1988).
16
Supersensitization of 5-HTlc receptors produced by 5,7-DHT
lesion, suggested that these receptors are present on nonserotonergic neurons (Conn et al., 1987),
The postnatal
development of 5-HTIC receptors has been studied in rat
brain choroid plexus, although little is known about their
ontogeny in other brain regions.
The number of binding
sites increases markedly between day 3 and 20 after birth
(Zilles et al., 1986), and these receptors appear to develop
earlier than other 5-HT, receptor subtypes.
Accompanying a
2-fold increase in 5-KTlc binding sites in rat brain between
day 17 and 27 after birth, there is a 5-fold increase in
corresponding mRNA levels (Roth et al., 1991).
The
mechanisms responsible for regulating 5-HT,c receptors by
exogenous or endogenous compounds are not currently clear
(Roth et al., 1990).
5-HTj receptors have been found mainly in brain
postrema and other brain areas including cortex, amygdala
and hippocampus, rather than striatum (Kilpatrick et al.,
1987; Barnes et al., 1990).
When compared to other
serotonergic receptors, 5-HTj receptors have a particularly
low density in the brain.
5-HT4 receptors might belong to
another functionally important 5-HT receptor family in the
CNS.
So far, the recognition sites for these receptors have
not been identified because they have been hampered by the
absence of a high affinity radioligand (Bockaert et al.,
1992).
17
6-Hvdroxvdopamine and Dopamine Receptor SupersensItigation
6-hydroxydopamine (6-OHDA), a neurotoxin, selectively
destroys brain catecholamine-containing neurons and their
terminals (Breese and Traylor, 1970; Uretsky and Iversen,
1970; Kostrzewa and Jacobowitz, 1974).
6-OHDA provides a
pharmacological means to explore central dopaminergic
function in adult fUngerstedt and Arbuthnott, 1970; Kelly et
al., 1975; Creese et al., 1977) and developing rats (Breese
et al., 1984; 1985a; 1985b).
In many brain regions, the
reduction or depletion of a dopaminergic afferent input has
been shown to result in a functional postsynaptic receptor
supersensitivity.
Lesions of nigrostriatal pathway in adult
rats produces a motor dysfunction (Breese and Traylor, 1970;
Ungerstedt, 1971c; Zigraond and Strieker, 1973) and
behavioral supersensitivity (Ungerstedt, 1971a,b), which is
accompanied by an increase in the density of DA D3 receptors
in the denervated striatum (Creese et al., 1977; Staunton et
al., 1981).
Although neonatal lesioned rats possess the capacity to
develop apparently normal motor and ingestive behaviors in
the virtual absence of the emerging DA system (Erinoff et
al., 1979; Pearson et al., 1980; Bruno et al., 1986), the
enhanced behavioral responsiveness towards DA D, and D2
agonists and antagonists has also been well documented after
lesion with the toxin (Breese et al., 1984; 1985; 1987;
Duncan et al., 1987).
In a series of studies, Breese and
co-workers (1985) demonstrated that the sensitivity of
neonatal 6-OHDA lesioned rats is often enhanced, with the
animals becoming particularly sensitive to DA D, agonists.
This enhanced behavioral responsiveness was not accompanied
by a change in density (B^) and affinity for either D, or D2
receptors in the striatum (Breese et al., 1987; Luthman et
al., 1990), suggesting that the enhanced response to DA
agonists in neonatal 6-OHDA-lesioned rats could be
reflecting a change in the sensitivity of secondary and
tertiary messenger systems linked to the DA recognition
sites; or an alteration in
non-doparainergic neural
mechanisms which influence the response to DA receptor
stimulation.
Furthermore, results from the same laboratory showed
that repeated treatment of neonatal 6-OHDA-lesioned animals
with SKF 38393, a specific D, receptor agonist (Setler et
al., 1978), led
to a progressive increase in locomotor
activity and several other behaviors in adulthood, a
phenomenon referred to as 'priming' (Breese et al., 1985b;
Criswell et al., 1989).
These actions of SKF 38393 were
blocked by SCH 23390, a specific D( antagonist (Breese et
al., 1985b; Criswell et al., 1989, 1990).
Therefore,
priming of Dt receptor responsiveness in neonatal 6-OHDAlesioned rats represents a model of a long-term alteration
in neural function related to repeated activation of a
receptor system.
Recently, Simson et al. (1992) found that behavioral
supersensitivity in neonatally lesioned rats was not related
to the alteration in the activation of adenylate cyclase,
because no enhancement of cAMP production was observed with
activation of DA D, receptors in lesioned animals.
These
investigators, however, suggested that differences of
regionally enhanced electrophysiological sensitivity of
striatal neurons to Dt agonists and regional distribution
pattern of tyrosine hydroxylase-like immunoreactivity and cfos-like immunohistochemical expression within the striatum
of neonatal lesioned rats, might contribute to the unique
behavioral pattern seen in lesioned rats when they are
challenged with DA D, agonists in adulthood (Johnson et al.,
1992; Simson et al., 1992).
6-Hvdroxvdopamlne and Serotonin System
Another feature of the 6-OHDA lesion in neonatal rats
is a substantial increase in the adult striatal levels of 5hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5HIAA) (Breese et al., 1984; Stachowiak et al., 1984).
This
lesion-induced elevation in 5-HT content is associated with
a corresponding increase in [3H]5-HT uptake (Stachowiak et
al.,1984) and with a proliferation of 5-HT containing fibers
in the rostral portion of striatum (Berger et al., 1985;
Snyder et al., 1986).
The same 6-OHDA denervation of
striatum associated with 5-HT hyperinnervation has also been
20
observed in adult lesioned rats and thus it is proposed as
heterotypic terminal outgrowth, in the sense that it
involves excessive growth of a neuronal system different
from that which was lesioned (Zhou et al., 1991).
Autoradiographic study by Radja et al, (1993) demonstrated
that 5-HT1B, 5-HTIoeoAB (including 5-HTlc and 5-HT1D), 5-HT2
receptor subtypes were upregulated in adult rat brain after
a neonatal 6-OHDA lesion to striatum that is associated with
5-HT hyperinnervation in that brain region.
This
upregulation of several 5-HT receptor subtypes was likely to
represent increases in receptor number (B^) rather than the
affinity of the receptors (Kd) (Radja et al., 1993).
Little is known about the functional implications of
such striatal 5-HT hyperinnervation and its corresponding 5HT receptor subtype upregulation in lesioned animals.
In
other studies on neonatal 6-OHDA-lesioned rats it was
reported that the sprouted 5-HT fibers did not seem to have
an inhibitory control on endogenous 5-HT- and acetylcholinecontaining neurons (Jackson et al., 1988).
Towle et al.
(1989) found that 5-HT hyperinnervation was not responsible
for supersensitized locomotor and stereotyped responses to
either a DA or 5-HT agonist in neonatally lesioned rats,
although their emphasis was focused on 5-HT2 receptors.
Dopamine System and^Animal_Bohavloral_Models
Denervation supersensitivity models have been widely
used to study central dopaminergic function, to elucidate
mechanisms of action of DA related agents, and to develop
models of neurological and psychiatric diseases.
As
measured by a variety of enhanced behavioral changes, the
mechanism responsible for this phenomenon of denervation is
supposedly associated with either receptor up-regulation
or
functional changes in second messenger systems with regard
to these receptors.
The best studied animal models are as
follows.
1. Rotational model.
This was first described by Ungerstedt
(1971a), and was produced by injection of 6-OHDA into one
substantia nigra of the rat.
The resultant unilateral
lesion of the nigrostriatal pathway leads to a permanent
depletion of DA in the ipsilateral striatum.
Behavioral
supersensitivity (i.e. contralateral circling) is seen when
the rat is challenged with a DA agonist.
This increased
sensitivity to agonist administration has been correlated
with an increase in striatal DA D2 receptor density
ipsilateral to the lesion (Creese et al., 1977; Hishra et
al., 1980; Heikkila et al., 1981).
2. Stereotypy model.
Tarsy and Baldessarini (1974)
demonstrated that apomorphine-induced stereotyped behavior
was increased in rats after prolonged pretreatment with the
DA synthesis inhibitor, o-methyl-p-tyrosine.
This was
comparable to the effect after chronic administration of
antipsychotic drugs, suggesting that this behavioral
22
stereotypy is probably specifically related to compensatory
supersensitivity of OA receptors.
This stereotypy includes
sniffing, rearing, licking, gnawing, chewing and locomotion
behaviors.
3. oral movement model.
Rats chronically treated with
neuroleptics show an increased incidence of purposeless or
vacuous chewing movements, when challenged by DA receptor
agonists (Clow et al., 1979; Klawans et al., 1972;
Waddington et al., 1980).
This oral activity is similar to
the oral-bucial-masticatory movements classically described
in patients with tardive dyskinesia.
The phenomenology of
this oral activity in rats is defined as 'vacuous (or
abortive or spontaneous) chewing, whereby what appear to be
robust chewing sequences are manifested, but are not
directed onto any evident physical material' (Waddington,
1990).
In neonatal 6-OHDA-lesioned rats, the effects of a
D, agonist or D2 antagonist on oral responses are greatly
potentiated, even though the
and Kd for striatal Dt and
Dj receptor binding do not accompany the marked reduction in
DA content (Kostrzewa and Hamdi, 1991).
This indicates that
the enhanced oral movements in these neonatally lesioned
rats are due to supersensitized DA receptors.
4.Catalepsy model.
It is one of the classic rodent models
to induce a state of transient immobility in testing DA
antagonist activity, which is characterized by placing the
forepaw of the rat on a testing bar or on an inclined wire
mesh screen and measuring the cataleptic time on the bar or
the screen (Costall and olley, 1971, Kostrzewa and Kastin,
1993).
This effect is mediated by blockade of striatal DA
receptors (Costall and Olley, 1971; Koffer et al., 1978;
Heller et al., 1985, Kostrzewa and Kastin, 1993).
Brain Dopamine System and Clinical Diseases
Many clinical diseases are known to be specifically
related to malfunctioning of DA systems.
Parkinson's disease is a progressive neurodegenerative
disorder of the basal ganglia.
Although characterized
pathologically by loss of pigmented neurons in the
substantia nigra, the basic biochemistry of the disease was
not known until the 1960s.
It was found that there is a
substantial loss of DA in the striatum (Hornykiewicz, 1966).
It is clear now that the most striking degenerative loss of
DA neurons occurs in the nigrostiatal system.
Studies have
also been carried out on the relationship between the
sensitivity of postsynaptic DA receptors and the supply of
available DA in this disease.
Hornykiewicz (1975) reported
that increased activity of dopaminergic neurons may occur in
Parkinsonism as a compensation for the decrease of available
DA at the receptor.
Altered function of mesolimbocortical DA system has
been implicated in the pathophysiology of schizophrenia.
The disease was once considered to be related to a relative
24
excess of central dopaminergic neuronal activity, in view of
the conviction that antipsychotic agents act therapeutically
through blockade of central DA transmission (Matthysse,
1973; Snyder, 1982).
Increased brain dopaminergic activity
in schizophrenia could occur through several mechanisms such
as increased synthesis and release of DA or altered
sensitivity of DA receptors (Losonczy et al., 1987).
Tardive dyskinesia, characterized by involuntary mouth
movements, tongue fibrillation and thrusting, occurs in some
patients who have been taking neuroleptics for long periods.
This motor disturbance is believed to result from
compensatory supersensitivity of striatal DA receptors
(Klawans, 1973).
Gilles de la Tourette' syndrome is
characterized by childhood onset of chronic motor tics and
involuntary vocalization.
The often dramatic response to
treatment with haloperidol, a DA Dz antagonist, the
similarity of the movements to those seen in patient with LDOPA intoxication and the disturbed decrease in DA
metabolism (Snyder et al., 1970; Butler et al., 1979), have
suggested that excessive DA transmission may have occurred
in the syndrome.
Likewise, changes in DA receptor binding
and coupling have been reported in Huntington's disease
(Reisine et al., 1977; Cross and Rossor, 1983; De Keyser et
al., 1989), which is a genetically determined neurological
disorder, and associated with a progression of involuntary
choreiform movements, psychological change and dementia.
25
Lesch-Nyhan disease, which is associated with an inborn
deficiency of the enzyme hypoxanthine-guanine phosphoribosyl
transferase (Seegrailler et al., 1967) and characterized by
choreoathetoid movement and hypertonicity (Lesch and Nyhan,
1964; Wilson et al., 1983), has been found to be related to
reduced brain DA in children (Lloyd et al., 1981).
Because
treatment of neonatal rats with 6-OHDA resulted in increased
susceptability for self-mutilation behavior due to the
destruction of dopaminergic neurons, this useful
neurochemical model has been proposed for screening of a
variety of pharmacological agents that could minimize the
self-mutilation behavior observed in Lesch-Nyhan patients
(Breese et al., 1984; Breese et al., 1985a,b).
5-HTr Receptor-Related Animal Behaviors and Clinical
Implications
Recent evidence suggests that although 5-HT,c receptors
are at high density in the choroid plexus, they are widely
distributed in the brain and that their activation may have
numerous behavioral and physiological effects.
They may
thus be implicated in a number of CNS disorders (Julius et
al., 1988; Holineaux et al., 1989; Mengod et al,, 1990).
1. Locomotor effects: A clear role of 5-HTlc in behavior has
not yet been established due to the lack of a specific
agonist for the receptor.
However, motor effects were
reported to be associated with the activation of 5-HT]C
26
receptors.
The piperazine agonists TFMPP [l-(m-
trifluoromethylphenyl)piperazine), m-CPP [l-(mchlorophenyl)piperazine] and non-selective ligands, such as
MK 212 [6-chloro-2-(l-piperazinyl)pyrazine], dosedependently suppress spontaneous locomotor activity in rats
(Kennett and Curzon, 1988a; Lucki et al., 19S9; Klodzinska
et al., 1989).
This effect was found to be prevented only
by 5-HT antagonists with high affinity for 5-HTtc receptors,
such as metergoline and mianserin, suggesting specific
involvement of these receptors.
2. Feeding: Systemic administration of m-CPP and TFMPP
reduces food intake in freely feeding rats (Kennett et al.,
1991).
Studies with 5-HT antagonists suggested that this
hypophagic effect is mediated by activation of 5-HT,c
receptors, though activation of 5-HTtB sites was also needed
for the response to occur (Kennett and Curzon, 1988b).
Dourish et al. (1989) proposed that postsynaptic 5-HTtc
receptors located in the ventromedial hypothalamus play an
important role in regulating feeding.
3. Psychiatric disorders: Administration of m-CPP in normal
human volunteers has been reported to increase feelings of
anxiety, dysphoria and panic (Charney et al., 1987; Murphy
et al., 1989).
Anxiogenesis was indicated by both decreased
social interaction and decreased activity in rats (Kennett
et al., 1989).
These effects might be related to 5-HT]c
receptors, because they are blocked by mianserin,
metergoline and cyproheptadine (i.e., compounds with high
affinity for the 5-HT,c receptor) .
4. Hypertonic saline consumption: Administration of
fenfluramine, fluoxetine, m-CPP, or MK 212 decreases
hypertonic saline intake at doses that have negligible
effects on water or physiological saline consumption (Neil
and Cooper, 1989).
Since the more selective 5-HT,,, agonist
CGS 12066B (7-trifluoromethyl-4(4-ethyl-l-pipera2inyl)pyrrolofl,2-alquinoxaline] has no such an effect, 5-HT,c
receptors have been suggested to be involved in mediating
salt-drinking behavior.
Further pharmacological
characterization of this effect is necessary.
5. Migraine effect: Recent evidence indicates that
ergotamine and dihydroergotamine, two effective antimigraine
agents, are potent 5-HT,c receptor antagonists in piglet
choroid plexus (Brown et al., 1991).
Therefore, it is
suggested that 5-HT,c receptors may play a role in
initiating migraine attacks (Fozard and Gray, 1989).
The above findings of so many potential roles for 5HT,C receptors suggest that the effects of many drugs acting
on brain 5-HT systems might have been targeting on these
particular receptor sites.
The availability of more
selective tools will clarify the roles of 5-HT,c receptors
in behavior and in CNS diseases.
28
Rational*
The availability of 6-OHDA to destroy brain DAcontaining neurons in both adult and neonatal rat provides a
pharmacological means to study motor functions and lesionrelated biochemical changes that may depend on the age at
which the dopaminergic pathway is destroyed (Breese and
Traylor, 1970, 1972; Smith et al., 1973; Breese et al.,
1984, 1985b).
Treatment of adult rats with 6-OHDA produces
a profound motor dysfunction such as aphagia, adipsia and
akinesia (Breese and Traylor, 1970; Ungerstedt, 1971c;
Zigmond and Strieker, 1973).
In contrast, neonatal
treatment of rats with the toxin does not result in such an
acute motor syndrome despite the fact that near-total
destruction of this emerging DA system is produced (Erinoff
et al., 1979; Bruno et al., 1986; Duncan et al., 1987).
However, these rats exhibit an increased susceptibility for
self-mutilation behavior (Breese et al., 1984, 1985a,b).
Clinically, Parkinson's disease is associated with
reduced brain DA and motor dysfunction (Hornykiewicz, 1973),
and Parkinsonian patients display tremor, bradykinesia and
rigidity.
Another disorder related to the same brain
biochemical difficiency is Lesch-Nyhan syndrome.
The latter
syndrome is characterized by choreoathetoid movement,
hypertonicity and unique compulsive self-mutilation of
digits and tissue about the mouth (Lesch and Nyhan, 1964).
It is believed that the adult 6-OHDA-treated rat serves as a
29
neurochemical model for Parkinson's disease (Ungerstedt,
1971), whereas the neonatal lesioned rat provides a model
for central dysfunctions observed in childhood diseases, one
of which is Lesch-Nyhan syndrome (Breese et al., 1984,
1985a,b).
Neonatal 6-OHDA-lesioned rats are also known to be
supersensitized to DA D, receptor agonists in the absence of
an altered density and affinity of the receptor.
SKF 38393,
a selective D, agonist, produces marked changes in
stereotyped and locomotor behaviors in the neonatal lesioned
rats (Breese et al., 1984, 1985a,b, 1987).
This change in
sensitivity is a latent one, which must be unmasked by
repeated doses of SKF 38393.
This is referred to as a
'priming' phenomenon (Criswell et al., 1989).
Priming of
neonatal 6-OHDA-lesioned rats has been implicated in
learning, development of craving following repeated intake
of some drugs, or the appearance of motor disturbances
following prolonged administration of drugs that influence
dopamine receptor functions (Criswell et al., 1989, 1990).
Hamdi and Kostrzewa (1991) demonstrated that the D, receptor
could be ontogenetically sensitized by repeated treatments
with a D, agonist during development.
As in adult-primed
rats, there were enhanced D, agonist-induced stereotyped
behaviors in adulthood.
In order to further investigate
ontogenetic sensitization of neonatal 6-0HDA-treated rats
and examine its underlying biochemical association, the
30
selective D, receptor agonist, SKF 38393, was used alone, or
in combination with neonatal 6-OHDA lesion during postnatal
ontogeny in this study.
This project was thus undertaken with the objective of
(1) assessing the degree or extent of ontogenetic
sensitization of neonatal 6-OHDA-lesioned rats by repeated
treatments of the animals with a DA D, agonist in
development
(2) investigating the status of dynamics of the
DA Dt receptors, such as high and low affinity binding sites
in the neonatal 6-OHDA-lesioned rat model, or in neonatal 6OHDA-lesioned rats repeatedly treated in postnatal ontogeny
with SKF 38393
(3) evaluating the association of D,
receptors with second messenger systems in the above animal
models, and also
(4) determining mRNA levels for D,
receptors in these rat models.
The above investigations can lead to a better
understanding of whether ontogenetic DA D, agonist
treatments modulate the behavioral responses in adult rats
that were lesioned neonatally with 6-OHDA.
These studies
would also resolve the problem of whether several
biochemical markers would be associated with the enhanced
behavioral responses generated by a neonatal chemical lesion
or combined neonatal lesion plus ontogenetic DA D, agonist
treatments.
Moreover, in neonatal 6-OHDA-lesioned rats, acute
treatment with a DA D, receptor agonist results in a great
potentiation of the incidence of oral activity, even though
the
and Kd for striatal Dj receptor binding do not
accompany the marked reduction in DA content (Kostrzewa and
Hamdi, 1991}.
It is likely that the enhanced oral response
in neonatal 6-OHDA-lesioned rats is due to supersensitized
D, receptors,
since the striatal DA depletion in neonatal
6-OHDA-treated rats is accompanied by elevation in 5-HT
levels (Breese et al., 1984; Stachowiak et al., 1984;
Luthman et al., 1987),
hyperinnervation of rostral striatum
by 5-HT-containing fibers (Berger et al., 1985; Snyder et
al., 1986), and upregulation of several 5-HT receptor
subtypes in striatum (Radja et al., 1993), it is reasonable
to study possible interactions between DA and 5-HT
neurochemical systems in the Dt supersensitized induction of
oral activity by using this animal model.
Another aim in the present project, therefore, was to
evaluate whether 5-HT receptors were involved in the
induction of supersensitized oral responses in neonatal 6OHDA-lesioned rats.
The piperazine compound, m-CPP, was
used in this study, because this 5-HT receptor agonist was
found to induce oral activity in intact rats (Stewart et
al., 1989).
A series of other 5-HT agonists and antagonists
was also used to determine which probable 5-HT receptor
subtype would be related to such involvement.
This study
should provide evidence of whether there is a functional
correlation between DA D, receptor and 5-HT fiber
innervation of the striatum.
Chapter 2
Materials and Methods
Animals and Treatment
Timed-pregnant Sprague-Dawley derived albino rats were
obtained from Charles River Laboratories (Research Triangle,
NC) approximately 1 week before delivery.
Animals were
housed singly in clear Perspex cages in a room with
controlled temperature (22 ± 1 °C) and lighting (12 h-12 h
light-dark cycle, lights on at 07:00), and had free access
to food and water.
Date of litter delivery was noticed
within 12 hours and the date of birth was considered as day
0.
At birth, litters were reassigned, so that each dam had
rats from several litters, with each reconstituted litter
consisting of a maximum of ten pups.
Starting at birth all pups were treated once a day for
28 consecutive days by intraperitoneal (i.p.) injection of
SKF 38393 HCl (l-phenyl-2,3,4,5-tetrahydro-[lH]-3benzazepine-7,8-diol hydrochloride; 3.0 mg/kg, salt form;
Research Biochemicals, Inc., Natick, MA) or saline vehicle.
At 3 days from birth all rats
also received a bilateral
intracerebroventricular (i.c.v.) injection of 6-OHDA
hydrobromide (100 /jg, salt form, on each side; Regis
Chemical Co., Chicago, IL) or the vehicle, saline-ascorbic
acid (0.1%).
Neonates were pretreated with desipramine (20
mg/kg i.p.) 1 hr before 6-OHDA injection to prevent
33
34
destruction of noradrenergic neurons,
starting 3 days after
birth, all animals were divided into four treatment groups:
(1) saline (2) SKF 38393 (3) 6-OHDA (4) SKF 38393 plus 6OHDA.
At the age of 6 weeks, the first portion of the rats
from each group was challenged with SKF 38393, to study (1)
locomotor activity
and (2) stereotyped behaviors (see
behavioral observations). These rats were continuously
tested with a weekly challenge dose of SKF 38393 for another
three weeks.
At 9 weeks of age, a second portion of the
rats from all treatment groups without weekly challenge dose
of SKF 38393 in adulthood, was sacrificed for biochemical
analyses only.
At 3 and 9 months of age, another two separate groups
of rats consisting of neonatal saline and 6-OHDA treatment,
respectively, were used for determination of oral activity
response to DA D, agonist and antagonist as well as a series
of 5-HT receptor agents.
All animals were sacrificed by decapitation.
Brains
were immediately removed and the striata were dissected
free, quickly frozen on dry ice, and stored at -70 °c for
later in vitro neurochemical assessments (see biochemical
assays below).
Behavioral Observations
1. Locomotor activity.
Between 6 and 9 weeks of age, rats
were placed singly in donut-shaped test chambers (46 cm in
35
diameter) with six equidistant markings along the
circumference. After a 60 min habituation period in a quiet
and well-lighted room, a challenge dose of SKF 38393 HCl
(3.0 mg/kg i.p., salt form) or vehicle was administered.
Locomotor activities were then videotaped by a camcorder for
60 min starting 10 min after the challenge agent.
Tapes
were replayed and the distance traveled by each rat was thus
determined.
2. Stereotyped behaviors.
At 8 weeks of age, rats were
placed singly in clear Perspex cages (48 x 26 x 18) with
wood chip bedding, in a quite and well-illuminated room, and
were acclimated to the testing surroundings for 30 min.
An
individual rat was challenged first with saline vehicle 10
min before behavioral testing.
The rat was then observed
for stereotyped behaviors for 10 min.
SKF 38393
A challenge dose of
HCl (3.0 mg/kg i.p., salt form) was then
administered and rats were again observed for 10 min for
stereotyped behaviors, starting 10 min after injection.
Behaviors were quantified as seconds in which each
particular behavior was performed by a rat.
Therefore, the
total time an individual rat spent in performing or not
performing an activity was 600 seconds (10 min observation
period).
The behaviors observed were locomotion, sniffing,
grooming, eating, taffy pulling (coordinated movement of
front paws toward the mouth and then away from the body),
digging, gnawing, licking, jumping, paw treading and
locomotion.
Yawning and rearing were counted as numbers of
incidents, not seconds.
Immobility was defined as the time
period in which a rat was not observed in any stereotypic
activity.
3. Oral activity: Rats were observed for oral responses to
test agents between 3 and 9 months of age.
For each test
session, rats were placed in individual clear plastic cages
(48 x 26 x 18 cm) with steel grid floors in a quiet, wellventilated and well-lighted room.
Rats were allowed to
acclimate to the new environment for at least 1 hr.
Between 9:00 A.M. and 4:00 P.M. each rat was given a
single i.p. or s.c. treatment with vehicle, SKF 38393 HC1
{0.03-3.0 mg/kg i.p.),* m-CPP 2HC1 [l-(3chlorophenyl)piperazine dihydrochloride, o.5-8.0 mg/kg
i.p.]; CGS 12066B maleate {7-trifluoromethyl-4(4-ethyl-lpiperazinyl)-pyrrolo[l,2-alquinoxaline], 1:2 maleate salt,
3.0 mg/kg i.p.}; or 8-0H-DPAT HBr [(±)-8hydroxydipropylaminotetralin hydrobromide, 0.1 mg/kg s.c.].
Each rat was then observed one at a time, for 1 min every 10
min, over a 60-min period, beginning 10 min after any of
these treatments.
counted.
Numbers of rapid jaw movements were
Some rats were pretreated with mianserin HC1 (1.0
mg/kg s.c., 30 min); ketanserin tartrate (5.0 mg/kg i.p., 30
min); MDL 72222 (3-tropanyl-3,5-dichlorobenzoate, 10.0 mg/kg
s.c., 30 min); or SCH 23390 HCl [R-(+)-7-chloro-8-hydroxy-3methyl-l-phenyl-2,3,4,5-tetra-hydro-lH-3-benzazepine
37
hydrochloride, 0.30 mg/kg i.p., 1 hr].
At least 1 week
intervened between these observation sessions.
Oral activity in these studies is of the type described
by Haddington (1990) as "vacuous (or abortive or
spontaneous) chewing, whereby what appear to be robust
chewing sequences are manifested, but are not directed onto
any evident physical material."
No differentiation between
lateral and vertical jaw movements was made.
Also, there
was occasional tongue thrusting noted in the these rats.
Oral activity that occurred in eating, grooming or taffy
pulling was not counted.
petemination of Pa,_5-HT_*na_Thelr_Hetabolites in the
Striatum
Striatal concentrations of DA, 5-HT and their
metabolites were measured by liquid chromatography with
electrochemical detection as follows.
The striata were
sonicated in 1 ml of 0,1 H trichloroacetic acid containing
0.2 mg/ml cysteine as a stabilizing agent and an internal
standard which was 0.2 nmol/ml 5-hydroxyindole carboxylic
acid (5HICA).
The resulting homogenate was centrifuged at
12,000 x g for 5 min and DA, homovanillic acid (HVA), 3,4dihydroxyphenylacetic acid (DOPAC), 5-HT and 5hydroxyindoleacetic acid (5-HIAA) were assayed by injecting
30 nl of supernatant directly onto the C18 analytical
column.
The column was an Econosphere CIS (5 micron, 4.6 x
38
150 ram) from Alltech Associates.
The mobile phase was 0,1 M
monochloroacetic acid, 1 raM EDTA, 220 mg/1 sodium
octanesulfonic acid, 8% acetonitrile, pH 2.6 at a flow rate
of 1.3 ml/min and temperature of 40 °C.
The instrumentation consisted of a Bioanalytlcal System
LCEC Analyzer and a Waters WISP automatic sample injector
with a refrigrated sample compartment (samples kept at 5°C).
The glassy carbon electrode was used at a potential of +0.75
volts.
Peak heights and sample concentration were
calculated with a Hewlett-Packard HP1000 chromatography data
system.
Determination of
da
competition for f^iscH 23390 Binding to
striatal Homogenates
To assess DA competition for [3H]SCH 23390 binding, a
modified method of Hess et al. (1986) was used.
Briefly,
striata were homogenized with a Teflon on glass mortar and
pestle, in 100 volumes of ice cold 50 mM Tris buffer (pH
7.4).
This gentle homogenizing procedure was used to avoid
destruction of Dt receptors during this tissue preparation
step (Norman et al., 1989).
Homogenates were then
centrifuged at 35,100 x g for 10 min at 4 °C using a Beckman
J2-21M centrifuge and JA-20.1 rotor.
After one wash, the
homogenates were preincubated at 37 °c for 15 min, chilled
in ice and centrifuged as before.
Tissue pellets were
finally resuspended in 200 volumes of fresh buffer.
Aliquots (300 pi) of homogenates were added to 450 pM
of [3H]SCH 23390 (specific activity 294 mCi/mg; Amersham,
Arlington Heights, IL) in Tris buffer containing 120 mM
NaCl, 5 mM KCl, 2 mM CaCl2, 0.1% ascorbic acid, 10 pM
pargyline (final concentrations) and varying concentrations
of DA as competitor (1 nM - 1 mM), to yield a 2.5 ml final
assay volume.
Samples were incubated for 40 min at 37 °C in
a shaking water bath, and then were rapidly filtered under
partial vacuum on Whatman GF/F glass fiber filters using a
Millipore filtration unit.
Filters were washed 3 times with
5 ml of ice-cold Tris-salt solution each time.
After drying
overnight, filters were placed in 9 ml of fluor, and tritium
activity was determined in a Beckman LS 9800 liquid
scintillation spectrometer.
A GraphPad program (GraphPad
Software Inc., San Diego, CA), using a nonlinear regression
model for complex ligand-receptor binding systems, was used
to fit the agonist-antagonist competition curves matching
two binding sites.
The ICjo values for DA at the high and
low affinity site and the percentage of each receptor
binding component were estimated by the computer program.
The K, for each ICi0 was determined according to following
equation:
ICm
K,
=
--------------
1 + L / Kj
where L is the concentration of [JH]SCH 23390 and Kd is its
affinity for the receptors.
40
Determination of mRNA Levala for Pt and 5-HT,,. Receptors in
the Striatum
Two mixtures of 48-base oligonucleotide probes for the
respective rat DA D, and 5-HT,c receptor mRNA were used (NEN
Research Products, Boston, MA). The sequences for D(
receptor mRNA are complementary to bases 13-60 (5'-GAA ATC
CCT CTC CGC TGG CAG CCC GGC CTC ATC CAT
GGT AGA AGTGTT-3'),
520-567 (5/-GGA CTG CTG CCC TCT CCA AGG
CTG AGA TGC CCC GGA
TTT GCT TCT GGG-3') and 664-711 (5'-TGT CAC ACT TGT CAT CCT
CGG TGT CCT CCA GGG AGG TAA AAT TGC CAT-3'), while the
sequences for 5-HTlc receptor mRNA are complementary to
bases 4-51 (5'-GCC GAT TAG GTG CAT CAG GAG CGA GCG CAC CGC
GTT GCC AAG GTT CAC-3'), 721-768 (5'-CTC CTC GGT GTG ACC TCG
AAG TAA CAT CAG AGT TTG ACG GCG CAG GAC-3')
GGT GCC TCT GGG ACG CTT TTG TTT CTT CTT
and 856-903
(5'-
TCG ACG TGG TTT CTG
ATC-3').
Rat striatal tissues from above treatment groups were
homogenized in denaturing solution containing 4.0 M
guanidine thiocyanate, 42 mM sodium citrate, 0.83% N-lauroyl
sarcosine and 0.2 mM B-mercaptoethanol, using a Tekmar
Tissuraizer (setting 5, 20 sec).
After adding 2.0 M sodium
acetate (pH 4.0), the homogenates were extracted with equal
volume of phenol:chloroform:isoamyl alcohol mixture and then
centrifuged at 10,000 x g (20 min at 4 °C).
The upper
aqueous phase was collected and the total RNA was
precipitated with isopropanol by centrifugation.
RNA was
41
quantified by UV spectrophotometry at 260 nm wavelength.
A sample representing 5-10 fig of striatal RNA denatured
at 95 °C for 2 min, was fractionated by electrophoresis on
1% agarose gel containing 2% formaldehyde, 20 mM 3-(Nmorpholino)propanesulfonic acid, 5 mM sodium acetate and 1
mM EDTA.
The gel was stained with ethidium bromide and
visualized under UV light to check the integrity and loading
of the RNA in each lane.
The RNA was then transferred to
nylon membranes (Schleicher & schuell, Keene, NH), which
were subsequently crosslinked under UV light for 5 min and
baked at 80 °C for half an hour.
Some of the striatal RNA
samples from each treatment group were denatured in 7.4%
formaldehyde at 65 °C and then transferred directly to nylon
membrane on an apparatus for dot blot analysis.
The
membranes were probed with respective D, and 5-HTlc receptor
mRNA probe according to following procedure.
Membranes were incubated for l hr at 58 °C in 20 ml of
prehybridization buffer containing 5 x SSC, 20 mM Na2HP04, 7%
SDS, l x Denhardt's solution and 100 fig/ml denatured salmon
sperm DNA.
Following prehybridization, the buffer was
replaced with the same new fresh buffer and the membranes
were hybridized with oligonucleotide probes at 58 °C for 24
hr, which were end-labelled with [r-HP]ATP using
oligonucleotide 5'-end labelling system (Promega
Corporation, Madison, WI).
The membranes were washed twice
in 2 x SSC, 1% SDS and 20 mM Na2HP04 at room temperature for
15 rain.
They were finally washed twice in 0.5 x SSC and 1%
SDS at 58 °C for 15 min and then were exposed to Kodak XOMAT AR film (Sigma Chemical Co., St. Louis, MO} at -70 °C
with two intensifying screens for 48-72 hr to ensure that
the exposure time was within the linear range of the film.
The optical densities of the bands or the dots were measured
by a Bio Image Analysis system (Millipore Corporation
Imaging systems, Ann Arbor, MI).
The probes were removed
from the membranes by washing in solution containing 50%
formamide and 2 x SSPE buffer at 65 °C for 1 hr before
rehybridization with cDNA probe for B-actin mRNA.
The B-
actin mRNA probe was labelled with [a-JIP]dCTP by a Prime-aGene labelling system (Promega Corporation, Madison, WI) to
give a specific activity of 1-2 x 109 cpm per fig of DNA.
The B-actin mRNA measurements were used mainly for
correcting errors that occurred in preparation or loading of
the RNA samples.
Determination of DA-fltimulated Adenvlate Cyclase Aetivitv in
the Striatum
The striatal tissues from all above treatment groups
were homogenized with a Teflon on glass mortar and pestle,
in 50 volumes of ice-cold 10 mM Tris maleate/2 mM EGTA
buffer (pH 7.4).
Homogenates were then centrifuged at
35,100 x g for 10 min in Beckman model J2-21M superspeed
centrifuge, using a JA-20.1 rotor.
After one wash with the
43
same buffer and centrifugation as before, the resultant
pellet was resuspended in 70 volumes of fresh buffer and
kept on ice prior to use for adenylate cyclase activity
assays.
Adenylate cyclase activity was assayed in a 200-jil
incubation mixture containing 80 mM Tris maleate, 0.4 mM
EGTA, 4.0 mM MgS04, 1.0 mM isobutylmethylxanthine, 5.0 mM
phosphocreatine, 50 unit/ml creatine phosphokinase, 0.02%
ascorbic acid, 10 juM pargyline, 0.1 mM GTP, 50 nM spiperone,
and varying concentrations of DA (0-10° M).
In some
instances, 12.2 /iM forskolin or 10 mM NaF in combination
with 10 jiM Alcl3 ([A1F4)‘), was added to the mixture instead
of DA, in order to stimulate the catalytic subunit of the
enzyme or the Ns protein, respectively.
Spiperone was
included in all incubation solutions to block DA D2
receptor-mediated inhibition of adenylate cyclase activity.
Membrane suspensions (30-40 /ig protein) were preincubated at
30 °C for 5 min in the reaction mixture.
The reaction was
then started by adding ATP (final concentration: 1.0 mM) and
the mixtures were incubated at 30 °c for 15 min.
After
termination of the reaction in boiling water for 4 min, the
mixtures were centrifuged at 13,000 x g for 5 min.
Aliquots
of the supernatant were assayed for cAMP content using an
Amersham cAMP enzymeimmunoassay kit (RFN 225, Amersham, UK;
sensitivity: 38.4 pg/ml).
Protein content of striatal
homogenates was determined by the method of Lowry et al.
44
(1951).
Data Mialvaia
Locomotor activity: Data are expressed as the mean
distance (meters) ± S.E.M. in different groups of rats
traveling within activity testing chambers.
The statistical
significance of the data was determine by a two-way analysis
of variance (ANOVA), followed by a post-ANOVA Newman-Keuls
test.
A p value of < 0,05 was considered to be
statistically significant.
Stereotyped behaviors: Results are expressed as the
mean time (seconds) ± S.E.M. in which rats from different
treatment groups perform a particular behavior.
The
statistical significance of the results was evaluated by a
one-way ANOVA, followed by a post-ANOVA Newman-Keuls test. A
p value of < 0.05 or smaller was considered to be
statistically significant.
Oral activity: Results are expressed as the mean number
of oral movements ± S.E.M. of rats receiving neonatal 6-OHDA
treatment or vehicle.
The statistical significance was
determined by a one-way ANOVA, followed by a post-ANOVA
Newman-Keuls test.
A p value of < 0.05 or smaller was
considered to be statistically significant.
Monoamine determinations: The mean nanomoles of
monoamine or metabolite per gram of tissue ± S.E.M. in
different treatment groups were analyzed by the ANOVA,
followed by a post-ANOVA Newman-Keuls test, in order to
45
determine the statistical significance among treatment
groups.
A p value of < 0.05 was considered to be the level
for statistical significance.
Binding studies: Competition curves were analyzed
according to models for the competition of radioligand and
competitor, DA, to one or two binding sites.
The
statistical difference between models was tested by
comparing the sum of squares which resulted from different
fits of the data.
A sequential F-test was used according to
the following equation:
SS, - SS,
F =
df, - df,
------------
ssa
df,
where SS refers to the sum of squares, and df refers to the
number of degree of freedom.
The subscrit 1 and 2 refer to
the model for one, and two binding sites, respectively.
A
model for two binding sites was retained only when it fitted
the data statistically significantly better than a model for
one binding site (p < 0.05).
Also, the mean (+ S.E.M.)
percent of high affinity binding sites (%RH), and the
respective mean (± S.E.M.) KH and KL in each treatment group
were analyzed by one-way ANOVA.
A p value of < 0.05 was
considered to be the level for statistical significance.
mRNA analyses and adenylate cyclase activity assays:
The mean {± S.E.M.) ratio of D( or 5-HTIC receptor mRNA to JJ-
46
actin mRNA, and mean (± S.E.M.) picomoles of cAMP per
milligram of protein per min, respectively, were analyzed by
ANOVA, followed by a post-ANOVA Newman-Keuls test, in order
to evaluate the statistical significance among different
treatment groups.
A p value of < 0.05 was considered to be
the level for statistical significance.
Chapter 3
Results
DA D1 Receptor Bupersensltlvitv
Locomotor activity of rats
Starting at 6 weeks adult rats from the different
treatment groups were tested at weekly intervals for
spontaneous and SKF 38393-induced locomotor activity.
At 6,
7 and 9 weeks there was little locomotor activity in each
group after administration of saline, and no statistically
significant difference between the groups (p > 0.05, left 4
bars in figs. l, 2 and 3).
At 6 , 7 and 9 weeks, SKF 38393 HC1 treatment (3.0 mg/kg
i.p.) did not increase locomotor activity in the vehicle
control rats, nor in the rats treated repeatedly during
development with SKF 38393 HCl (3.0 mg/kg per day x 28 days
from birth, i.p.; Fig. 1, 2 and 3, 5th and 6th bars).
At 6
weeks acute SKF 38393 HCl treatment (3.0 mg/kg i.p.)
produced a slight increase in locomotor activity in the
group of rats that was lesioned at 3 days after birth with
6-OHDA HBr (200 jig, i.c.v.), although this effect was not
different from that after saline treatment (fig. 1, 7th
bar).
However, in the group of rats that was both lesioned
at 3 days after birth with 6-0HDA and treated repeatedly in
development with SKF 38393 HCl (3.0 mg/kg per day x 28
47
48
days), acute SKF 38393 treatment at 6 weeks produced a large
increase in locomotor activity (p < 0.05, fig. 1, 8th bar).
Rats in this group traveled about 210 meters in 1 hr, vs.
about 40 meters for the 6-OHDA-lesioned group (p < 0.05, 8th
vs. 7th bar of fig. 1).
The greater SKF 38393-induced
activity in this group suggests that ontogenic SKF 38393
treatments produced homologous sensitization of the DA
receptors.
One week later when rats were challenged again with SKF
38393, there was an increase in locomotor activity in the 2
groups of rats lesioned at 3 days after birth with 6-OHDA.
In the group treated at 3 days with 6-OHDA, locomotor
activity increased substantially from one week before, now
155 meters in the one hour following the challenge dose of
SKF 38393.
In the group that was lesioned with 6-OHDA at 3
days and also treated repeatedly in development with SKF
38393, the challenge dose of SKF 38393 at 7 weeks increased
the distance traveled to more than 590 meters, nearly triple
that from the week before (8th bar of fig. 2 vs. 8th bar of
fig. 1).
In the 8th week rats were tested for stereotyped
activities following the SKF 38393 injection, so there is no
locomotor activity data.
In the 9th week the challenge dose
of SKF 38393 produced more than a 2-fold increase in
locomotor activity in the group of rats lesioned at 3 days
after birth with 6-OHDA (p < 0.05, 7th bar of fig. 3 vs 7th
49
bar of fig. 2).
However, in rats receiving both 6-OHDA and
SKF 38393 treatments in development, the challenge dose of
SKF 38393 at 9 weeks increased the distance traveled only
slightly and not significantly from that at 7 weeks (8th bar
of fig. 3 vs. 8th bar of fig. 2).
Nevertheless, SKF 38393-
induced locomotor activity in this group was higher at 9
weeks vs. 6 weeks (p < 0.05, 8th bar of fig. 3 vs. 8th bar
of fig. 1).
Stereotyped behaviors of rata
At 8 weeks, following a saline injection, there was
little stereotyped activity in the 4 groups of rats, with
immobility time being 340 to 490 seconds (i.e., 600 seconds
of observation, table 1 ).
Following the challenge dose of
SKF 38393 HCl (3.0 mg/kg i.p.) activity increased in all
groups (table 2).
However, for rats treated repeatedly in
development with SKF 38393, there was no difference from
control for time spent in sniffing, locomotion, grooming,
eating, taffy pulling, digging, gnawing and paw treading,
following the challenge dose of SKF 38393 at 8 weeks.
Also,
numbers of yawns and rearings was not different between
these two groups.
In the group of rats lesioned at 3 days with 6-OHDA,
the challenge dose of SKF 38393 at 8 weeks substantially
increased time in locomotion (351.7 ± 59.6 vs. 1.7 ± 0.9
sec) and paw treading (89.0 ± 50.2 vs. 0.0 sec), compared to
50
the vehicle control group (all p < 0.05, table 2).
Conversely, in the 6-OHDA vs. control group, the challenge
dose of SKF 38393 decreased time in grooming (4.3 ± 4.3 vs.
135.4 ± 21.8 sec), gnawing (1.1 ± 0.7 vs. 12.1 ± 2.3 sec)
and immobility (84.1 ± 56.3 vs. 396.1 ± 34.4 sec) (all p <
0.05, table 2).
In the group of rats lesioned at 3 days with 6-OHDA and
also treated daily for the first 28 days from birth with SKF
38393 HCl (3.0 mg/kg per day, i.p.), the challenge dose of
SKF 38393 at 8 weeks produced several changes in stereotyped
behaviors.
Time in locomotion (228.9 ± 74.8 sec), taffy
pulling (172.5 ± 87.8 sec) and paw treading (140.3 ± 75,8
sec) was increased vs. the control group (p < 0.05); taffy
pulling was different vs. the 6-OHDA group (p < 0.05, table
2).
Time in grooming (3.5 ± 3.5 sec) and immobility
(0.0
sec) were different vs. control (p < 0.05); while immobility
time was different vs. the 6-OHDA group (p < 0.05).
Striatal levels of DA. DOPAC. HVA, 5-HT and 5-HIAA
As shown in table 3, neonatal 6-OHDA treatment produced
a profound depletion of DA (>99% reduction), DOPAC (>99%
reduction) and HVA (>99% reduction) in the striatum of rats.
Conversely, neonatal 6-OHDA treatment was associated with a
70% increase in striatal 5-HT and 35% increase in 5-HIAA
content (all p < 0.001).
SKF 38393 treatments for the first
28 days after birth did not produce an effect on striatal
51
levels of DA, 5-HT and their metabolites, nor did this
treatment modify the effect of 6-OHDA.
Agonist competition for PA D1 receptor binding in striatal
baBpq<nn**g
Computer-modeled DA agonist-[sH]SCH23390 antagonist
competition binding curves are shown in figs. 4-7 for the
respective groups receiving saline, ontogenetic SKF 38393
and/or neonatal 6-OHDA treatments.
site model.
Each curve fits a two-
Dissociation constants to high affinity (RH)
and low affinity (RL) have been designated KH and KL,
respectively.
Table 4 summarizes computer-assisted binding
parameters for DA competition curves, demonstrating that
these curves exhibit high and low affinity binding
components.
No apparent difference was observed in the
distribution of high and low affinity agonist binding sites
between 6-OHDA vs. saline group.
Also, there was no
significant effect of ontogenetic SKF 38393 treatments on
the affinity of DA at either high or low affinity states of
the receptor.
Postnatal treatment of 6-OHDA lesioned rats
with SKF 38393 for 28 consective days likewise did not
affect high or low affinity agonist binding sites.
Striatal levels of DA Dl receptor mRNAs
Dot blot analysis revealed that in the first portion of
rats that did not receive weekly challenge doses of SKF
52
38393 in adulthood, striatal mRNA levels for DA D, receptor
were significantly reduced (p < 0.05) in neonatal 6-0HDAlesioned rats, regardless of whether ontogenetic SKF 38393
treatments were given (fig. 8A).
SKF 38393 treatments for
the first 28 days after birth per se did not produce an
effect on striatal mRNA content of the D, receptor (fig.
8A).
In the second portion of rats receiving weekly adult
challenge doses of SKF 38393, the striatal mRNA levels for
D, receptors in neonatal 6-OHDA-treated rats were restored
to the control level, so that there was no statistically
significant difference in D, receptor mRNA content among the
treatment groups (fig. 8B).
Striatal DA-atimulated adenylate cyclase activity
No significant difference in basal adenylate cyclase
activity was observed between treatment groups (fig. 10).
Stimulation of D( receptors by DA significantly increased
adenylate cyclase activity in the striatum at higher doses
of DA (lO^-lO*1 M) in all treatment groups.
The relative
increase among saline, SKF 38393, 6-OHDA, and /SKF 38393 +
6-OHDA' treatment groups was similar (fig. 9).
To evaluate
the effects of neonatal 6-OHDA and ontogenetic SKF 38393
treatments on the receptor coupling with G-protein, NaF was
used to stimulate Ns.
The adenylate cyclase activity was
enhanced by NaF, but there were no statistical differences
among the treatment groups (fig. 10, p=0.05l).
To examine
53
the effects of neonatal 6-OHDA and ontogenetic SKF 38393
treatments on the activity of adenylate cyclase, the enzyme
was stimulated directly with forskolin.
The activity of
adenylate cyclase was markedly increased by forskolin.
However, no significant difference was found in the
treatment groups of saline, SKF 38393, 6-OHDA, and SKF 38393
plus 6-OHDA (fig. 10, p=0.759).
Mediation of PA Agonist-induced Oral Behavior
bv the Serotonin System
Dose-response study of SKF 38393-induced oral activity
To determine the optimal doses of SKF 38393 for
inducing oral activity, a dose-response curve was
constructed (fig. 11).
In the saline control group, SKF
38393 at a dose of 0.1 mg/kg and higher resulted in only a
slight increase in oral activity.
In the 6-OHDA group, the
threshold dose of SKF 38393 was 0.03 mg/kg, with oral
activity increasing to 15 ± 2.9 movements (p < 0.01).
At
higher doses of SKF 38393 there were about 40 oral movements
during the session (p < 0.005).
Effect of combined D1 aoonlat and D1 antagonist treatments
To determine whether a DA D1 receptor antagonist would
attenuate the oral responses of rats to a D1 agonist, rats
were treated with
sch
23390 (0.3 mg/kg, i.p.) 1 hr before
54
SKF 38393 (0.3 mg/kg, i.p.}*
As shown in fig. 12, SCH 23390
attenuated the action of SKF 38393 (p < 0.005), indicating
that SKF 38393-induced oral activity is a Dl-receptormediated event.
Dose-responsa study of m-CPP-lnduced oral activity
To determine the optimal dose of m-CPP for induction of
the oral response, a series of doses of m-CPP was
administered to rats.
As shown in fig. 13, the oral
response was increased in both the control and 6-0HDAlesioned groups of rats after m-CPP
doses inthe range of
0.5 to 8.0 mg/kg i.p..
responsewas greater in
The overall
the 6-OHDA vs. the control group of rats (p < 0.001), with
the apparent maximal response in the 6-OHDA-lesioned rats
occurring at the 4.0 mg/kg dose of m-CPP.
Effects of S-HT1A and 5-HT,P receptor
agonistson oral
activity
To determine the possible involvement of 5-HT(
receptors on induction of oral activity, rats were treated
with either the 5-HTtA receptor agonist, 8-OH-DAPT HBr (0.1
mg/kg s.c., 10 min before observation) or the 5-HT,0
receptor agonist, CGS-12066B maleate (7-trifluoromethyl-4(4methyl-l-piperazinyl)pyrrolo[l,2-alquinoxaline], 1:2 maleate
salt, 3.0 mg/kg i.p., 10 min before observation).
As shown
in fig. 14, 8-OH-DPAT did not induce an oral response in
55
rats.
Also, CGS-12066B did not produce a statistically
significant increase in oral activity in control and 6-OHDAlesioned rats (fig. 14}.
Effects of 5-HT receptor antagonists on m-CPP-lnduced oral
activity
To assess whether m-CPP might produce enhanced oral
activity through an action on several different types of 5HT receptors, a variety of 5-HT receptor antagonists were
administered in conjunction with m-CPP (1.0 mg/kg i.p.).
After pretreatment with mianserin (1.0 mg/kg s.c.), a mixed
antagonist for 5-HT)c and 5-HT2 receptors, there was complete
attenuation of the oral response to m-CPP (p < 0.001, fig.
15).
Ketanserin (5.0 rag/kg i.p.), a relatively selective
antagonist for the 5-HT2 receptor, failed to modify the oral
response to m-CPP (fig. 15).
Similarly, MDL 72222 (10.0
mg/kg s.c.), a relatively selective antagonist for the 5-HT3
receptor, failed to alter the response to m-CPP (fig. 15).
Effect of a DA D, receptor antagonist on m-CPP-lnduced oral
To determine whether a DA Dt receptor antagonist would
attenuate the oral response of rats to the 5-HT agonist, mCPP, rats were treated with SCH 23390 (0.3 mg/kg i.p.).
As
shown in fig. 16, SCH 23390 failed to attenuate the enhanced
oral response to m-CPP, indicating that 5-HT agonist-induced
56
oral activity is not mediated through the DA p! receptor.
Effect of a 5rHTir receptor antagonist on
da
D,_acrQnlgt^
induced oral activity
To determine whether a 5-HTiC receptor antagonist would
attenuate the oral response to a DA D, receptor agonist,
rats were treated with mianserin (1.0 mg/kg s.c.) 30 min
before SKF 38393 (1.0 mg/kg i.p.).
As shown in fig. 17,
pretreatment with mianserin attenuated the enhanced oral
response to SKF 38393, in both control (p < 0.05) and 6OHDA-lesioned rats (p < 0.001),
This finding indicates that
the oral response of rats to a D( agonist is mediated via a
5-HT system involving 5-HTJC receptors.
Effect of neonatal 6-OHDA treatment on concentrations of_PA^
5-HT and their metabolites in the striatum
As expected, neonatal 6-OHDA treatment profoundly
reduced striatal DA, HVA and DOPAC contents (>98% reduction,
p < 0.001; table 5).
Accompanying this change was an
elevation in striatal 5-HT content by 77% (p < 0.001), and a
slight but not statistically significant increase in 5-HIAA
content (table 5).
57
Effect of neonatal 6-QHPA and ontogenet_ic_8KF_3a393
treatmentg_on mRNA levels for B-HT^ recaptors of the
striatum
To evaluate the effect of neonatal 6-OHDA and
ontogenetic SKF 38393 treatments on 5-HTlc receptor gene
transcription, dot blot analysis was also employed.
There
was no significant difference in the mRNA levels for 5-HT,c
receptors in the striatum between neonatal 6-OHDA-lesioned
and intact rats (fig. ISA).
Ontogenetic treatments of
either intact or lesioned rats with SKF 38393 did not alter
the mRNA level for the 5-HTlc receptor (fig. 18A). After
repeated administration
of SKF 38393 in adulthood, there
was still no change in 5-HT(C receptor mRNA in saline, 6OHDA, SKF 38393, and 'SKF 38393 + 6-OHDA' treatment groups
(fig. 18B).
58
TABLE 1
Behavioral effects of acute saline challenge in rats treated
developmentally with SKF 38393 or saline, in combination with
single i.c.v. injection of 6-OHDA or its vehicle.
Group
Behavior
Saline
Sniffing
SKF 38393
49.7 ± 10.7
Locomotion
1.5 ±
1.0
38.2 ± 12.1
Grooming
Eating
174.0 ± 50.6
5.2 ±
6-OHDA
6-OHDA + SKF
66.8 + 34.3
93.6
± 25.0
2.6
1.7 ± 0.9
3.0
37.4 ± 24.7
21.6 + 7.8
57.6
± 31.4
26.4 + 16.1
7.5 ± 4.7
10.1
±
4.8
0.0
±
0.0
±
1.2
4.2 ±
4.2
Taffy pulling 0.0 ±
0.0
0.0 ±
0.0
0.0 ±
0.0
Digging
1.0 ±
0.4
1.8 ±
1.2
0.8 +
0.4
1.1 ±
0.6
Gnawing
6.5 ±
0.9
7.8 ±
3.1
13.4 +
2.8
15.2 ±
2.6
Paw treading
0.0 ±
0.0
0.0 ±
0.0
0.0 +
0,0
0.0 ±
0.0
Yawning'
0.3 ±
0.3
0.0 ±
0.0
0.6 +
0.3
0.2 ±
0.1
Raring'
5.6 ±
2.3
10.2 ±
3.8
5.3 +
2.9
3.8 ±
1.4
Immobility
493.0 ± 20.7 337.2 :t 64.2
482.3 ± 38.6
414.9 ± 36.2
Behavioral activity was determined in rats that were treated during
postnatal development with a DA D, agonist, SKF 38393 HCl (3.0 mg/kg
per day x 28 days, i.p., from day of birth and/or the catecholamine
neurotoxin 6-OHDA HBr (200 /ig i.c.v., salt form, at 3 days from
birth; desipramine pretreatment, 1 hr). Rats were challenged with
saline at 8 weeks from birth and were immediately observed for
stereotyped behaviors for 10 min. Behaviors were thus quantified
as seconds in which each individual behavior was performed by a
rat. The total observation time was 600 seconds.
lYawning and
rearing behaviors were counted as numbers of incidents, not
seconds. Each group is the mean of 10 rats.
59
TABLE 2
Behavioral
effects
of
acute SKF
38393
in rats
treated
developmentally with SKF 38393 or saline, in combination with
single i.c.v. injection of 6-OHDA or its vehicle.
Group
Behavior
Sniffing
Locomotion
Grooming
Eating
Saline
SKF 38393
49.5 ± 14.2
1.8 ±
0.9
135.4 ± 21.8
0.5 ±
Taffy pulling 0.0 ±
95.2 ± 24.0
0.8 ±
0.5
95.6 ± 22.0
6-OHDA
22.8 ±
6-OHDA + SKF
7.6
54.8 ± 34.6
351.7 ± 59.6* 228.9 ± 74.8*
4.3 +
4.3*
3.5 ±
1.8 ±
1.0
0.0 ±
0.0
0.0
0.0 ±
0.0
0.0 i
0.0 172.5 ± 87.8*+
0.5
0.0 ±
3.5*
0.0
Digging
0.4 ±
0.3
2.1 +
1.2
2.3 ±
1.9
0.0 ±
0.0
Gnawing
12.1 ±
2.3
7.7 ±
1.8
1.1 ±
0.7*
0.0 +
0.0*
Paw treading
0.0 ±
0.0
0.0 ±
0.0
Yawning*
0.1 ±
0.1
0.0 ±
0.0
Raring*
4.1 ±
1.8
4.3 ±
1.7
Immobility
396.1 ± 34.4 392.5 ± 38.0
89.0 ± 50.2* 140.3 ± 75.8*
0.0 ±
7.2 +
0.0
2.9
84.1 ± 56.3*
0.0 ±
0.0
13.3 ±
5.5
0.0 ±
0.0*+
Behavioral activity was determined in rats that were treated during
postnatal development with a DA D, agonist, SKF 38393 HCl (3.0 mg/kg
per day x 28 days, i.p., from day of birth) and/or the
catecholamine neurotoxin 6-OHDA HBr (200 nq i.c.v., salt form, at
3 days from birth; desipramine pretreatment, 1 hr). Rats were
challenged with SKF 38393 HCl (3.0 mg/kg i.p.) at 8 weeks from
birth and were immediately observed for stereotyped behaviors for
10 min.
Behaviors were thus quantified as described in Table 1
legend. Each group is the mean of 10 rats.
*p < 0.000 vs. saline control group; +p < 0.01 vs. 6-OHDA group.
60
TABLE 3
Effect of neonatal 6-OHDA and ontogenetic SKF 38393 treatments on
concentrations of DA, 5-HT and their metabolites in the striatum
of adult rats.
MONOAMINES (nmol/g)
GROUP
n
Saline
6
SKF 38393
5
6-OHDA
SKF 38393
+
6-OHDA
10
9
DA
HVA
DOPAC
5-HT
5-HIAA
79.2±2.3
8.6±0,5
16.110.4
4.010.1
5.210.2
74.1±3.0
8.210.3
13.810.6
3.910.2
5.210.3
0.6+0.07* 0.110.03*
0 .110.02* 6.810.2* 7.010.3*
0.6±0.08*
0.210.08*
0.210.05*
6.210.2* 6,810.2*
Rats were treated daily with vehicle or SKF 38393 HCl (3.0 mg/kg
i.p.) for 28 consecutive days from birth in combination with
i.c.v. injection of 6-OHDA (200 jxg, salt form, 3 days after
birth; desipramine pretreatment, l hr) or its vehicle. Striata
were removed for assay at 11 weeks. Values are mean nanomoles
per gram of tissue ± S.E.M.
*p < 0.001 when compared to control group.
61
TABLE 4
Computer-modeled parameters for dopamine inhibition of [3H)SCH 23390
binding in the striatal homogenates of saline, 6-OHDA, SKF 38393
and 'SKF 38393 + 6-OHDA' treatment groups of rats.
Group
% RH
K h (nM)
Kl (/iM)
Saline
75.1 ± 2.6
212.2 ± 26.8
30.9 ± 9.9
SKF38393
75.3 ± 3.2
231.6 ± 26.6
29.2 ± 6.8
6-OHDA
68.7 ± 2.6
272.5 ± 38.5
24.4 ± 4.0
SKF38393
+
6-OHDA
73.2 ± 2.0
275.0 ±59.2
30.5 ± 6.8
Rats were treated daily with vehicle or SKF 38393 HCl (3.0 mg/kg
i.p.) for 28 consecutive days from birth in combination with i.c.v.
injection of 6-OHDA HBr (200 /ig, salt form, 3 days after birth;
desipramine pretreatment, 1 hr) or its vehicle.
Striata were
removed for assay at 11 weeks. Competition curves were constructed
by using 14 concentrations of inhibitor, DA, to compete for 0.45 nM
[ HJSCH 23390 binding in rat striatum and were fit best by a twosite computer derived model where the affinity of [3H]SCH 23390 was
constrained to be equal (Kd » 0.61 nM) at both high (RH) and low
(RL) affinity sites. The dissociation constants for high (KH) and
low (Kt) affinity agonist-binding sites were thus determined by
using computer program (GraphPad Software Inc., San Diego, CA).
Data are expressed as mean values or percent ± S.E.M. of
8 determinations in each treatment group.
62
TABLE 5
Effect of neonatal 6-OHDA treatment on concentrations of DA, 5-HT
and their metabolites In the striatum of rats
Honoamine
or Metabolite
Treatment
Saline (n=6)
% of Control
6-OHDA (n=9)
DA
67.83 ± 1.80
0.95 ± 0.16*
1.4
HVA
7.50 ± 0.53
0.14 ± 0.04*
1.9
DOPAC
14.45 ± 0.87
0.27 ± 0.05*
1.9
5-HT
3.52 ± 0.15
6.23 ± 0.55*
5-HIAA
6.53 ± 0.30
7.50 ± 0.60
177
115
Rats were treated at 3 days after birth with 6-OHDA HBr (200 pg,
l.c.v. salt form; deslpramine pretreatment, 1 hr) or Its vehicle.
Striata were removed for assay at 9 months.
Values are mean
nanomoles per gram of tissue 1 S.E.M. Numbers In parentheses are
numbers of samples per group.
*p < 0.001 when compared to vehicle control group.
63
EFFECT OF SKF-38393 ON LOCOMOTOR ACTIVITY IN RATS
1000
800
First Injection
C_
CD
-M
CD
600
0)
S
ro
400
in
200
0
Acute Saline
Treatment
Acute SKF 38393
Treatment
Figure 1. Effect of first acute weekly SKF 38393 HC1 (3.0
mg/kg i.p.) injection on locomotor activity in rats. Rats
were treated during postnatal ontogeny with either saline
O f SKF 38393 Hcl (3,0 mg/kg i.p, daily x 28 days;0H|),
6-OHDA HBr (200 jig i.c.v., 3 days after birth; desipramine
pretreatment, 1 hr;H) or combined SKF 38393 + 6-OHDA
treatments (|). Rats were challenged at 6 weeks from birth
with SKF 38393 HCl (3,0 mg/kg i.p.), 10 min before
observation, and then were videotaped for their locomotor
response for 60 min. Locomotor activity represents the
distance (meters) traveled by each rat. Each bar indicates
the mean of 7 to 10 rats. * p < 0.05, vs. control group
challenged with SKF 38393 HC1; + p < 0.05, vs. same group
challenged with saline.
64
EFFECT OF SKF-38393 ON LOCOMOTOR ACTIVITY IN RATS
1000
800
c_
<D
A->
Q>
<D
(J
C
td
-M
Second Injection
600
400
V)
200
0
Acute Saline
Treatment
Acute SKF 38393
Treatment
Figure 2. Effect of second acute weekly SKF 38393 HCl (3.0
mg/kg i.p.) injection on locomotor activity in rats. Rats
were treated during postnatal ontogeny with either saline
(□)/ SKF 38393 HC1 (3.0 mg/kg i.p.) daily x 28 days; QQ),
6-OHDA HBr (200 pg i.c.v., 3 days after birth; desipramine
pretreatment 1 hr;g) or combined SKF 38393 + 6-OHDA
treatments (»• Rats were challenged at 7 weeks from birth
with SKF 38393 HCl (3.0 mg/kg i.p.), 10 min before
observation, and then were videotaped for their locomotor
response for 60 min. Locomotor activity represents the
distance (meters) traveled by each rat. Each bar indicates
the mean of 7 to 10 rats. * p < 0.05, vs. control group
challenged with SKF 38393 HC1; + P < 0.05, vs. same group
challenged with saline.
65
EFFECT OF SKF-38393 ON LOCOMOTOR ACTIVITY IN RATS
1000
*+
gOOh
C
QJ
-M
(U
Fourth Injection
600
<U
U
C
(0
in
400
200
0
Acute Saline
Treatment
Acute SKF 38393
Treatment
Figure 3. Effect of fourth acute weekly SKF 38393 HC1 (3.0
mg/kg i.p.) injection on locomotor activity in rats. Rats
were treated during postnatal ontogeny with either saline
O , SKF 38393 HC1 (3.0 mg/kg i.p. daily x 28 days; Dffl) ,
6-OHDA HBr (200 jig i.c.v., 3 days after birth; desipramine
pretreatment, 1 hr; H) or combined SKF 38393 + 6-OHDA
treatments (■). Rats were challenged at 9 weeks from birth
with SKF 38393 Hcl (3.0 mg/kg i.p.), 10 min before
observation, and then were videotaped for their locomotor
response for 60 min. Locomotor activity represents the
distance (meters) traveled by each rat. Each bar indicates
the mean of 7 to 10 rats. * p < 0.05, vs. control group
challenged with SKF 38393 HC1; + p < 0.05, vs. same group
challenged with saline.
66
DOPAMINE INHIBITION OF [3H]SCH 23390 BINDING
IN RAT STRIATUM
120
100
Saline
CD
E
z
D
I— I e
□
*rH
z
X
I— I t
□Q Ed
_J
<
I—
O
MO
9
6 -5 -4
7
-8
log [DOPAMINE] (M)
-3
-2
Figure 4. Competition of DA for 0.45 nM [3H]SCH 23390
binding in the striatum of the saline group of rats. Rats
were treated with vehicle i.c.v. injection (bilateral;
desipramine pretreatment; 3 days after birth) and the
striata were removed 11 weeks from birth. The competition
curve in striatal homogenates was fit best by a two-site
computer derived model where the affinity of (JH]SCH 23390
was constrained to be 0.61 nM. Each data point represents
the mean of 8 determinations + S.E.M.. The competition curve
was analyzed as in table 4.
67
DOPAMINE INHIBITION OF [3 H]SCH 23390 BINDING
IN RAT STRIATUM
120
SKF 38393
100
CD
2:
1— 1 €
□
■H
2
I— I X
(0
CO
e
_J
-<
I—
□
o
a-s
80
60
40
20
0
9
4
6
7
5
log [DOPAMINE] (M)
8
-3
-2
Figure 5. Competition of DA for 0.45 nM [JH]SCH 23390
binding in the striatum of the ontogenetic SKF 38393 group
of rats. Rats were treated ontogenetically with SKF 38393
HC1 (3.0 mg/kg i.p. daily x 28), the striata were removed at
11 weeks from birth. The competition curve was constructed
as in figure 4.
DOPAMINE INHIBITION OF [3H]SCH 23390 BINDING
IN RAT STRIATUM
120 r
■ ■■■
t ....
-9
I . . ..... I . . . .
I .
I . 1 1 1 I I I I 1 I I I I I I
-8 -1 -6 -5 -4
log [DOPAMINE] (M)
-3
-2
Figure 6. Competition of DA for 0.45 nM (3H]SCH 23390
binding in the striatum of the neonatal 6-OHDA group of
rats. Rats were treated neonatally with 6-OHDA HBr (200 jig
i.c.v., 3 days after birth; desipramine pretreatment), and
the striata were removed at 11 weeks from birth. The
competition curve was constructed as in figure 4.
69
DOPAMINE INHIBITION OF [3H]SCH 23390 BINDING
IN RAT STRIATUM
120
SKF 38393 +
6-OHDA
00
80
CD
Z
i— i
□
z
h-H
CD
■C
1—
o
X
60
03
o
40
20
0
9
5 -4
6
7
log [DOPAMINE] (M)
8
-3
-2
Figure 7. Competition of DA for 0.45 nM [JH)SCH 23390
binding in the striatum of the neonatal 6-OHDA plus
ontogenetic SKF 38393 group of rats. Rats were treated
ontogenetically with SKF 38393 HC1(3.0 mg/kg i.p. daily x
28) and lesioned neonatally with 6-OHDA HBr (200 /ig i.c.v.,
3 days after birth; desipramine pretreatment). The striata
were removed at 11 weeks from birth. The competition curve
was constructed as in figure 4.
70
STRIATAL DOPAMINE D1 RECEPTOR mRNA LEVELS
BEFORE TREATMENTS WITH SKF 38393 IN ADULT RATS
3.0 r
z:
cr
2.5
2.0
u
i
<
cc
e
5
1.5
1. 0
0.5
0.0
Saline
SKF 38393
6-OHDA
SKF 38393 +
6-OHDA
Figure 8A. Effects of neonatal 6-OHDA and ontogenetic SKF
38393 treatments on striatal mRNA levels for DA D, receptors
before treatments with SKF 38393 in adulthood. Each bar
represents the mean (± s.E.M.) ratio of optical density for
D, receptor mRNA vs. that for ft-actin mRNA. * p < 0.05 vs.
saline group.
71
STRIATAL DOPAMINE Dl RECEPTOR mRNA LEVELS
AFTER TREATMENTS WITH SKF 38393 IN ADULT RATS
3.0
<
OH
2.5
c
2.0
A-»
U
<
I
1.5
a:
e
5
1.0
0.5
0.0
Saline
SkF 38393
6-OHDA
SKF 38393 +
6-OHDA
Figure 8B. Effects of neonatal 6-OHDA and ontogenetic SKF
38393 treatments on striatal mRNA levels for DA D, receptors
after treatments with SKF 38393 in adulthood. Each bar
represents the mean (± S.E.M.) ratio of optical density for
D, receptor mRNA vs. that for fl-actin mRNA. No significant
differences were observed among saline, SKF 38393, 6-OHDA
and 'SKF 38393 + 6-OHDA' treatment groups.
72
DOPAMINE-STIMULATED STRIATAL ADENYLATE CYCLASE
ACTIVITY IN RATS
° Saline
150
c
*iH
C
o
E
S
« SKF 38393
• 6-OHDA
♦ SKF 38393
+ 6-OHDA
125
c
*S
-M
O
c_
o CL
CO)
Q_
E
N
■c <D
(J I 1
100
(J
"O
oe
Q.
0
-6
-5
-4
-3
log [DOPAMINE] (M)
-2
Figure 9. Effects of neonatal 6-OHDA and ontogenetic
SKF 38393 treatments on DA-stimulated adenylate cyclase
activity. Each point represents the mean (± S.E.M.) cAMP
production (pmoles/mg protein/min) of 5 to 6 determinations.
The basal enzyme activities for saline, SKF 38393, 6-OHDA
and SKF 38393 + 6-OHDA treatment groups are: 19.0 ± 2.7,
19.1 ±3.6, 14.7+2.3, and 19.6 ± 2.1 pmoles/mg
protein/min, respectively. No significant differences in
stimulation were observed between the treatment groups.
* p < 0.05, vs. basal activity in the same group.
73
STRIATAL BASAL, NaF- OR FORSKOLIN-STIMULATED
ADENYLATE CYCLASE ACTIVITY IN ADULT RATS
° Saline
■ SKF 38393
■ 6-OHDA
■ SKF 38393
+ 6-OHDA
5000
4000
3000
2000
1000
BASAL
FORSKOLIN
Figure 10. Effects of neonatal 6-OHDA and ontogenetic
SKF 38393 treatments on basal adenylate cyclase activity and
NaF- and forskolin-stimulated adenylate cyclase activity.
No significant differences were observed among saline,
SKF 38393, 6-OHDA and 'SKF 38393 + 6-OHDA' treatment groups
on basal enzyme activity. NaF and forskolin increased
adenylate cyslase activity in all treatment groups, but the
relative increase was not statistically significant among
these four groups of rats.
74
SKF 38393-INDUCED ORAL ACTIVITY IN RATS
60
tfi
-M
C
50
QJ
□ Control
6-OHDA
« 40
ro
30
c_
a
a
a
20
10
0.0
0.03
0.1 0.3 1.0
Dose of SKF 38393 (mg/kg)
3.0
Figure IX. Dose-response relationship for SKF 38393-induced
oral activity in rats. Numbers of oral movements were
determined for one minute every 10 minutes over a 60-min
period, starting 10 minutes after SKF 38393 HC1 (0.03 to
3.0 mg/kg i.p.). Each group is the mean of 4 to 6 rats.
The number of oral movements in 6-OHDA vs. control rats is
different at each respective dose of SKF 38393 (p < 0.01).
75
ATTENUATION OF SKF 38393-INDUCED ORAL ACTIVITY
IN RATS BY SCH 23390
50
03
4-J
c
03
E
03
>
O
40
□ Saline
■ 6-OHDA
30
(13
C,
o
20
o
*
o
X
10
0
Saline
SCH 23390
SKF 30393
SKF 38393 +
SCH 23390
Figure 12. Attenuation of SKF 38393-induced oral activity in
rats by SCH 23390. Numbers of oral movements were
determined for one minute every 10 minutes, for 60 minutes,
starting 10 minutes after SKF 38393 HC1 (0.3 mg/kg i.p.) and
60 minutes after SCH 23 390 HCl (0.3 mg/kg i.p.). Each group
is the mean of 4 to 6 rats. * p < 0.005 vs. other 6-OHDA
groups.
76
m-CPP-INDUCED ORAL ACTIVITY IN RATS
tt
in
-M
c
CD
E
CD
>
O
**
□ Control
■ 6-OHDA Lesioned
n)
C_
O
o
o
0.0
0.5
1.0
2.0
4.0
8.0
Dose of m-CPP (mg/kg)
Figure 13. Dose-response relationship for m-CPP-induced oral
activity in control and 6-OHDA-lesioned rats. Rats were
studied for oral activity as in figure 11. Each bar
represents mean number of oral movements ± S.E.M.. Each
m-CPP dose produced a greater response in the 6-OHDA vs.
control group. * p < 0.01; ** p < o.ooi vs. respective
control or 6-OHDA group challenged with saline.
77
EFFECTS OF SEROTONIN IA AND IB RECEPTOR AGONISTS
ON ORAL ACTIVITY IN RATS
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8-OH-DPAT
CGS-12066B
Figure 14. Effects of 8-OH-DPAT, a 5-HT,A receptor agonist,
and CGS-12066B, a 5-HTin receptor agonist, on oral activity
in control and 6-OHDA-lesioned rats. Rats were observed for
oral activity, as described in figure 11. The number of
oral movements was determined in the l-hr interval, starting
10 min after challenge treatment with saline, or 8-OH-DPAT
(0.1 mg/kg s.c.}, or CGS-12066B (3.0 mg/kg i.p.). Each
group is the mean of 8 to 9 (control) or 11 to 14 (6-OHDA
lesioned) rats. There was no statistically significant
increase in oral activity after 8-OH-DPAT or CGS-12066B
treatments.
78
EFFECTS OF SEROTONIN RECEPTOR ANTAGONISTS
ON m-CPP-INDUCED ORAL ACTIVITY IN RATS
80 r
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6-OHDA Lesioned
o
cd
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Figure 15. Effects of 5-HT receptor antagonists on m-CPPinduced oral activity in control and 6-OHDA-lesioned rats.
Rats were studied for oral activity as in figure 11.
Mianserin HC1 (1.0 mg/kg s.c.), ketanserin tartrate
(5.0 mg/kg i.p.) and MDL 72222 (10.0 mg/kg i.p.) were
administered 30 min before m-CPP 2HC1 (1.0 mg/kg i.p.) or
its vehicle. Each group is the mean (± S.E.M.) of 6 to 11
rats. * p < 0.001 vs. m-CPP effect in the 6-OHDA-lesioned
rats.
79
LACK OF EFFECT OF SCH 23390
ON m-CPP-INDUCED ORAL ACTIVITY IN RATS
70
in
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40
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30
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20
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10
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Saline
SCH 23390
m-CPP
CH 23390 +
m-CPP
Figure 16. Lack of effect of SCH 23390 on m-CPP-induced oral
activity in control and 6-OHDA-lesioned rats. Rats were
studied for oral activity as in figure 11. SCH 23390
(0.3 mg/kg i.p.) was administered 1 hr before m-CPP 2HC1
(1.0 mg/kg i.p.) or its vehicle. Each group is the mean
(± S.E.M.) of 4 to 11 rats. SCH 23390 did not attenuate the
effect of m-CPP (p > 0.05).
80
ATTENUATION OF SKF 38393-INDUCED ORAL ACTIVITY
IN RATS BY MIANSERIN
45
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40
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Saline
Mianserin
SKF 38393 Mianserin +
SKF 38393
Figure 17. Attenuation of SKF 38393-induced oral activity in
control and 6-OHDA-lesioned rats by mianserin. Rats were
studied for oral activity as figure 11. Mianserin HC1 (1.0
mg/kg s.c.) was administered 30 min before SKF 38393 HC1
(1.0 mg/kg i.p.) or its vehicle. Each group is the mean
(± S.E.M.) of 4 to 11 rats. * p < o.ooi vs. other groups.
81
STRIATAL SEROTONIN 1C RECEPTOR mRNA LEVELS
BEFORE TREATMENTS WITH SKF 38393 IN AOULT RATS
z
cc
4
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<c
1
3
cc
E
o
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in
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Saline
SKF 38393
6-OHDA
SKF 38393 +
6-OHDA
Figure 18A. Effects of neonatal 6-OHDA and ontogenetic
SKF 38393 treatments on striatal mRNA levels for 5-HTlc
receptors before treatments with SKF 38393 in adulthood.
Each bar represents the mean (± S.E.M.) ratio of optical
density for 5-HTir receptor mRNA vs. that for 0-actin mRNA.
No significant differences were observed among saline,
SKF 38393, 6-OHDA and 'SKF 38393 + 6-OHDA' treatment groups.
82
STRIATAL SEROTONIN IC RECEPTOR mRNA LEVELS
AFTER TREATMENTS WITH SKF 38393 IN ADULT RATS
2.5 r
•c
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4-1
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0.5
594
0.0
Saline
SKF 38393
6-OHDA
SKF 38393 +
6-OHDA
Figure 18B. Effects of neonatal 6-OHDA and ontogenetic
SKF 38393 treatments on striatal mRNA levels for 5-HTIC
receptors after treatments with SKF 38393 in adulthood.
Each bar represents the mean (± S.E.M.) ratio of optical
density for 5-HTlt: receptor mRNA vs. that for B-actin mRNA.
No significant differences were observed among saline,
SKF 38393, 6-OHDA and 'SKF 38393 + 6-OHDA' treatment groups.
Chapter 4
Discussion
DR D. Receptor supersensitization
Neonatal treatment of rats with a catecholamine
neurotoxin, 6-OHDA, results in the supersentization of DA
receptors.
That is, doses of L-dihydroxyphenylalanine (L~
DOPA), apomorphine or SKF 38393 which have little effect in
intact rats, produce marked changes in stereotyped and
locomotor behaviors in the lesioned rats that were tested as
adults (Breese et al., 1984, 1985a,b, 1987; Hamdi and
Kostrzewa, 1991).
The DA Di receptor antagonist, SCH 23390,
attenuates the enhanced stereotyped and locomotor behaviors
by the D: agonist, SKF 38393, in the lesioned rats,
indicating that Dx receptors are specifically involved in
the induced receptor sensitization (Breese et al., 1985a,b,
1987).
The ability to sensitize D: receptors appears to be
related to immaturity of the DA system at the time of
lesioning, since 6-OHDA treatment of adult rats is not
associated with such receptor sensitization (Breese et al.,
1984).
Actually, a latent supersensitization of Dt
receptors occurs with this lesion, and the sensitization
process is only unmasked by repeated DA D: agonist
treatments in adulthood (Criswell et al., 1989, 1990;
Johnson et al., 1992).
This process of induction of long-
lived Dj receptor supersensitivity is known as 'priming'
83
(Breese et al., 1985b; Criswell et al., 1989).
Recently,
Hamdi and Kostrzewa (1991) reported that the D, receptors
could be ontogenetically sensitized by repeated Dt agonist
treatments during the first 33 days after birth.
As in
adult-primed rats, there was an enhancement of the D,
agonist-induced stereotyped behaviors in adulthood.
Also,
as in studies by Breese and co-workers, there was no change
in the B,^ or Kj for D, binding in the striatum of these rats
(Breese et al., 1987).
The status of the DA Dt receptor during postnatal
ontogeny is not completely resolved.
There are some reports
of only slight change in the B ^ and Kd for []H]SCH 23390
binding to striatal homogenates in the first 1 to 2 months
after birth (Broaddus and Bennett, 1990), and other reports
of a dramatic increase in B„„ during the first 4 to 5 weeks
after birth (Giorgi et al., 1987; Zeng et al., 1988) and
even transient increases in the Kd in this interval (Zeng et
al., 1988).
Using histochemical methods, however, it has
been clearly demonstrated that DA D, receptors develop in
patches in the striatum, being closely associated with DA
nerve terminal input to these patches (Zeng et al., 1988).
As D, receptors increase in number, the striosomal pattern
of DA D, receptors becomes obscured by the global increase
in DA D| receptors in the matrix of the striatum.
This
pattern of DA D, receptor development indicates that the
early series of SKF 38393 treatments in the present study
would initially interact only with the population of DA D,
receptors that had developed up to that point in tine.
Interaction of SKF 38393 with striatal DA D, receptors would
change during development, as the population of the receptor
continued to increase in number during ontogeny.
In theory,
each subsequent dose of SKF 38393 was able to act at greater
numbers of Dt receptors, which would mean that there was
unequal exposure of the DA D, receptor population to the DA
D, agonist.
Perhaps this relates to the inability of SKF
38393 to fully sensitize DA D, receptors to later SKF 38393induced locomotor activity effects.
Although many SKF 38393-induced responses of neonatalsensitized and adult-primed rats are similar, there are some
differences in degree of response for many behaviors, which
possibly are related to the extent of sensitization.
The
extent of DA D, sensitization in those rats treated
repeatedly in postnatal ontogeny with the DA D, agonist, SKF
38393 was investigated in the present study.
For that
reason the locomotor responses to SKF 38393 were determined
after the first 2 doses and after the last dose of the D(
agonist.
It is clear from effects observed in rats treated
only with neonatal 6-OHDA, that adult priming was essential
for unmasking of the latent supersensitivity of D,
receptors.
In essence, the first dose of SKF 38393 produced
only a slight increase in the locomotor response of rats,
while subsequent doses produced increasingly greater effects
86
(7th bars of figs. 1-3; p < 0.05, ANOVA).
This finding in
the present study is in full agreement with those earlier
reports (Breese et al., 1985b; Criswell et al., 1989, 1990).
In contrast to this effect, sensitization of DA Dt
receptors was evident with the first challenge dose of SKF
38393 at 6 weeks, in neonatal 6-OHDA lesioned rats that were
treated daily with SKF 38393 during the first 4 weeks of
postnatal ontogeny.
That is, an enhanced locomotor response
was observed after initial SKF 38393 treatment in adulthood
(8th bar of fig, l, p < 0.05).
Subsequent SKF 38393
challenge doses increased locomotor activity to a level
ultimately about 3-fold greater than that seen after the
first adult challenge dose (8th bars of figs. 2-3).
Therefore, it appears that ontogenetic treatments of
neonatal 6-OHDA-lesioned rats with SKF 38393 produces
partial but not complete DA D, receptor supersensitization.
Additional adult SKF 38393 treatments were necessary to
produce the maximal level of D, sensitivity.
Because locomotor activity is only one of several kinds
of behaviors induced by DA agonists, we observed rats for
SKF 38393-induced stereotypies at 8 weeks.
DA Dt agonists
produce exaggerated stereotyped behavioral responses in
neonatal 6-OHDA-lesioned rats (Breese et al., 1984, 1985a,b,
1987; Hamdi and Kostrzewa, 1991).
It was important to
determine if several SKF 38393 challenge treatments would
substantially modify the stereotypy effect of a DA Dt
87
agonist in adult rats.
We found that ontogenetic SKF 38393
treatments did not markedly influence stereotyped responses
in neonatal 6-OHDA-lesioned rats.
Virtually all of the
behaviors were expressed in both the 6-OHDA group and 'SKF
38393 + 6-OHDA' group, except for taffy pulling, which was
expressed in only the latter group.
Also, immobility time
was less in this group vs. the 6-OHDA group.
These findings
suggest that ontogenetic SKF 38393 treatments are able to
partially modify later stereotyped behavioral effects of a
DA D| agonist.
Despite the fact that a different method was used in
the present study to evaluate stereotyped behavior,
virtually all of the SKF 38393-induced responses are similar
to those observed previously in rats that were lesioned
neonatally with 6-OHDA and ontogenetically sensitized with
SKF 38393 (Hamdi and Kostrzewa, 1991}.
The one exception is
that SKF 38393-induced grooming is reduced in the present
study, compared to an enhanced grooming response in the
previous study (Hamdi and Kostrzewa, 1991).
This difference
may relate to additional priming in adulthood in the present
study.
It is likely that, as D(-related behaviors became
more prominent with additional SKF 38393 treatments in
adulthood, the competing behaviors resulted in less grooming
behavior.
In essence, a rat can only exhibit so many
behaviors at one time, so an increase in performing of one
behavior might be expected to diminish the time spent in
88
another behavior.
Given the behavioral aberrations observed in both
neonatal 6-OHDA-lesioned rats and those receiving combined
neonatal 6-OHDA and ontogenetic SKF 38393 treatments in the
present study, a DA competition for [3H]SCH 23390 binding
analysis was performed from striatal homogenates of the
rats.
This was done because earlier reports showed a lack
of change of D, receptor number and affinity by either using
striatal homogenates (Breese et al., 1987; Duncan et al.,
1987; Dewar et al., 1990; Hamdi and Kostrzewa, 1991) or
performing autoradiography (Luthman et al., 1990; Simson et
al., 1992), after neonatal 6-OHDA treatment.
As yet, very
little is known about whether numbers of high affinity D,
receptor would be altered in these rats or in rats given
both neonatal 6-OHDA and ontogenetic D, agonist treatments.
No change in high affinity binding sites for DA D, receptors
was noted in neonatal 6-OHDA vs. saline rats when using DA
competition for radiolabeled D, antagonist binding (table
4).
Also, in the present study postnatal treatment of
neonatal 6-OHDA-lesioned rats with SKF 38393 did not affect
the distribution of high and low affinity agonist binding
sites (table 4).
Collectively, these results demonstrate
that behavioral supersensitivity to systemic administration
of DA D| agonist in adulthood, or priming of rats lesioned
as neonates and those given neonatal lesion plus ontogenetic
treatment with the D, agonist, is neither due to increased D,
89
receptor number in general, nor is it due to altered high
affinity receptor binding in particular.
With the techniques of recombinant DNA technology being
used, specific sequences encoding different types of DA
receptor including rat (Zhou et al., 1990) and human D,
receptors (Dearry et al., 1990; Sunahara et al., 1991), have
been cloned .and sequenced, and thereby can be used to
analyze the receptor gene expression in the brain.
Therefore, availability of the receptor mRNA probe has made
it possible to analyze the specific mRNA coding for DA D(
receptor and thus to have a better understanding of the
receptor regulation and supersensitivity after neonatal 6OHDA lesion.
In order to investigate whether the enhanced
locomotor and stereotyped responsiveness of the lesioned
rats to adult challenge doses of D, agonist is attributable
to an altered rate of transcription of the gene or the mRNA
for DA D, receptors, we also evaluated the effect of SKF
38393 treatments on expression of the gene coding D,
receptors.
The finding that the level of D, receptor mRNA
after neonatal 6-OHDA was reduced, regardless of whether
ontogenetic SKF 38393 was co-administered, suggests that 6OHDA chemical lesion influences the receptor gene
expression.
Gelbard et al. (1990) demonstrated that
neonatal development of DA D, receptors in rat forebrain was
impaired after 6-OHDA lesion.
These investigators suggested
that in the absence of endogenous DA, the Dt receptors may
possibly either fail to develop adequately or fail to be
maintained.
The present data reveal possible molecular
mechanisms underlying this biochemical event.
Our finding
indicates that neonatal 6-OHDA lesion impairs ontogeny of DA
Dj receptors due to failure of receptor gene expression.
After repeated treatments with SKF 38393 in adulthood (adult
priming), there was no change in striatal Dt mRNA levels
among the treatment groups, indicating that priming restores
D, receptor gene expression in the lesioned rats.
These
results are in accord with previous studies, demonstrating
that priming of striatal D, receptors in neonatal 6-OHDA
lesioned rats occurred in the absence of altered D, receptor
density and affinity (Breese et al., 1987; Duncan et al.,
1987; Dewar et al., 1990; Luthman et al., 1990; Hamdi and
Kostrzewa, 1991).
Moreover, these findings are fully
consistant with the above agonist competition for
radiolabeled DA D, antagonist binding data, indicating that
the DA D| receptor recognition site after neonatal 6-OHDA
lesion may not be a critical factor in the development of
behavioral supersensitivity to DA D, agonist.
Based on pharmacological manipulation of adult animals,
it has been proposed that alterations in DA D, receptor mRNA
by repeated reserpine treatments (Butkerait and Friedman,
1993) or by chronic DA D, receptor blockade (Schettini et
al., 1992) are associated with an increased level of mRNA
coding for G protein alpha subunit (Nsa). The present data
showed that after adult priming with a D, agonist, the level
of Dt receptor mRNA in neonatally lesioned or combined
neonatally lesioned plus ontogenetically sensitized rats did
not differ from that of the control at a time when
behavioral sensitization of the animals is known to occur.
Taken together, these studies suggest that a site distal to
the DA D| receptor recognition site, or possibly, a site
rather than in DA neurochemical system but in a system or
systems modulating DA-mediated effects, accounts for the
apparent supersensitization of D, receptor-related function.
The present study, using DA to stimulate adenylate
cyclase in the presence of spiperone, indicates that the
link of DA D, receptors to this enzyme is not changed in the
striatum by lesioning neonates with 6-OHDA, in spite of
profound depletion of striatal DA content and
supersensitized behavioral responses.
Moreover, we found
that forskolin- and NaF-stimulated adenylate cyclase
activity did not differ in lesioned and intact rats.
Additionally, ontogenetic treatments with SKF 38393 failed
to affect basal and DA-stimulated adenylate cyclase activity
in both neonatal lesioned and intact animals.
This finding
supports that of Sirason et al. (1992), who found that
agonist-stimulated adenylate cyclase activity did not change
in neonatal 6-OHDA lesioned rats that were primed in
adulthood.
It appears that DA Dt receptor linked adenylate
cyclase is also not a major determinant of supersensitized
92
behavioral responses to D( agonists in neonatal 6-OHDAlesioned rats.
It is well established that neonatal 6-OHDA treatment
of rats is associated with a marked reduction in the
t
striatal level of DA, due to destruction of nigrostriatal
neurons (Breese and Traylor, 1971, 1972; Breese et al.,
1984), and an elevation in 5-HT content (Breese et al.,
1984; Stachowiak et al., 1984), due to sprouting and ensuing
hyperinnervation by 5-HT-containing fibers (Stachowiak et
al., 1984; Berger et al., 1985; Luthman et al., 1987; Towle
et al., 1989).
The findings in this study are in full
agreement with those earlier reports.
That is, DA content
of the striatum in 6-OHDA-lesioned rats is profoundly
reduced by nearly 99%, and DOPAC and HVA contents are
reduced by a similar amount (table 3).
Accompanying these
changes is the elevation of striatal 5-HT content by 70%,
and 5-HIAA by 35% (table 3).
Additionally, ontogenetic
treatments of rats with SKF 38393 in the present study, did
not produce an effect on striatal DA, 5-HT and their
metabolites, nor did this treatment modify the effect of 6OHDA (table 3).
Based on above striatal contents of DA, 5-
HT and their metabolites found in this study, we feel that
the animal models are similar to those in earlier studies.
93
Mediation of DR Agonist-Induced oral Behavior bv the 5-HT
System
In neonatal 6-OHDA-lesioned rats a supersensitized
response to DA D, agonists is observed in adulthood (Breese
et al., 1985a,b, 1987; Hamdi and Kostrzewa, 1991), despite
the lack of change in number or affinity of D, receptors
(Breese et al., 1987; Hamdi and Kostrzewa, 1991).
The oral
activity response to D, receptor agonists and D2 antagonists
is also enhanced in the neonatal lesioned rats (Kostrzewa
and Hamdi, 1991).
In the present study, the augmented oral
response to SKF 38393 in lesioned rats is attenuated by SCH
23390, a DA D, receptor antagonist, indicating that the Dt
receptor subtype is primarily involved in the enhanced
response (fig. 12).
Recently, it was found that 5-HT agonists also can
induce oral activity in rats (Stewart et al., 1989).
Since
the striatal DA depletion in neonatal 6-OHDA-treated rats is
accompanied by elevated 5-HT levels (Breese et al., 1984;
Stachowick et al., 1984; Luthman et al., 1987; table 5 in
the present study) and hyperinnervation of striatum by 5-HT
fibers (Berger et al., 1985; Snyder et al., 1986), it was of
interest to determine whether the induction of oral activity
by 5-HT agonists would be potentiated in these rats.
The
enhanced response to m-CPP over a wide dose range in
lesioned rats in this study, demonstrates that 5-HT
receptors are also supersensitized in this group of rats.
94
Production of overt supersensitization of 5-HT receptors in
neonatal 6-OHDA-lesioned rats is suggested by the fact that
the maximal oral activity response is 2- to 3-fold higher in
this group vs. control (fig. 13).
It has also been shown
that m-CPP acts directly as a 5-HT agonist that lacks 5-HT
uptake inhibitor properties (Fuller et al., 1981).
Therefore, the most likely explanation for m-CPP action
relates to 5-HT receptor supersensitization.
The series of studies with 5-HT receptor agonists and
antagonists indicate that 5-HTu and 5-HT,B receptors are not
associated with oral activity.
The agonist, 8-OH-DPAT, is
relatively selective for 5-HT,A receptors (Arvidsson et al.,
1981; Hiddlemiss and Fozard, 1983}, and the dose used in the
present study is known to produce a response in vivo (Fuller
and Snoddy, 1987; Stewart et al., 1989).
CGS 12066B, a
selective 5-HT,B receptor agonist with little activity at D,
sites (Neale et al., 1987), did not induce oral activity.
Although we have no direct evidence that CGS 12066B acted at
the 5-HT|B site in the brain, the dose used in this study is
more than 10-fold greater than that found by others to alter
the firing of dorsal raphe neurons (Neale et al., 1987).
Ketanserin and MDL 72222, antagonists with high
affinity for 5-HT2 (Leysen et al., 1981, 1982) and 5-HTj
(Fozard, 1984) receptors, respectively, were ineffective in
attenuating the enhanced oral response to m-CPP.
The doses
of ketanserin and MDL 72222 are similar or higher than that
95
known to produce antagonism of 5-HT2 and 5-HTj receptors in
vivo (Fozard, 1984; Awouters et al., 1990; Nash, 1990).
These findings suggest that 5-HT2 and 5-HT3 receptors are not
involved in the effect of m-CPP.
Attenuation of the
enhanced oral response to m-CPP by mianserin, an antagonist
with affinity for both 5-HTlc and 5-HT2 receptors, suggests
that it is the 5-HTlc receptor subtype that becomes
supersensitized after loss of DA input to the striatum.
It was not practical to use a wide range of doses for
the different agonists and antagonists in these studies, and
it is conceivable that a different dose and/or different
observation time for some of these agents might have
resulted in an effect on the oral response.
However, based
on findings with 5-HT receptor agonists and antagonists in
this study, there is an apparent correlation between 5-HT,c
receptors and the oral responses of rats.
In the present study, the other major finding
is the
lack of effect of SCH 23390 on m-CPP-induced oral activity.
This indicates that the DA system is not the final pathway
in the behavioral event.
This finding is in agreement with
our biochemical studies in which there was no alteration in
DA D, receptor high affinity binding sites, no change in
basal and DA-stimulated adenylate cyclase activity, and no
change in Dt receptor mRNA level after priming in adulthood.
SCH 23390 has a high affinity for the D, receptor, with an
ICH
for [3H]SCH 23390 binding being about 150 times less
than that for [3H)mesulergine binding (Billard et al., 1984;
Hoyer et al., 1988).
The dose of SCH 23390 in this study is
similar or identical to that used to specifically attenuate
oral activity responses to a D, agonist (Levin et al., 1989;
Rosengarten et al., 1986); to attenuate Dj agonist-induced
behaviors (Breese et al., 1985a,b; Criswell et al., 1989);
and to attenuate the development of D, receptors (Saleh and
Kostrzewa, 1988; Kostrzewa and Saleh, 1989).
The effectiveness of mianserin in attenuating the
enhanced oral response to SKF 38393, suggests that D,
receptor-mediated oral activity relies on a 5-HT
neurochemical system for transduction of the response.
The
DA and 5-HT pathways associated with oral activity may
operate in sequence, with the 5-HT system representing a
later stage in this circuit.
It is also possible, however,
for these two neurochemical systems to operate in parallel
but with the 5-HT system exerting greater control.
Antagonists of the 5-HT system could conceivably attenuate
D, agonist-induced oral responses in either design.
The
observed oral response may be a consequence of 5-KT fiber
sprouting and hyperinnervation in rostral striatum.
Additional studies are needed to resolve these aspects of
interaction between the DA and 5-HT neurochemical systems.
In other studies on neonatal 6-OHDA-lesioned rats it was
reported that the sprouted 5-HT fibers did not seem to
possess an inhibitory control on endogenous 5-HT- and
97
acetylcholine-containing neurons (Jackson et al., 1988), and
that 5-HT innervation did not appear to be responsible for
supersensitized locomotor and stereotyped responses to a DA
agonist (Towle et al., 1989).
The present findings
demonstrate that 5-HT neurons can be intricately involved in
mediation of supersensitized responses to DA agonists.
To study the effects of neonatal 6-OHDA lesion and
ontogenetic SKF 38393 treatments on gene expression for 5HTIC receptors, the mRNA levels for the receptor subtype
were also determined in the present study.
Unlike DA D,
receptors, neither neonatal 6-OHDA nor ontogenetic SKF 38393
treatments affect mRNA coding for the 5-HTlc receptor.
Also, repeated treatments of both intact and lesioned rats
with SKF 38393 in adulthood did not influence the levels of
mRNA for 5-HT,c receptors in the striatum.
Therefore, the
biochemical change responsible for the enhanced oral
response in 6-OHDA-lesioned rats, other than altered gene
expession for 5-HTlc receptors, is yet to be determined.
It
has been known that 5-HT,c receptors are coupled to the
breakdown of phosphatidylinositides (Conn and Sanders-Bush,
1986).
An increase in inositol trisphosphates (IP3) was
reported to be related to the supersensitization of 5-HTlc
receptors generated by chemical denervation (Conn et al.,
1987).
It may be that coupling of the receptor with this
second messenger system could be different in the
supersensitized 5-HT,c receptors in our neonatal 6-OHDA rat
model.
Alternatively, balance and adjustment among striatal
DA, 5-HT and other neuronal systems after neonatal 6-OHDA
lesion may be responsible for the supersensitization of
multiple neural receptor systems (Kostrzewa and Heely,
1993).
The present series of studies helps to identify the
altered 5-HT receptor responses that occur in those rats
with greatly increased oral activity after neonatal
destruction of DA-containing fibers in the brain.
This
animal model should be useful in studying functional
associations between central dopaminergic and serotonergic
neurons.
Also, the discovery of a 5-HT neurochemical
component in enhanced oral dyskinetic activity implicates
another direction that may be taken toward the discovery of
agents that modulate dyskinetic oral activity in assorted
human clinical disorders.
Chapter 5
Summary
The present series of studies has demonstrated the
following:
1. Ontogenetic treatments of neonatal 6-OHDA-lesioned rats
with a DA Dt agonist, SKF 38393, can partially sensitize Dj
receptors in adulthood.
2. Neonatal 6-OHDA chemical lesions impair DA Di receptor
ontogeny due to failure of gene expression for the receptor,
while repeated treatments of the lesioned rats with SKF
38393 in adulthood result in restoration of the mRNA level
for D! receptors.
3. Changes in DA Dj receptor high affinity binding, Dx
receptor mRNA levels, and
receptor-stimulated adenylate
cyclase activity, can not account for supersensitized
behavioral responses in neonatally lesioned rats.
4. The 5-HTlc receptor subtype becomes functionally
supersensitized after neonatal destruction of DA input to
the striatum, in the absence of a change in striatal mRNA
level for this receptor subtype.
Moreover, induction of
oral activity by a DA D! agonist is mediated via a
serotonergic system.
5. The neonatal 6-OHDA rat model should be useful in
exploring functional and neurochemical associations between
central dopaminergic and serotonergic systems.
99
BIBLIOGRAPHY
101
Bibliography
Abdel-Latif, A. A., Yau, S.-J., and Smith, J. P. 1974
Effects of neurotransmitters on phospholipid metabolism
in rat cerebral cortex slices- cellular and subcellular
distribution. J. Neurochem., 22:383-393.
Albert, P. R., Zhou, Q. Y., Van Tol, H. H., Bunzow, J. R.,
and Civelli, 0. 1990 Cloning functional expression
and mRNA tissue distribution of the rat 5hydroxytryptaminelA receptor gene. J. Biol. Chem.,
265:5825-5832.
Andersen, P. H., Groenvald, F. C., and Jansen, J. A. 1985
A comparison between dopamine-stimulated adenylate
cyclase and JH-SCH 23390 binding in rat striatum. Life
Sci., 37:1971-1983.
Andersen, P. H.and Groenvald, F. C. 1986 Specific
binding of 3H-SCH 23390 to dopamine D, receptors in
vivo. Life Sci., 38:1507-1515.
Andersen, P. H. and Nielsen, E. B. 1986
The dopamine D,
receptors: biochemical and behavioral aspects. In
Neurobiology of central Dt dopamine receptors, vol.
204, Advances in experimental medical biology, eds.
Breese, G. R. and Creese, I., pp.73-91. New York:
Plenum Press.
Arluison, M. and Delamanche, I. S. 1980
High resolution
radioautographic study of the serotonin innervation of
the rat corpus striatum after intra-ventricular
administration of [JH ]5-hydroxytryptamine. Neurosci.,
5:229-240.
Arvidsson, L.-E., Hacksell, U., Nilsson, J. L. G., Hjorth,
S., Carlsson, A., Lindberg, P., Sanchez, D., and
Wikstrom, H. 1981 8-hydroxy-2-(di-npropylamino)tetralin, a new centrally acting 5hydroxytryptamine receptor agonist. J. Med. Chem.,
24:921-923.
Awouters, F., Niemegeers, C. J. E., Megens, A. A. H. P., and
Janssen, P. A. J. 1990 Functional interaction between
serotonin-Sj and dopamine-Dj neurotransmission as
revealed by selective antagonism of hyper-reactivity to
tryptamine and apomorphine. J. Pharmacol. Exp. Ther.,
254:945-951.
Barnes, J. M., Barnes, N. M., Champaneria, s., Costall, B.,
and Naylor, R. J. 1990 Characterization and
102
autoradiographic localisation sites identifified with
[3H]-(S)-zacopride in the forebrain of the rat.
Neurophannacol., 29:1037-1045.
Beal, M. F. and Martin, J. B. 1985 Topographical dopamine
and serotonin destribution and turnover in rat
striatum. Brain Res., 358:10-15.
Berger, T. W., Haul, S., Strieker, E. M., and Zigmond, M. J.
1985 Hyperinnervation of the striatum by dorsal rephe
afferents after dopamine-depleting brain lesions in
neonatal rats. Brain Res., 336:354-358.
Billard, W., Ruperto, V., Crosby, G., Iorio, L. c., and
Barnett, A. 1984 Characterizationof the binding
of3H-SCH 23390, a selective D, receptor antagonist
ligand, in rat striatum. Life Sci., 35:1885-1893.
Bockaert, J., Fozard, J. R., Dumuis, A., and Clarke, D. E.
1992 The 5-HT4 receptor: a place in the sun. Trend
Pharmacol. Sci., 13:141-145.
Boeynaems, J. M. and Dumont, J. E. 1975 Quantitative
analysis of the binding of ligands to their receptors.
J. Cycl. Nucl., 1:123-142.
Boeynaems, J. M. and Dumont, J. E. 1977
The two-step model
of ligand-receptor interaction. Mol. Cell.
Endocrinol., 7:33-47.
Bogdanski, D. F., Weissbach, H., and Udenfriend, S. 1957
The distribution of serotonin, 5-hydroxytryptophan
decarboxylase, and monoamine oxidase in brain. J.
Neurochem., 1:272-278.
Bouthenet, M.-L., Souil, E., Martres, M.-P., Sokoloff., P.,
Giros, B., and Schwartz, J.-C. 1991 Localization of
dopamine D} receptor mRNA in the rat brain using in
situ hybridization histochemistry comparison with
dopamine D2 receptor mRNA. Brain Res., 564:203-219.
Brasel, J. A., Ehrenkranz, R. A., and Winick, M. 1970
polymerase activity in rat brain during ontogeny.
Biol., 23:424-432.
dna
Dev.
Breese, G* R., Baumeister, A. A., McCown, T. J., Emerick, S.
G., Frye, G. D., Crotty, K., and Mueller, R. A. 1984
Behavioral differences between neonatal and adult 6hydroxydopamine-treated rats to dopamine agonists:
Relevance to neurological symptoms in clinical
syndromes with reduced brain dopamine. J Pharmacol.
103
Exp. Ther., 231:343-354.
Breese, 6. R., Baumeister, A. A., Napier, T. C., Frye, G.
D., and Mueller, R. A. 1985a Evidence that D,
dopamine receptors contribute to the supersensitive
behavioral responses induced by Ldehydroxyphenylalanine in rats treated neonatally with
6-hydroxydopamine. J. Pharmacol. Exp. Ther., 235:287295.
Breese, G. R., Duncan, G. E., Napier, T. C., Bondy, S. C.,
Xorio, L. C., and Mueller, R. A. 1987 6hydroxydopamine treatment enhance behavioral responses
to intracerebral microinjection of D, and D2 dopamine
agonists into nucleus accumbens and striatum without
changing dopamine antagonist binding. J. Pharmacol.
Exp. Ther., 240:167-176.
Breese, G. R*, Napier, T. C., and Mueller, R. A. 1985b
Dopamine agonist-induced locomotor activity in rats
treated with 6-hydroxydopamine at differing ages:
Functional supersensitivity of D, dopamine receptors in
neonatally lesioned rats. J. Pharmacol. Exp. Ther.,
234:447-455.
Breese, G. R. and Traylor, T. D. 1970 Effects of 6hydroxydopamine in brain norepinephrine and dopamine
evidence for selective degeneration of catecholamine
neurons. J. Pharmacol. Exp. Ther., 174:413-420.
Breese, G. R. and Traylor, T. D. 1971 Depletion of brain
noradrenaline and dopamine by 6-hydroxydopamine. Br.
J. Pharmacol., 42:88-99.
Breese, G. R. and Traylor, T. D. 1972 Developmental
characteristics of brain catecholamines and tyrosine
hydroxylase in the rat; Effects of 6-hydroxydopamine.
Br. J. Pharmacol., 44:210-222.
Broaddus, W. C. and Bennett, J. P. 1990 Postnatal
development of striatal function. I. An exmination of
D| and Dj receptors, adenylate cyclase regulation and
presynaptic dopamine markers. Dev. Brain Res., 52:265271.
Brown, A. M., Patch, T. L., and Kaumann, A. J. 1991 The
antimigraine drugs ergotamine and dihydroergotamine are
potent 5-HTlc receptor agonists in piglet choroid
plexus. Br. J. Pharmacol., 104:45-48.
Bruno, J. P., Zigmond, M. J., and Strieker, E. M.‘ 1986
104
Rats given dopamine depleting brain lesions as neonates
do not respond to acute homeostatic imbalances as
adults. Behav. Neurosci. 100:125-128.
Butkerait, P. and Friedman, E. 1993 Repeated reserpine
increases striatal dopamine receptor and nucleotide
binding protein RNA. J. Neurochem., 60:566-571.
Butler, I. J., Koslow, S. H., Seifert, Jr, W. E., Caprioli,
R. M., and Singer, H. S. 1979 Biogenic amine
metabolism in Tourette syndrome. Ann. Neurol., 6:3739.
Carlsson, A., Lindqvist, M., Magnusson, T., and Waldeck, B.
1958 On the presence of 3-hydrxytryptamine in brain.
Science, 127:471.
Carlsson, A. 1959 The occurrence, distribution and
physiological role of catecholamines in the nervous
system. Pharmcol. Rev., 11:490-493.
Chalmers, D. T. and Watson, S. J. 1991 Comparative
anatomical distribution of 5-HTu receptor mRNA and 5HTU binding in rat brain - a comparative in situ
hybridization/in vitro receptor autoradiographic study.
Brain Res., 561:51-60.
Charney, D. s., Woods, S. W., Goodman, W. K., and Heninger,
G. R. 1987 Serotonin function in anxiety: II. Effects
of the serotonin agonist mCPP in panic disorder
patients and healthy subjects. Psychopharmacol.,
92:14-24.
Cheronis, J. C., Erinoff, L., Heller, A., and Hoffmann, P.
C, 1979 Pharmacological analysis of the functional
ontogeny of the nigrostriatal dopaminergic neurons.
Brain Res., 169:545-560.
Clow, A., Jenner, P., and Marsden, c. D. 1979 changes in
dopamine-mediated behavior during one year's
neuroleptic administration. Eur. J. Pharmacol.,
57:365-375.
Conn, P. J., Janowsky, A., and Sanders-Bush, E. 1987
Denervation supersensitivity of 5-HTlc receptors in rat
choroid plexus. Brain Res., 400:396-398.
Conn, P. J. and Sanders-Bush, E. 1986 Agonist-induced
phosphoinositide hydrolysis in choroid plexus. J.
Neurochem., 47:1754-1760.
105
Costall, B. and Olley, J., E. 1971 Cholinergic- and
neuroleptic-induced catalepsy: modification by lesions
in the caudate-putamen. Neuropharmacol., 10:297-306.
Coyle, J. T. and Axelrod, J. 1972 Tyrosine hydroxylase in
rat brain: developmental characteristics. J.
Neurochem., 19:1117-1123.
Coyle, J. T. and Campochiaro, P. 1976 Ontogenesis of
dopaminergic-cholinergic interactions in the rat
striatum: a neurochemical study. J. Neurochem.,
27:673-678.
Creese, I. and Iversen, S. 1973 Blockage of amphetamine
induced motor stimulation and stereotypy in the adult
rat following neonatal treatment with 6hydroxydoparaine. Brain Res., 55:369-382.
Creese, I., Burt, D. R., and Snyder, s. H. 1977 Dopamine
receptor binding enhancement accompanies lesiondinduced behavioral supersensitivity. Science, 197:596598.
Creese, I and Leff, S. E. 1982 Dopamine receptors: A
classification. J. Clin. Psychpharmacol., 2:329-335.
Creese, I., Sibley, D. R., Hamblin, H. W., and Leff, S. E.
1983 The classification of dopamine receptors:
Relationship to radioligand binding. Annu. Rev.
Neurosci., 6:43-71.
Criswell, H., Mueller, R. A., and Breese, G. R. 1989
Priming of D, dopamine receptor responses: long-lasting
behavioral supersensitivity to a D, dopamine agonist
following repeated administration to neonatal 6-OHDAlesioned rats. J. Neurosci., 9:125-133.
Criswell, H. E., Mueller, R. A., and Breese, G. R. 1990
Long-term Dt-dopamine receptor sensitization in
neonatal 6-OHDA-lesioned rats is blocked by an NMDA
antagonist. Brain Res., 512:284-290.
Cross, A. and Rossor, M. 1983 Dopamine D( and D3 receptors
in Huntington's disease. Eur. J. Pharmacol., 88:223229.
Dahlstrom, A. and Fuxe, K. 1964 Evidence for the existence
of monoamine-containing neurons in the central nervous
system. Acta Physiol. Scand., 62 (suppl. 232):1-55.
Dal Toso, R., Sommer, B., Ewert, K., Herb, A., Pritchett, D.
106
B., Bach, A., Shivers, B. D., and Seeburg, P. H. 1989
The dopamine D2 receptor: two molecular forms generated
by alternative splicing. EMBO J., 8:4025-4034.
Dawson, T. M., Gehlert, D. B., McCabe, R. T., Barnett, A.,
and Wamsley, J. K. 1986 D, dopamine receptors in the
rat brain: a quantitative autoradiographic analysis.
J. Neurosci., 6:2352-2365.
Dearry, A., Gingrich, J. A., Falardeau, P., Fremeau, R. T.,
Bates, M. D., and Caron, M. G. 1990 Molecular cloning
and expression of the gene for a human D, dopamine
receptor. Nature, 347:72-76.
De Keyser, J., De Backer, J.-P., Ebinger, G. and Vauquelin,
G. 1989 Coupling of D, dopamine receptors to the
guanine nucleotide binding protein G, is deficient in
Huntington's disease. Brain Res., 496:327-330.
Dewar, K. M., Soghomonian, J.-J., Bruno, J. P. Descarries,
L., and Reader, T. A. 1990 Elevation of Dopamine D3
but not D, recetpors in adult rat neostriatum after
neonatal 6-hydroxydopamine denervation. Brain Res.,
536:287-296.
Doucet, G., Descarries, L., and Garcia, S. 1986
Quantification of the dopamine innervation in adult rat
neostriatum. Neurosci., 19:427-445.
Dourish, C. T., Clark, M. L., Iversen, S. D. 1989 Evidence
that blockade of post-synaptic 5-HT, receptors elicits
feeding in satiated rats. Psychopharmacol., 97:54-58.
Duncan, G. E., Criswell, H. E., McCown, T. J., Paul, A.,
Mueller, R. A., and Breese, G. R. 1987 Behavioral and
neurochemical responses to haloperidol and SCH 23390 in
rats treated neonatally or as adults with 6hydroxydopamine. J. Pharmacol. Exp. Ther., 243:10271034.
El Mestikawy, S., Fargin, A., Raymond, J. R., Gozlan, H.,
and Hnatowich, M. 1991 The 5-HT1A receptor: an
overview of recent advances. Neurochem. Res., 16:1-10.
Erinoff, L., Macphail, R. C., Heller, A., and Seiden, L. S.
1979 Age dependent effects of 6-hydroxydopamine on
locomotor activity in the rat. Brain Res., 164:195205.
Fischette, C. T., Nock, B., and Renner, K.
1987
Effects of
107
5,7-dihydroxytryptamine on serotonin( and serotoninj
receptors throughout the rat central nervous system
using quantitative autoradiography. Brain Res.,
421:263-279.
Fozard, J. R. 1984 MDL 72222: A potent and highly
selective antagonist at neuronal 5-hydroxytryptamine
receptors. Naunyn-Schmiedeberg's Arch. Pharmacol.,
326:36-44.
Fozard, J. R. and Gray, J. A. 1989 5-HTlc receptor
activation: a key step in the initiation of migraine?
Trends Pharmacol. Sci., 10:307-309.
Frazer, A*, Maayani, S., and Wolfe, B. B. 1990 Subtypes of
receptors for serotonin. Annu. Rev. Pharmacol.
Toxicol., 30:307-348.
Fremeau, Jr., R. T., Duncan, G. E., Fornaretto, M.-G.,
Dearry, A., Gingrich, J. A., Breese, G. R. and Caron,
M. G. 1991 Localization of Dt dopamine receptor mRNA
in brain supports a role in cognitive, affective, and
neuroendocrine aspects of dopaminergic
neurotransmittion. Proc. Natl. Acad. Sci. USA,
88:3772-3776.
Fuller, R. W. and Snoddy, H. D. 1987 Influence of route of
administration on potency of selective 5-HTlA agonist,
8-hydroxy-2-(di-n-propylamino)tetralin, in rats. Res.
Commun. Chem. Pathol. Pharmacol., 58:409-412.
Fuller, R. W., Snoddy, H. D., Mason, N. R., and Owen, J. E.
1981 Disposition and pharmacological effects of mchlorophenylpiperazine in rats. Neuropharmacol.,
20:155-162.
Fuxe, K., Hokfelt, T., Agnati, L. F., Johansson, 0.,
Goldstein, M., Perez la Mara, M., Possani, L., Tapia,
R., Teran, L., and Palacios, R. 1978 Mapping out
central catecholamine neurons: immunohistochemical
studies on catecholammine-synthesizing enzymes. In
Psychpharmacology: A generation of progress, eds.
Lipton, M. A., Dimascio, A., and Killam, K. F., pp. 6794, New York: Raven Press.
Gelbard, H. A., Teicher, M. H., Baldessarini, R. J.,
Gallitano, A., Marsh, E. R., Zorc, J., and Faedda, G.
1990 Dopamine D, receptor development depends on
endogenous dopamine. Dev. Brain Res., 56:137-140.
Gelbard,
H. A., Teicher,
M. H.,Faedda, G., and
108
Baldessarini, R. J. 1989 Postnatal development of
dopamine Dt and D2 receptor sites In rat striatum.
Dev. Brain Res., 49:123-130.
Gerfen, C. R., Herkenham, M., and Thibault, J. 1987 The
neostriatal mosaic. II. Patch- and matrix-directed
mesostriatal dopaminergic and non-dopaminergic systems.
J. Neurosci., 7:3915-3934.
Giorgi, 0., DeMontis, G., Porceddu, M. L., Mele, s.,
Calderini, G., Toffano, G., and Biggio, G. 1987
Developmental and age-related changes in D, dopamine
receptors and dopamine content in the rat striatum.
Dev Brain Res., 35:283-290.
Giros, B., Sokoloff, P., Martres, M.-P., Riou, J.-F.,
Emorine, L. J., and Schwartz, J.-c. 1989 Alternative
splicing directs the expression of two D2 dopamine
receptor isoforms. Nature, 342:923-926.
Glennon, R. A. and Dukat, M. 1991 Sertonin receptors and
their ligands: a lack of selective agents. Pharmacol.
Biochem. Behav., 40:1009-1017.
Hamblin, M. W., Leff, S. E., and Creese, I. 1984
Interaction of agonists with D2 dopamine receptors:
evidence for a single receptor population existing in
multiple agonist affinity-states in rat striatal
membrane. Biochem. Pharmacol., 33:877-887.
Hamblin, M. W, and Metcalf, M. A. 1991 Primary structure
and functional characterization of a human 5-HT,D-type
serotonin receptor. Mol. Pharmacol., 40:143-148.
Hamdi, A. and Kostrzewa, R. M. 1991 Ontogenic homologous
supersensitization of dopamine D, receptors. Eur. J.
Pharmocol., 203:115-120.
Heikkila, R. E., Shapiro, B. S., and Duvoisin, R. C. 1981
The relationship between loss of dopamine nerve
terminals, striatal [3HJ-spiroperidol binding and
rotational behavior in unilaterally 6-hydroxydopaminelesioned rats. Brain Res., 211:285-292.
Herrick-Davis, K., Maisonneuve, I. M., and Titeler, M. 1989
Postsynaptic localization and up-regulation of
serotonin 5-HT10 receptors in rat brain. Brain Res.,
483:155-157.
Herrick-Davis, K. and Titeler, M. 1988 Detection and
characterization of the serotonin 5-HT1D receptor in
109
rat and human brain.
J. Neurochem., 50:1624-1631.
Hess, E. J., Battaglia, G., Norman, A., lorio, L., and
Creese, I. 1966 Guanine nucleotide regulation of
agonist interaction at [3H]SCH 23390-labeled D,
dopamine receptors in rat striatum. Eur. J.
Pharmacol., 121:31-38.
Ho, B. T. and Huang, J. T. 1975 Role of dopamine in damphetamine-induced discriminitive responding.
Pharmacol. Biochem. Behav., 3:1085-1092.
Hoffman, B. J. and Mezey, E. 1989 Destribution of sertonin
5-HTlc receptor mRNA in adult rat brain. FEBS Lett.,
247:453-462.
Hokln, H. R. 1970 Effects of dopamine, gamm-aminobutyric
acid and 5-hydroxytryptamine on incorporation of 3JP
into phosphatides in slices from the guinea pig brain.
J. Neurochem., 17:357-364.
Hornykiewicz, 0. 1966 Dopamine (3-hydroxytyramine) and
brain function. Pharmacol. Rev., 18:925-964.
Hornykiewicz, 0., 1973 Parkinson's disease: From
brainhomogenate to treatment. Fed. Proc., 32:183-190.
Hornykiewicz, 0. 1975 Parkinsonism induced by dopaminergic
antagonists. Adv. Neurol., 9:155-164.
Hoyer, D. 1988 Molecular pharmacology and biology of 5HT1C receptors. Trends Pharmacol. Sci., 9:89-94.
Hyttel, J. 1978 Effects of neuroleptics on 3H-haloperidol
and 3H-cis-(Z)-flupenthixol binding and on adenylate
cyclase activity in vitro. Life Sci., 23:551-556.
Hyttel, J. 1981 Similarities between the binding of 3Hpiflutixol and 3H-flupentixol to rat striatal dopamine
receptors in vitro. Life Sci., 28:563-569.
Hyttel, J. 1983 SCH 23390 - the first selective dopamine
D, antagonist. Eur. J. Pharmacol., 91:153-154.
Iorio, L. C., Barnett, A., Leitz, F. H., Houser, V. P., and
Korduba, C. A. 1983 SCH 23390, a potential
benzazepine antipsychotic with unique interactions on
dopaminergic systems. J. Pharmacol. Exp. Ther.,
226:462-468.
Jackson, D., Bruno, J. P., Stachowiak, M. K. and Zigmond, M.
110
J. 1988 Inhibition of striatal acetylcholine release
by serotonin and dopamine after the intracerebral
administration of 6-hydroxydopamine to neonatal rats.
Brain Res., 457:267-273.
Jacobs, S. and Cuatrecasas, P. 1976 The mobile receptor
hypothesis and "cooperativity" of hormone binding;
application to insulin. Biochim. Biophys. Acta,
433:482-495.
Johnson, K. B., Criswell, H. E., Jensen, K. F., Simson, P.
E., Mueller, R. A., and Breese, G. R. 1992 Comparison
of the D,-dopamine agonists SKF-38393 and A-68930 in
neonatal 6-hydroxydopamine-lesioned rats: Behavioral
effects and induction of c-fos-like immunoreactivity.
J. Pharmacol. Exp. Ther., 262:855-865.
Julius, D., Mac Dermott, A. B., Axel, R., and Jessel, T. M.
1988 Molecular characterization of a functional cDNA
encoding the serotonin 5-HTIC receptor. Science,
241:558-564.
Kebabian, J. W. and Caine, D. B. 1979
for dopamine. Nature, 277:93-95.
Multiple
receptors
Kelly, P. H., Seviour, P. W., and Iversen, S. D. 1975
Amphetamine and apomorphine responses in the rats
following 6-OHDA lesions of the nucleus accumbens septi
and corpus striatum. Brain Res., 94:507-522.
Kennett, G. A. and Curzon, G. 1988a Evidence that mcpp may
have behavioral effects mediated by central 5-HTlc
receptors. Br. J. Pharmacol., 94:137-147.
Kennett, G. A. and Curzon, G. 1988b Evidence that
hypophagia induced by mCPP and TFMPP requires 5-HTlB
receptors and 5-HT,c receptors; hypophagia induced by
RU 24969 only requires 5-HTIB receptors.
Psychpharmacol., 96:93-100.
Kennett, G. A. and Curzon, G. 1991 Potencies of
antagonists indicate that 5-HTlc receptors mediate
mCPP-induced hypophagia. Br. J. Pharmacol,, 103:20162020 .
Kennett, G. A., Whitton, P., Shah, K., and Curzon, G. 1989
Anxiogenic effects of mCPP and TFMPP in animal models
are opposed by 5-HTlc receptor antagonists. Eur. J.
Pharmacol., 164:445-454.
Kilpatrick, G. J., Jones, B. J., and Tyers, M. B.
1987
Ill
Identification and distribution of 5-HT3 receptors in
rat brain using radioligand binding. Nature, 330:746748.
Klawans, H. L. and Rubovits, R. 1972 An experimental model
of tardive dyskinesia. J. Neural Transm., 33:235-246.
Klawans, H. L. 1973 The pharmacology of tardive
dyskinesias. Am. J. Psychiatr., 130:82-86.
Klodzinska, A., Jaros, T., Chojanacka-Wojcik, E., and Maj,
J. 1989 Exploratory hypoactivity induced by TFMPP and
mCPP. J. Neural Transm., 1:207-218.
Koffer, K. B., Berney, S., and Hornykiewicz, 0. 1978 The
role of the corpus striatum in neuroleptic- and
narcotic-induced catalepsy. Eur. J. Pharmacol., 47:8186 .
Kostrzewa, R. M. and Hamdi, A. 1991 Potentiation of
spiperone-induced oral activity in rats after neonatal
6-hydroxydopamine. Pharmacol. Biochem. Behav., 38:215218.
Kostrzewa, R. M. and Jacobowitz, D. H. 1974
Pharmacological actions of 6-hydroxydopamine.
Pharmacol. Rev., 26:199-288.
Kostrzewa, R. M. and Kastin, A. J. 1993 Tyr-MIF-1
attenuates development of tolerance to spiperoneinduced catalepsy in rats. Brain Res. Bull., 31:707712.
Kostrzewa, R. M. and Neely, D. 1993 Enhanced pilocarpineinduced oral activity responses in neonatal 6-OHDA
treated rats. Pharmacol. Biochem. Behav., 45:737-740.
Kostrzewa, R. M. and Saleh, M. I. 1989 Impaired ontogeny
of striatal dopamine D, and D2 binding sites after
postnatal treatment of rats with SCH-23390 and
spiroperidol. Dev. Brain Res., 45:95-101.
Leff, S. E., Hamblin, H. W,, and Creese, I. 1985
Interaction of dopamine agonists with brain D,
receptors labeled by ’H-antagonists: evidence for the
presence of high and low affinity agonist-binding
states. Mol. Pharmacol., 27:171-183.
Lesch, M. and Nyhan, W. L. 1964 A familial disorder of
uric acid metabolism and central nervous system
function. Am. J. Med., 36:561-570.
112
Levin, E. D., See, R. E., and South, D. 1989 Effects of
dopamine D, and D2 receptor antagonists on oral
activity in rats. Pharmacol. Biochem. Behav., 34:4348.
Leysen, J. E., Awouters, F., Kennis, L., Laduron, P. K.
Vandenberk, J., and Janssen, P. A. J. 1981 Receptor
binding profile of R 41 468, a novel antagonist at 5HT2 receptors. Life Sci., 28:1015-1022.
Leysen, J. E., Niemegeers, C. J. E., Van Nueten, J. M., and
Laduron, P. M. 1982 [*H]Ketanserin (R41 468), a
selective JH-ligand for serotoni^ receptor binding
sites. Binding properties, brain distribution and
functional role. Mol. Pharmacol., 21:301-314.
Lidov, H. G. W. and Molliver, M. E. 1982 An
immuohistochemical study of serotonin neuron
development in the rat: ascending pathways and terminal
fields. Brain Res. Bull., 8:389-430.
Limbird, L. E. 1981 Activation and attenuation of
adenylate cyclase: the role of GTP-binding protein as
macromolecular messengers in receptor-cyclase coupling.
Biochem. J., 194:1-13.
Lloyd, K. G., Hornykiewicz, 0., Davidson, L., Shannak, K.,
Farley, I., Goldstein, M., Shibuya, M., Kelley, W. N.,
and Fox, I. H. 1981 Biochemical evidence of
dysfunction of brain neurotransmitters in the LeschNyhan syndrome. Mew Engl. J. Med., 305:1106-1111.
Loizou, L. A. and Salt, P. 1970 Regional changes in
monoamines of the rat brain during postnatal
development. Brain Res., 20:467-470.
Losonczy, M. F., Davidson, M., and Davis, K. L. 1987 The
dopamine hypothesis of schizophrenia. In
Psychpharmacology: The third generation of progress,
ed. Meltzer, H. Y., pp. 715-726. New York: Raven
Press.
Lowry, o. H., Rosebrough, M. J., Farr, A. L., and Randall,
R. J. 1951 Protein measurement with the Folin phenol
reagent. J. Biol. Chem., 193:265-275.
Luki, I., Ward, H. A., Frazer, A. 1989 Effect of l-(mchlorophenyl)piperazine and l-(mtrifluromethylphenyl)piperazine on locomotor activity.
J. Pharmacol. Exp. Ther., 249:155-164.
113
Luthman, J., Bolioli, B., Tustsurai, T., Verhofstad, A., and
Jonsson, G. 1987 Sprouting of striatal nerve
terminals following selective lesions of nigro-striatal
dopamine neurons in neonatal rat. Brain Res. Bull.,
19:269-274.
Luthman, J., Lindqvist, £., Young, D., and Cowburn, R. 1990
Neonatal dopamine lesion in the rat results in enhanced
adenylate cyclase activity without altering dopamine
receptor binding or dopamine- and adenosine 3',5'monophosphate-regulated phosphoprotein (DARPP-32)
immunoreactivity. Exp. Brain Res., 83:85-95.
Mahan, L. C., Burch, R. M., Monsma, Jr, F. J., and Sibley,
D. R. 1990 Expression of striatal Dx dopamine
receptors coupled to inositol phosphate production and
C a + mobilization in Xenopus oocytes. Proc. Natl.
Acad. Sci. USA, 87:2196-2200.
Marcinkiewicz, M., Verge., D., Gozlan, H., and Hamon, M.
1984 Autoradiogrphic evidence for the heterogeneity of
5-HTL site in the rat brain. Brain Res., 291:159-163.
Maricq, A. V., Peterson, A. S., Brake, A. J., Myers, R. M.,
and Julius, D. A. 1991 Primary structure and
functional expression of the 5-HT3 receptor, a
serotonin-gated ion channel. Science, 254:432-437.
Matthysse. S. 1973 Antipsychotic drug actions: a clue to
the neuropathology of schizophrenia. Fed. Proc.,
32:200-205.
Memo, M. C., Missale, C., Carruba, M. 0., and Spano, P. F.
1986 02 dopamine receptors associated with inhibition
of dopamine release from rat neostriatum are
independent of cyclic AMP. Neurosci. Lett., 71:192196.
Meller, E., Kuga, S., Friedhoff, A. J., and Goldstein, M.
1985 Selective D2 dopamine receptor agonists prevent
catalepsy induced by SCH 23390, a selective Dj
antagonist. Life sci., 36:1875-1864.
Mengod, G., Nguyen, H., Le, H., Weber, C., Lubbert, H., and
Palacios, J. M. 1990 The distribution and cellular
localization of the serotonin lc receptor mRNA in the
rodent brain examined by in situ hybridization
histochemistry. Comparison with receptor binding
distribution. Neurosci., 35:577-591.
Middlemiss, D. N. and Fozard, J. R.
1983
8-Hydroxy-2-(di-
114
n-propyl-amino)-tetralin discriminates between subtypes
of the 5-HT, recognition sites. Eur. J. Pharmacol.,
90:151-153.
Miquel, M. C., Doucet, E., Boni, C., El Mestikawy, S.,
Hatthiessen, L., Daval, G., Verge, D., and Hamon, M.
1991 Central serotonin,A receptors: respective
distributions of encoding mRNA, receptor protein and
binding sites by in situ hybridization histochemistry,
radioimmunohistochemistry and autoradiographic mapping
in the rat brain. Neurochem. int., 19:453-465.
Hishra, R. K., Marshall, A. M., and Varrouza, S. L. 1980
Supersensitivity in rat caudate nucleus: effect of 6hydroxydopamine on the time course of dopamine receptor
and cyclic AMP changes. Brain Res., 200:47-57.
Molineaux, S. M., Jessell, T. M., Axel, R., and Julius, D.
1989 5-HTlc receptor is a prominent serotonin receptor
subtype in the central nervous system. Proc. Natl.
Acad. Sci. USA, 86:6793-6797.
Montagu, K. A. 1957 Catechol compounds in rat tissues and
in brain of different animals. Nature, 180:244-245.
Moon Edley, S. and Herkenham, M. 1984 Comparative
development of striatal opiate receptors and dopamine
revealed by autoradiography and histofluorescence.
Brain Res., 305:27-42.
Murphy, D. L., Mueller, E. A., Hill, J. L., Tolliver, T. J.,
and Jacobsen, F. M. 1989 Comparative anxiogenic,
neuroendocrine and other physiologic effects of mchlorophenylpiperazine given intravenously or orally to
healthy volunteers. Psychopharmacol., 98:275-282.
Murrin, L. C. 1983 Characteristics of ]H-cis-flupenthixol
binding in rat striatum. Life Sci., 33:2179-2186.
Napier, T. C., Coyle, S., and Breese, G. R. 1985 Ontogeny
of striatal unit activity and effects of single or
repeated haloperidol administration in rats. Brain
Res., 333:35-44.
Nash, J. F. 1990 Ketanserin pretreatment attenuates MDMAinduced dopamine release in the striatum as measured by
in vivo microdialysis. Life Sci., 47:2401-2408.
Neale, R. F., Fallon, S. L., Boyar, W. C., Wasley, J. W, F.,
Martin, L. L., Stone, G. A., Glaeser, B. S., Sinton, C.
M., and Williams, M. 1987 Biochemical and
115
pharmacological characterization of CGS 12066B, a
selective serotonin-lB agonist. Eur. J. Pharmacol.,
136:1-9.
Neil, J. C. and Cooper, S. J. 1989 Selective reduction by
serotonergic agents of hypertonic saline consumption in
rats: evidence for possible 5-HTlc mediation.
Psychopharmacol., 99:196-201.
Norman, A. B., Borchers, M.
L., and Sanberg, P. R.
dopamine receptors and
in rat brain following
homogenization. Brain
T., Wachendorf, T. J., Price, A.
1989 Loss of D, and D2
muscarinic cholinergic receptors
in vitro polytron
Res. Bull., 22:633-636.
Odarjuk, J., Maretzki, D., Gross, J., and Gerber, G. 1986
Influence of postnatal hypoxia on 32-p-labeling of
polyphosphoinositides and phosphatidic acid in striatum
synaptosomes from rat brain. Biomed. Biochem. Acta,
45:593-599.
Offord, S. J., Ordway, G. A., and Frazer, A. 1988
Application of [12iI] iodocyanopindolol to measure 5hydroxytryptamine,B receptors in the brain of the rat.
J. Pharmacol. Exp. Ther., 244:144-153.
Olson, L., Seiger, A., and Fuxe, K. 1972 Heterogeneity of
striatal and limbic dopamine innervation: highly
fluorescent islands in developing and adult rats.
Brain Res., 44:283-288.
Onali, P., Olianas, M. C., and Gessa, G. L. 1984 Selective
blockade of dopamine Dt receptors by SCH 23390
discloses striatal dopamine D2 receptors mediating the
inhibition of adenylate cyclase in rats. Eur. J.
Pharmacol., 99:127-128.
Onali, P., Olianas, M. C., and Gessa, G. L. 1985
Characterization of dopamine receptors mediating
inhibition of adenylate cyclase activity in rat
striatum. Hoi. Pharmacol., 28:138-145.
Palacios, J. H. and Dietl, H. M. 1988 Autoradiographic
studies of serotonin receptors. In The Serotonin
Receptors, Sanders-Bush, E., ed. pp. 89-183, Humana
Press, Clifton.
Parent, A., Descarries, L., and Beaudet, A. 1981
Organization of ascending serotonin systems in the
adult rat brain. A radioautographic study after
intraventricular administration of [JH]5-
116
hydroxytryptamine.
Neurosci., 6:115-138.
Pazos, A., Cortes, R., and Palacios, J. M. 1985a
Quantitative autoradiographic napping of serotonin
receptors in the rat brain. II. Serotonin-2 receptors.
Brain Res., 346:231-249
Pazos, A., Hoyer, D., and Palacios, J. M. 1985b The
binding of serotonergic ligands to the porcine choroid
plexus: characterization of a new type of serotonin
recognition site. Eur. J. Pharmacol.,106:539-546.
Pazos, A. and Palacios, J. H. 1985 Quantitative
autoradiographic mapping of serotonin receptors in the
rat brain. I. Serotonin-1 receptors. Brain Res.,
346:205-230.
Pearson, D. E., Teicher, M. H., Shaywitz, B. A., Cohen, D.
J., Young, J. G., and Anderson, G. H. 1980
Enviromental influences on body weight and behavior in
developing rats after neonatal 6-hydroxydopamine,
Science, 209:715-717.
Peroutka, S. J,, Schmidt, A. W., Sleight, A. J., and
Harrington, M. A. 1990 serotonin receptor 'families'
in the central nervous system: an overview. In The
Neuropharmacology of Serotonin, Whittaker-Azmitia, P.
M. and Peroutka, s. J. eds. Ann. N.Y. Acad. Sci.,
600:104-112.
Peters, D. A. V., McGeer, P. L., and McGeer, E. G. 1968
The distribution of tryptophan hydroxylase in cat
brain. J. Neurochem., 15:1431-1435.
Phillips, A. G. and Fibiger, H. C. 1973 Dopaminergic and
noradrenergic substrates of positive reinforcement:
Differential effects of d- and 1-amphetamine. Science,
179:575-576.
Pijnenburg, A. J. J., Honig, w. M. M., Van Der Heyden, J. A.
M., and Van Rossum, J. M. 1976 Effects of chemical
stimulation of the meoslimbic dopamine system upon
locomotor activity. Eur. J. Pharmacol., 35:45-58.
Pritchett, D. B., Bach, A. W. J., Wozny,
Toso, R., Shih, J. C., and Seeburg,
Structure and functional expression
serotonin 5-HT2 receptor. EMBO J.,
H., Taleb, O., Dal
P. H. 1988
of cloned rat
7:4135-4140.
Radja, F., Descarries, L., Dewar, K. M. and Reader, T. A.
1993 Serotonin 5-HT, and 5-HT3 receptors in adult rat
117
brain after neonatal destruction of nigrostriatal
dopamine neurons: a quantitative autoradiographic
study. Brain Res., 606:273-285.
Reisine, T. D., Fields, J. Z., Stern, L. Z., Johnson, P. C.,
Bird, E. D., and Yamamura, H. I. 1977 Alterations in
dopaminergic receptors in Huntington's disease. Life
Sci., 21:1123-1128.
Riad, M., El Mestikawy, S., Verge, D., Gozlan, H., and
Hamon, M. 1991 Visualization and quantification of
central 5-HTtA receptors with specific antibodies.
Neurochem. Int., 19:413-423.
Rodbell, M. 1980 The role of hormone receptors and GTP
regulatory protein in membrane transduction. Nature,
284::17-22.
Rosengarten, H. and Friedhoff, A. J. 1979 Enduring changes
in dopamine receptor cells of pups from drug
administration to pregnant and nursing rats. Science,
203:1133-1135.
Rosengarten, H., Schweitzer, J. W., Egawa, J., and
Friedhoff, A. J. 1986 Diminished D2 dopamine receptor
function and the emergence of repetitive jaw movements.
Adv. Exp. Med. Biol., 235:159-167.
Roth, B. L., Hamblin, M. W., and Ciaranello, R. D. 1990
Regulation of 5-HT2 and 5-HTlc serotonin receptor
levels-methodology and mechanisms.
Neuropsychopharmacol., 3:427-433.
Roth, B. L., Hamblin, M. W., and Ciaranello, R. D. 1991
Developmental regulation of 5-HT2 and 5-HTlc mRNA and
receptor levels. Dev. Brain Res., 58:51-58.
Saleh, M. I. and Kostrzewa, R. M. 1988 Impaired striatal
dopamine receptor development: Differential D,
regulation in adults. Eur. J. Pharmacol., 154:305-311.
Savasta, M., Dubois, A., and Scatton, B. 1986
Autoradiographic localization of Dt dopamine receptors
in the rat brain with 3H-SCH 23390. Brain Res.,
375:291-301.
Schettini, G., Ventra, C., Florio, T., Grimaldi, M., Meucci,
0., and Marino, A. 1992 Modulation by GTP of basal
and agonist-stimulated striatal adenylate cyclase
activity following chronic blockade of D, and D2
dopamine receptors: Involvement of G proteins in the
118
development of receptor supersensitivity.
Neurochem., 59:1667-1674.
J.
Schulz, D. w., Wyrick, S. D., and Mailman, R. B. 1984 3HSCH 23390 has the characteristics of a dopamine
receptor ligand in the rat central nervous system.
Eur. J. Pharmacol., 106:211-212.
Seamon, K. B., Padgett, W., and Daly, J. W. 1981
Forskolin: unique diterpene activator of adenylate
cyclase in membranes and in intact cells. Proc. Natl.
Acad. Sci. USA, 78:3363-3367.
Seegmiller, J. E., Rosenbloom, F. M., and Kelley, W. N.
1967 An enzyme defect associated with a sex-linked
human neurological disorder and excessive purine
synthesis. Science, 155:1682-1684.
Seeman, P. 1980 Brain dopamine receptors.
Rev., 32:229-313.
Pharmacol.
Setler, P. E., Sarau, H. M., Zirkle, c., and Saunders, H. L.
1978 The central effects of a novel dopamine agonist.
Eur. J. Pharmacol., 50:419-430.
Sibley, D. R. and Monsma, Jr., F. J. 1992 Molecular
biology of dopamine receptors. Trends Pharmacol. Sci.,
13:61-69.
Simson, P. E., Johnson, K. B., Jurevics, H. A., Criswell, H.
E., Napier, T. c., Duncan, G. E., Mueller, R. A., and
Breese, G. R. 1992 Augmented sensitivity of D,dopamine receptor in lateral but not medial striatum
after 6-hydroxydopamine-induced lesions in the neonatal
rat. J. Pharmacol. Exp. Ther., 263:1454-1463.
Smith, R. D., Cooper, B. R., and Breese, G. R. 1973 Growth
and behaviral changes in developing rats treated
intracisternally with 6-hydroxydopamine: Evidence for
involvement of brain dopamine. J. Pharmacol. Exp.
Ther., 185:609-619.
Snyder, A. M., Zigmond, M. J., and Lund, R. D. 1986
Sprouting of serotonergic afferents into striatum after
dopamine-depleting lesions in infant rats: a retrograde
transport and immunocytochemical study. J. Comp.
Neurol., 245:274-281.
Snyder, S. H., Taylor, M., Coyle, J. T., and Meyerhoff, J.
L. 1970 The role of brain dopamine behavioral
regulation and actions of psychotropic drugs'. Am. J.
119
Psychiatr., 127:199-207.
Snyder, S. H. 1982
Schizophrenia.
Neurotransmitter and CNS disease:
Lancet, 2:970-974.
Soghomonian, J.-J., Ooucet, G., and Descarries, L. 1987
Serotonin innervation in adult rat neostriatum. I.
Quantified regional distribution. Brain Res., 425:85100 .
Sokoloff, P., Giros, B., Martres, M.-P., Bouthenet, M.-L.,
and Schwartz, J. C. 1990 Molecular cloning and
characterization of a novel dopamine receptor (D3) as a
target for neuroleptics. Nature, 347:146-151.
Sourkes, T. L. and Poirier, L. J. 1968 Serotonin and
dopamine in the extrapyramidal system. Adv.
Pharmacol., 6A:335-346.
Stachowiak, M. K., Bruno, J. P., Snyder, A. M., Strieker, E.
M., and Zigmond, M. J. 1984 Apparent sprouting of
striatal serotonergic terminals after dopaminedepleting brain lesions in neonatal rats. Brain Res.,
291:164-167.
Staunton, D. A., Wolfe, B. B., Groves, P. M., and Molinoff,
P. B. 1981 Dopamine receptor changes following
destruction of the nigrostriatal pathway: Lack of a
relationship to rotation behavior. Brain Res.,
211:315-327.
Steinbusch, H. W. M. 1981 Distribution of serotoninimmunoreactvity in the central nervous system of the
rat. Cell bodies and terminals. Neurosci., 6:557-618.
Stewart, B. R., Jenner, P., and Marsden, C. D. 1989
Induction of purposeless chewing behaviour in rats by
5-HT agonist drugs. Eur. J. Pharmacol., 162:101-107.
Stevens, J. R. 1973 An anatomy of schizophrenia ? Arch.
Gen. Psychiatry, 29:177-189.
Stoof, J. C. and Kebabian, J. W. 1984 Two dopamine
receptors: biochemistry, physiology and pharmacology.
Life Sci. 35:2281-2296.
Sunahara, R. K., Guan, H.-C., O'Dowd, B. F., Seeman, P.,
Laurier, L. G., Ng, G., George, S., Torchia, J., Van
Tol, H. H. M., and Niznik, H. B. 1991 Cloning of the
gene for a human dopamine Dj receptor with higher
affinity for dopamine than D,. Nature, 350:614-619.
120
Tarsy, D. and Baldessarini, R. J. 1974 Behavioral
supersensitivity to apomorphine following chronic
treatment with drugs which interfere with synaptic
function of catecholamines. Neuropharmacol., 13:927940.
Tassin, J. P., Cheramy, A., Blanc, 6., Thierry, A.-M., and
Glowinski, J. 1976 Topographical distribution of
dopaminergic innervation and of dopaminergic receptors
in the rat striatum, I.. Microestimation of [JH]dopamine
uptake and dopamine content in microdiscs. Brain Res.,
107:291301.
Ternaux, J. P., Hery, F., Bourgoin, S., Adrien, J.,
Glowinski, J., and Hamon, M. 1977 The topographical
distribution of serotonergic terminals in the
neostriatum of the rat and the caudate nucleus of the
rat. Brain Res., 121:311-326.
Towle, A. C., Criswell, H. E., Maynard, E. H., Lauder, J.
M., Joh, T. H., Mueller, R. A., and Breese, G. R. 1989
Serotonergic innervation of the rat caudate following a
neonatal 6-hydroxydopamine lesion: An anatomical,
biochemical and pharmacological study. Pharmacol.
Biochem. Behav., 34:367-374.
Undie, A. and Friedman, E 1990 Stimulation of a dopamine
Dt receptor enhances inositol phosphates formation in
rat brain. J. Pharmcol. Exp. Ther., 253:987-992.
Ungerstedt, U. and Arbuthnott, G. W. 1970 Quantitative
recording of rotational behavior of rats after 6hydroxydopamine lesions of the nigrostriatal dopamine
system. Brain Res., 24:485-493.
Ungerstedt, U. 1971a Striatal dopamine release after
amphetamine or nerve degeneration revealed by
rotational behavior. Acta Physiol. Scand. Suppl.,
367:49-66.
Ungerstedt, U. 1971b Postsynaptic supersensitivity after
6-hydroxydopamine induced degeneration of the
nigrostriatal dopamine system. Acta Physiol, scand.
Suppl., 367:69-93.
Ungerstedt, u. 1971c Adipsia and aphagia after 6-OHDA
induced degeneration of the nigro-striatal dopamine
system. Acta Physiol. Scand. Suppl.,367:95-122.
Uretsky, N. J. and Iversen, L. L. 1970 Effects of 6hydroxydopamine on catecholamine containing neurons in
121
the rat brain.
J. Neurochem., 17:269-278.
Van Der Kooy, D. and Hattori, T. 1980 Dorsal raphe cells
with collateral projections to the caudate-putaman and
substantia nigra: a fluorescent retrograde double
labeling study in the rat. Brain Res., 186:1-7.
Van Tol, K. H. M., Bunzow, J. R. Guan, H.-C., Sunahara, R.
K., Seeman, P., Niznik, H. B., and Civelli, o. 1991
Clonine of the gene for a human dopamine D4 receptor
with high affinity for the antipsychotic clozapine.
Nature, 350:610-614.
Verge, D., Daval, G., Marcinkiewicz, M., Patey, A., El
Mestikawy, S., Gozlan, H,, and Hamon, M. 1986
Quantitative autoradiography of multiple 5-HT, receptor
subtypes in the brain of control or 5,7dihydroxytryptamine-treated rats. J. Neurosci.,
12:3474-3482.
Voight, M. M., Laurie, D. J., Seeburg, P. H., and Bach, A.
1991 Molecular cloning and characterization of a rat
brain cDNA encoding a 5-hydroxytryptamineIB receptor.
EMBO J., 10:4017-4023.
Voorn, P., Kalsbeek, A., Jorritsma-Byham, B. and
Groenewegen, H. J. 1988 The pre- and postnatal
development of the dopaminergic cell groups in the
ventral mesencephalon and the dopaminergic innervation
of the striatum of the rat. Neurosci., 25:857-887.
Haddington, J. L. and Gamble, S. J. 1980 Emergence of
apomorphine-induced "vacuous chewing" during 6 months
continuous treatment with fluphenazine decanoate. Eur.
J. Pharmacol., 68:387-388.
Haddington, J. L. 1990 Spontaneous orofacial movements
induced in rodents by very long-term neuroleptic drug
administration: Phenomenology, pathophysiology and
putative relationship to tardive dyskinesia.
Psychopharmacol., 101:431-447.
Haeber, C., Zhang, L., and Palacios, J. M. 1990 5-HT,D
receptors in the guinea pig brain: pre- and
postsynaptic localizations in the striatonigral
pathway. Brain Res., 528:197-206.
Heinshank, R. L., Zgombick, J. M., Macchi, M. J., Branchek,
T. A., and Hartig, P. R. 1992 Human serotonin-lD
receptor is encoded by a subfamily of 2 distinct genes:
122
5-HT(lD„} and S-HT(lDj),
89:3630-3632.
Proc. Nat. Acad. Sci, USA,
Widmann, R. and Sperk, G. 1986 Topographical distribution
of amines and major amine metabolites in the rat
striatum. Brain Res., 367:244-249.
Wilson, J. M., Young, A. B., and Kelley, W. N. 1983
Hypoxanthine-guanine phosphoribosyl transferase
deficiency: The molecular basis of the clinical
syndromes. New Engl. J. Med., 309:900-910.
Zeng, W., Hyttel, J., and Murrin, L. C. 1988 Ontogeny of
dopamine D, receptors in rat striatum. J. Neurochem.,
50:862-867.
Zhou, F. C., Bledsoe, S., and Murphy, J. 1991 Serotonergic
sprouting is induced by dopamine-lesion in substantia
nigra of adult rat brain. Brain Res., 556:108-116.
Zhou, Q. Y., Grandy, 0. K., Thambi, L., Kushner, J. A., Van
Tol, H. H. M,, Cone, R., Pribnow, D., Salon, J.,
Bunzow, J. R., and Civelli, 0. 1990 cloning and
expression of human and rat D, dopamine receptors.
Nature, 347:76-80.
Zigmond, M. J. and Strieker, E. M. 1973 Recovery of
feeding and drinking by rats after intraventricular 6hydroxydopamine or later hypothalamus lesion. Science,
182:717-719.
Zilles, K., Rath, M., Schleicher, A., Glaser, T., and
Traber, J. 1986 Ontogenesis of serotonin (5-HT)
binding sites in the choroid plexus of the rat brain.
Brain Res., 380:201-203.
VITA
Li Gong
Personal Data:
Date of Birth:
Place of Birth:
Marital Status:
Education:
Shanghai Medical University, Shanghai, China;
Medicine, Bachelor of Medicine, 1984.
East Tennessee State University, Johnson City,
Tennessee; Biomedical Science, M.S., 1990.
East Tennessee State University, Johnson City,
Tennessee; Pharmacology, Ph.D., 1993.
Professional
Experience:
Resident physician, Shanghai Cancer Hospital,
Shanghai, China, 1984-1987.
Graduate research assistant, Department of
Pharmacology, James H. Quillen College of
Medicine, ETSU, Johnson City, Tennessee,
1988-present.
Publications:
Papers:
September 7, 1960
Shanghai, China
Single
Gong, L., Daigneault, E.A., Acuff, R.V. and
Kostrzewa, R.M. Vitamin E supplements fail to
protect from acute MPTP neurotoxicity in mice.
NeuroReport, 2:544-546, 1991.
Kostrzewa, R.M. and Gong, L. Supersensitized Dl
receptors mediate enhanced oral activity after
neonatal 6-OHDA. Pharmacol. Biochem. Behav.,
39:677-682, 1991.
Gong, L. and Kostrzewa, R.M. Supersensitized
oral responses to a serotonin agonist in
neonatal 6-OHDA-treated rats. Pharmacol.
Biochem. Behav., 41:621-623, 1992.
Chi, D.S., Gong, L., Daigneault, E.A. and
Kostrzewa, R.M. Effects of MPTP and vitamin
E treatments on immune function in mice. Int.
J. Immunopharmacol., 14:739-746, 1992.
Gong, L., Kostrzewa, R.M. and Kalbfleisch, J.H.
MIF-1 fails to modify agonist-induced oral
activity in neonatal 6-OHDA-treated rats.
Peptides, 1993 (in press).
Gong, L., Kostrzewa, R.M., Perry, K.W. and
Fuller, R.W. Dose-related effects of a
neonatal 6-OHDA lesion on SKF 38393- and m123
124
chlorophenylpiperazine-induced oral activity
responses of rats. Dev. Brain Res., 1993 (in
press).
Abstracts:
Gong, L., Chi, D.S., Daigneault, E.A. and
Kostrzewa, R.M. Effects of MPTP and vitamin E
on interleukin-2 (IL-2) production in mice.
14th Society for Experimental Biology and
Medicine, Southeastern Section Meeting,
Johnson City, TN, 1989; Proc. Soc. Exp. Biol.
Med., 194:387, 1990.
Gong, L., Daigneault, E.A., Chi, D.S., Acuff,
R.V. and Kostrzewa, R.M. The effectiveness of
neurotoxin MPTP in mice treated with atocopherol. Joint llth Southeastern
Pharmacology Society and American College of
Clinical Pharmacology Meeting, Johnson City,
TN, 1990; J. Clin. Pharmacol.,31:276, 1991.
Gong, L., Daigneault, E.A. and Kostrzewa, R.M.
Mediation of enhanced SKF 38393-induced oral
activity by a serotonin system. 21st Society
for Neuroscience Meeting, New Orleans, LA,
Nov. 1991.
Gong, L., Kostrzewa, R.M., Fuller, R.W. and
Perry, K.W. Ontogenetic supersensitization of
dopamine D1 receptors. 13th Southeastern
Pharmacology Society Meeting, Charleston, SC,
September 1992.
Gong, L., Kostrzewa, R.M., Brus, R., Daigneault,
E.A., Fuller, R.W. and Perry, K.W.
Ontogenetic supersensitization of DA D1
receptors occurs without a change in D1 high
affinity sites. 23rd Society for
Neuroscience Meeting, Washington, D.C. Nov.,
1993.
Honors: Outstanding academic achievements, ETSU, 1987-1988; 19901991; 1991-1992; 1992-1993.
Society: Southeastern Pharmacology Society, student
member.