JOURNALOF NEUROPHYSIOLOGY
Vol. 70, No. 6, December 1993. Printed
in U.S.A.
Cholinergic Modulation
GijTZ
BRAUN
AND
BRIAN
of the Swimmeret Motor System in Crayfish
MULLONEY
Section of Neurobiology, Physiology and Behavior, and Neuroscience Doctoral Program,
Davis, California 95616
SUMMARY
AND
CONCLUSIONS
1. The muscarinic agonist pilocarpine induced the swimmeret
motor pattern in resting isolated preparations of the crayfish abdominal nerve cord and modulated the burst frequency in a dosedependent manner.
2. Nicotine did not elicit rhythmic activity in resting isolated
preparations but increased the burst frequency in active preparations. Nicotine produced higher burst frequencies than pilocarpine.
3. The acetylcholine ( ACh) analogue carbachol combined the
effects of pilocarpine
and nicotine. It activated isolated resting
preparations
and increased the burst frequency as effectively as
nicotine. The ACh-esterase inhibitor
eserine also increased the
burst frequency in active preparations.
4. Neither muscarinic nor nicotinic antagonists disrupted the
proctolin-induced
motor pattern, suggesting that proctolin and
cholinergic agonists affect two different pathways for the activation of the swimmeret system.
5. We conclude that cholinergic interneurons participate in initiation of the swimmeret motor pattern and can modulate its burst
frequency.
INTRODUCTION
The swimmerets are paired appendages on the abdominal segments of crayfish and other decapod crustaceans. In
the intact animal the swimmerets beat in a bilaterally synchronous metachronal wave that propagates caudally to
rostrally. These behavioral features are paralleled by the
activity of motor neurons innervating the swimmeret muscles. In the isolated abdominal nerve cord there are alternating bursts of return-stroke (RS) motor neurons and powerstroke (PS) motor neurons in the anterior and posterior
branches of the nerve Nl that innervates the swimmeret.
These bursts are bilaterally synchronous in each pair of N 1s
and are coordinated in a metachronal sequence from cauda1 to rostra1 ganglia (Hughes and Wiersma 1960). This
pattern can occur spontaneously, or can be induced by stimulation of axons in the connectives between the last thoracic and first abdominal ganglia (Wiersma and Ikeda
1964). Pharmacologically,
the swimmeret motor pattern
can be induced by application of the peptide proctolin
( Mulloney et al. 1987 ) . However, proctolin-induced
patterns display only a narrow range of burst frequencies
(Mulloney et al. 1987)) whereas the stimulation of axons in
the connective between the first and second abdominal ganglion in the lobster induces a broader range of burst frequencies (Davis and Kennedy 1972).
Acetylcholine (ACh) occurs as a neurotransmitter
in
arthropods, including crustaceans (Barker et al. 1972;
Florey 1973; Marder 1976), and receptors similar to vertebrate nicotinic and muscarinic receptors have been de-
University of California,
scribed pharmacologically
(Barker et al. 1986; Florey and
Rathmayer 1980; Freschi 199 1; Marder and PaupardinTritsch 1978; Pfeiffer-Lynn and Glantz 1990). Recently it
has been shown that the muscarinic agonist oxotremorine
also induces expression of the swimmeret motor pattern in
isolated crayfish abdominal nerve cords (Chrachri and Neil
1993). In contrast to proctolin, oxotremorine modulated
the burst frequency in a dose-dependent manner (Chrachri
and Neil 1993).
The ability of oxotremorine to modulate burst frequency
motivated us to investigate the roles of muscarinic and nicotinic receptors in initiation and modulation
of the swimmeret rhythm.
METHODS
Crayfish (Pac$zstacusleniusculus)of both sexes were obtained
from local fisherman and kept in freshwater tanks at 15OC.
Preparation
Before dissection, animals were cooled on ice. The ventral nerve
cord with the abdominal ganglia l-6 (A 1-A6) was dissected free
and pinned out in a Sylgard-coated dish containing modified van
Harreveld’s crayfish saline (in mM: 195 NaCl, 5.36 KCl, 2.6
MgCl, , 13.5 CaCl, , and 10 tris ( hydroxymethyl ) aminomethanemaleate buffer, pH 7.4). The sheath around the ganglia was removed to facilitate the diffusion of solutions into the tissue.
Extracellular
recordings
Each swimmeret is innervated by one nerve, Nl, which splits
into an anterior and a posterior branch. The axons of RS motor
neurons exit through the anterior branch; those of the PS motor
neurons exit through the posterior branch (Mulloney
et al. 1990;
Stein 197 1). Action potentials of both motor neuron populations
were recorded separately with extracellular Vaseline-insulated
pin
electrodes. Signals were amplified and stored on videotape for
later analysis.
Intracellular
recordings
Intracellular
recordings were made using conventional
techniques and equipment. Glass microelectrodes
were pulled (Sutter
Instruments) and filled with 3 M KCl. Their resistances were 1520 MQ. Signals were amplified (Axoclamp
2A, Axon Instruments) and stored on tape for later analysis.
Drugs and solutions
All drugs were purchased from Sigma and dissolved in-saline.
Drugs were added to the bath solution and washed out by superfusion with saline. When a series of concentration
steps was performed, increments in concentration
were separated by 5 min.
Between applications
of different drugs all preparations
were
washed in saline for r 1 h.
0022-3077/93 $2.00 Copyright 0 1993 The American Physiological Society
2391
2392
G. BRAUN
1. saline
es3_....
AND
B. MULLONEY
5. 1 h in saline
~
I
Is
PS4
9. IOpM proctolin
+ 1 OpM pilocarpine
2. IOpM
pilocarpine
(1.44
s-l)
3. 3OpM
pilocarpine
(2.14
s-l)
II1~1
I/(
’ 1’
4. IOOpM
’ /““i
‘/ !
pilocarpine
6. 4pM proctolin
I
7. 1 OJIM proctolin
(I .82 s-1)
(I .41 s-1)
(I .82 s-1)
10. + 3OpM
pilocarpine
(2.09
s-1)
1’
i
(2.31
s-1)
8. 5OpM proctolin (1.82
,
I
s-1)
11. + 1 OOpM pilocarpine
(I .89 s-1)
FIG. 1. Activity
patterns induced by pilocarpine
and by proctolin
in 1 isolated nerve cord. l-4: initiation
of the swimmeret motor pattern by pilocarpine.
Burst frequency
increased with the concentration
of pilocarpine.
5: after l-h wash in
saline, the preparation
returned
to a resting state. 6-8: different
concentrations
of proctolin
induced a smaller increase in
burst frequency
and generated bursts that differed in shape from the pilocarpine-induced
burst. 9- 11: after 1-h wash in saline
(not shown), the preparation
was exposed to different pilocarpine
concentrations
in the presence of 10 ,uM proctolin.
The
increase in burst frequency
was smaller than in pilocarpine
alone and bursts had a different
shape than in either pilocarpine
or proctolin
alone. Arrowheads:
set of large spikes that were absent from the pilocarpineand proctolin-induced
bursts [ PS3,
PS4: extracellular
recordings
of power-stroke
motor neurons in ganglia A3 and A4. The burst frequency
of the respective
pattern is shown in parentheses].
Data analysis
Burst frequency and phase were calculated from 20-s intervals
where the pattern showed little variation. The motor pattern was
normally very regular, so that in single preparations the SE of the
mean of the periods were 4% of the mean. Recording traces were
digitized and analyzed on a PC with software described elsewhere
(Mulloney and Hall 1987). Data were evaluated with t test statistics. For multigroup comparisons an analysis of variance and Student-Newman-Keuls
test were performed.
RESULTS
Pilocarpine initiated the swimmeret motor pattern in
isolated nerve cords and modulated the burst frequency
In resting preparations (i.e., there was either no or only a
little tonic firing of motor neurons observed in extracellular
recordings), pilocarpine elicited a swimmeret motor pattern at concentrations as low as 10 PM within 0.2- 1 min
after application to the bath (Fig. 1, l-2). Resting preparations either began to express intermittent
bouts of coordinated activity and then settled down to produce a continu-
ous pattern or began at once to express a continuous pattern. The periods and phases during intermittent
bouts
were in the same ranges as those observed during subsequent continuous activity. Pilocarpine concentrations < 10
,uM did not activate resting preparations (n = 3). With increasing doses of pilocarpine the burst frequency increased
(Fig. 1, 2-4; for a quantitative description on the modulation of burst frequency, see below). Superfusion with saline
stopped the rhythmic activity within a few minutes (not
shown).
Subsequent application of proctolin to the same preparations again activated the motor pattern, but the structure of
motor neuron bursts appeared to be different from those
during the pilocarpine-induced
activity (Fig. 1, 6). An increase in the proctolin concentration increased the burst
frequency less than pilocarpine (Fig. 1, 6-8 ) . When different pilocarpine concentrations were applied in the presence
of proctolin the burst frequency was lower than in pilocarpine alone, and the structure of bursts was different from
both the pilocarpine- and the proctolin-activated
patterns
(Fig. 1, 9-11).
CHOLINERGIC
MODULATION
Differences in the burst structure of motor patterns induced by different agonists and combinations
of agonists
were a consistent feature throughout this study. Basically,
the appearance of a burst could change 1) when one or
more units changed their intraburst firing frequency or 2)
when different or additional motor units were recruited.
We did not try systematically to discriminate between these
two possibilities. However, Fig. 1, 9-l 1, provides a clear
example of the additional recruitment of one or more motor units that were seen only in the presence of pilocarpine
plus proctolin but not with either drug alone.
Nicotine
cords
modulated
OF
MOTOR
ACTIVITY
2393
A
1. 1 OpM
proctolin
1. 1 O!M
I, II
PS 3 *
proctolin
I
PS 4
2.
+ 0.25pM
nicotine
2. +
IpM
carbachol
the burst frequency in isolated nerve
Nicotine, when applied to a resting preparation, did not
elicit a coordinated swimmeret
motor pattern but caused
only tonic activity in some motor neurons (not shown).
However, when added to an already active preparation, it
increased the burst frequency in a dose-dependent manner
(Fig. 2, A I-&).
The threshold for detectable effects of nicotine on the burst frequency in active preparations was -0.1
FM (not shown). Nicotine also changed the structure of
single bursts (Fig. 2A) but these changes were not consistent over the whole range of concentrations
tested. Lower
concentrations
(52 ,uM, Fig. 2, A2 and A3) increased the
number of larger motor neuron spikes, whereas higher concentration decreased the number of smaller spikes (Fig. 2,
n4 and A.5). At concentrations
>3 ,uM the motor pattern
became unstable (Fig. 2A5) and finally deteriorated into
tonic activity (not shown).
3. +
4.
IpM
nicotine
3. 1 h in saline
+ 3pM
nicotine
4. IpM
carbachol
tt-,
5. + ~,YM nicotine
5. IOpM
carbachol
Carbachol initiated the swimmeret motor pattern and
modulated the burstfiequency in isolated nerve cords
In a few experiments we applied ACh. However, doses
> 100 ,uM were necessary to activate resting preparations,
and the resulting changes were quite unstable over time
(not shown). Also, in a previous study, Mulloney et al.
( 1987) were unable to observe consistent effects of ACh
superfusion. We attribute this instability to the breakdown
of ACh by ACh esterases ( AChE; Maynard 197 1). Therefore we employed the cholinergic
analogue carbachol,
which acts on both nicotinic and muscarinic receptors and
is not affected by enzymatic breakdown.
Carbachol increased the burst frequency in preparations
already expressing the swimmeret motor pattern (Fig. 2, Bl
and B2). Carbachol also could initiate the swimmeret motor pattern in resting preparations with a threshold concentration - 1 ,uM (Fig. 2 B4). Higher doses led to an increase
in burst frequency and to changes in burst shape (Fig.
2 B.5). At concentrations
> 10 PM the pattern became more
likely to deteriorate (not shown).
Eficts ofnicotine
on individual
motor neurons
Nicotine not only modulated the burst frequency but of
all cholinergic agonists had the most variable effects on
burst structure. A small set of experiments (n = 4) with
intracellular recordings revealed several nicotine-induced
effects on PS motor neurons (Fig. 3).
In the first example, a PS motor neuron had displayed
large oscillations and generated phase-locked spikes during
FIG. 2. Activity
patterns induced by nicotine,
carbachol,
and proctolin. A : burst frequency
of a motor pattern activated by 10 PM proctolin
(I)
was increased by addition
(+) of nicotine.
At 0.25- 1 PM (2 and 3) the
burst shape indicated
the activation
of additional
motor units; at higher
concentrations
the number of active units decreased (4). With 4 PM nicotine, the pattern deteriorated
(5). B: burst frequency
of a motor pattern
activated by 10 FM proctolin
(I) was increased by addition of 1 PM carbachol(2).
After 1 -h wash in saline (3) the preparation
returned
to a resting
state and could be activated by 1 PM carbachol
(4). Increasing
the carbachol concentration
also increased the burst frequency
(5). (PS 3, PS 4:
extracellular
recordings
from PS motor neurons in A3 and A4. Different
preparations
in A and B).
rhythmic activity induced by proctolin alone (Fig. 3A).
Application of 1 ,uM nicotine increased the frequency of the
oscillations but caused only a small change in the average
membrane potential (Fig. 3A). Increasing the nicotine
concentration
to 2 ,uM further increased the frequency but
decreased the number of spikes (Fig. 3A).
Another PS motor neuron displayed only small oscillations with no spikes in the presence of proctolin alone (Fig.
3 B). Adding 1 ,uM nicotine depolarized the cell by - 15
mV, increased oscillation amplitude and frequency, and induced phase-locked action potentials (Fig. 3 B). After a l-h
wash, application of 10 FM proctolin together with 1.5 FM
nicotine caused phase-locked oscillations at a membrane
2394
G. BRAUN
AND
B MULLONEY
‘.
A
RS 3i
+ IpM nicotine
(1 .96s-1)
+ 2pM nicotine
(2.64s-1)
IS
R
II
RS 4J
PS MN
-66
l%%%L,L
,F&
1
mV’
proctolin
(1 .76s-1)
+ IpM nicotine
(2.33s-1)
+ 1.5pM
(3.49s~1)
nicotine
7,
,y
,3 L
-2.2nA
FIG. 3. Effects of nicotine
on individual
motor neurons. A: PS motor neuron in A3 showed rhythmic
oscillation
with
phase-locked
spikes during proctolin-induced
activity. With the addition of 1 PM nicotine,
the membrane
potential
depolarized slightly and phase-locked
spikes were still present. Increasing the nicotine concentration
to 2 FM depolarized
more and
reduced the number of spikes per burst. B: PS motor neuron in A4 showed only small oscillations
during proctolin-induced
activity. Adding 1 ,uM nicotine depolarized
the cell by 15 mV and produced
phase-locked
bursts of spikes. After washing, 10
PM proctolin
plus 1.5 PM nicotine
depolarized
the cell by 20 mV. The membrane
potential
still oscillated but no spikes
occurred.
Hyperpolarizing
pulses generated
rebound spikes that were reduced in number when release from hyperpolarization was out-of-phase
with the PS bursts. (PS 3, PS 4, RS 3, RS4, extracellular
recordings;
PS MN, intracellular
recording
from PS motor neuron in the same ganglion. Burst frequency
of the respective
pattern is shown in parentheses.)
potential more depolarized than during previous nicotine
application, and action potentials were absent (Fig. 3B).
The absence of spikes was probably due to adaptation
caused by the strong depolarization; after pulses of hyperpolarizing current the neuron generated rebound spikes,
which were reduced in number when the release from hyperpolarization occurred out-of-phase with the other PS motor
neurons (Fig. 3 B).
The accompanying extracellular recordings ( Fig. 3, A
and B) again illustrate the changes in burst structure already mentioned.
Eserine increased the burst frequency of the swimmeret
motor pattern in isolated nerve cords
The AChE inhibitor eserine also increased burst frequency when applied to preparations already activated by
proctolin (n = 4, Fig. 4). The onset of the response was later
than with cholinergic receptor agonists (> 1 min). Eserine
also prolonged the time required to terminate rhythmic activity using saline wash (Fig. 4), presumably because of the
different kinetics involved in enzyme-inhibitor
interaction
as opposed to receptor-ligand interaction.
In resting preparations, eserine had no effect (not
shown). This suggests that eserine enhanced ongoing cholinergic neurotransmission
during the proctolin-activated
state. A detailed dose-response relationship was not established but doses from 1 to 10 ,uM increased the burst frequency, whereas higher doses disrupted the rhythm, as with
nicotine and carbachol (not shown).
Eficts
ofcholinergic
agonists on burst frequency
During the present study, proctolin activated the motor
pattern only within a small frequency range. In 24 preparations activated by 10 PM proctolin, frequencies ranged
from 0.9 to 2.22 Hz, distributed around 1.58 per second
(Fig. 5A). We did not test other concentrations because
previous experiments ( Mulloney et al. 1987 ) indicated that
1 OpM proctolin
1 .92 s-1
+ 5,uM eserine
3.57
s-1
20 min
i.n saline
2.23 s-1
Is
FIG. 4. Eserine
increased the frequency
of motor neuron
bursts induced by 10 FM proctolin.
After 20-min wash in saline the motor pattern
persisted but the burst frequency
had decreased.
CHOLINERGIC
MODULATION
OF
[pilocarpine]
1 OuM
proctolin
0
MOTOR
uM
2395
ACTIVITY
rI
c
20406080100
0
I
-I
I
1 .o 1.2
burst
1.5
frequency
1.8
0246810
‘g
n=
4
4
6
5
L
l
0
0
[nicotine]
proctolin
pilocarpine
proctolin
uM
+ nicotine
[]
pilocarpine
+ pilocarpine
m
m
proctolin
carbachol
F7A proctolin
+ pilocarpine
+ nicotine
FIG. 5. Effects of proctolin and cholinergic drugs on burst frequency in isolated nerve cords. A : distribution of mean
burst frequencies induced by proctolin in 24 preparations. Bin width is 0.26 Hz, lowest frequency was 0.90 Hz, highest was
2.22 Hz. B: dose-frequency relations. For pilocarpine and proctolin plus pilocarpine n = 4; for proctolin plus nicotine n is
indicated in the figure because not all animals were tested at every concentration. Error bars: mean + SE. C: maximum
frequencies. Error bars: mean + SE. Dots: highest burst frequency ( * : P < 0.05, Student-Newman-Keuls test).
concentrations > 10 ,uM did not further increase the burst
frequency (see also Fig. 1, 7 and 8).
In contrast, all cholinergic agonists seemed to have dosedependent effects on the burst frequency. We established
dose-response curves for the burst frequency activated by
1) pilocarpine alone, 2) pilocarpine in the presence of 10
PM proctolin, and 3) nicotine in the presence of 10 FM
proctolin. The first and second experiments were performed with the same groups of animals; the third was obtained from a separate set. Concentrations were increased
every 5 min; changes in frequency were usually observed
within 1 min after application of a higher concentration.
Only preparations showing a stable swimmeret pattern
were evaluated.
With pilocarpine alone, the burst frequency increased up
to a concentration of 50 PM (~2 = 4, maximum burst frequency = 2.42 t 0.16 (SE) Hz; Fig. 5, B and C). The highest burst frequency reached in one preparation was 2.89 Hz.
When the same concentrations of pilocarpine were applied
in the presence of 10 FM proctolin, the increase in burst
frequency was attenuated (n = 4, maximum
burst frequency = 2.19 t 0.06 Hz; Fig. 5, B and C).
The effect of nicotine was examined in six preparations
activated with 10 PM proctolin. With increasing nicotine
doses between 0.25 and 3 PM the burst frequency increased
rapidly (n = 6, maximum burst frequency = 2.97 t 0.15
Hz; Fig. 5, B and C) . At higher concentrations the number
of preparations still showing patterned activity decreased.
The highest burst frequency reached was 3.49 Hz. Not all
preparations were tested with every concentration.
Because the maximum frequency, especially in the case
of nicotine, was not always reached at the highest concentration, we compared the maximum
burst frequency
evoked by the different drugs irrespective of the drug concentration (Fig. 5C). Pilocarpine alone was as effective as
pilocarpine plus proctolin (Fig. 5C), whereas nicotine in
the presence of proctolin was more effective than pilocarpine (P < 0.05, Student-Newman-Keuls
test; Fig. 5C).
We also compared the results obtained from five preparations that were exposed to 10 FM carbachol. The pattern
elicited by 10 PM carbachol had a frequency as high as that
elicited by proctolin plus nicotine, significantly greater than
the pilocarpine-induced
frequency (maximum
burst frequency = 3.12 t 0.16 Hz; P < 0.05, Student-NewmanKeuls test; Fig. 5C). In carbachol the highest burst frequency obtained was 3.64 Hz.
Independent of the pharmacological treatment and burst
frequency, the phase relation between ganglia remained relatively constant. Figure 6 shows the phase of PS bursts in
ganglion A4 relative to A5 in three experiments where different burst frequencies were obtained by varying the concentration of either pilocarpine, nicotine in the presence of
proctolin, or carbachol. The phases were within the range
observed in spontaneous, “command-driven,”
and proctolin-induced motor patterns described in a previous study
( Acevedo 1990).
Muscarinic antagonists did not disrupt proctolin-induced
activity
How does the pattern induced by cholinergic drugs relate
to that induced by proctolin? Can the proctolin-induced
pattern persist in the presence of cholinergic antagonists?
The muscarinic antagonists scopolamine and atropine
reliably blocked activity induced by carbachol or pilocarpine (n = 5). Whereas scopolamine was effective at doses as
low as 1 PM, > 10 PM atropine was necessary to block
rhythmic activity. Figure 7, 1 and 2, shows the blocking of
activity in 2 PM carbachol by 1 PM scopolamine. Washes of
0
0
g
zi 0.4 0.2 -
pilocarpine
nicotine
+ proctolin
carbachol
0
V
00
@ ov
V
00’
.
0.5
1.0
1.5
frequency-s
2.0
2.5
3.0
-1
6. Phase of PS motor neuron bursts in A4 relative to A5 did not
change with frequency or drug. Three different experiments with carbachol ( 1, 2, 4, 5, 7, and 10 PM), .pilocarpine ( 10, 20, and 70 PM), and 10
PM proctolin with nicotine (0.25, 0.5, an.d 1 PM).
FIG.
G. BRAUN AND B.MULLONEY
2396
1. 2pM
carbachol
4. 1OpM
proctolin
I
ps3
--w++++**+*
i
PS4
I
m
2. + 1pM
scopolamine
5. +
1OOpM
scopolamine
-P: :; : : :;
3. 2pM
carbachol
FIG. 7. Muscarinic
antagonists
did not block proctolin-induced
activity. Activity
induced by 2 PM carbachol
( 1) was blocked by 1 PM scopolamine within 2 min (2). After 2-h wash, activity was restored with carbachol ( 3 ) . Activity induced by 10 ,uM proctolin
(4) was not blocked by 100
PM scopolamine
( 5). The burst frequency
increased in the presence of
scopolamine.
l-2 h were required to restore the pattern in carbachol
again (Fig. 7, 3). Subsequent activity induced by 10 PM
proctolin persisted in the presence of 100 ,uM scopolamine
(Fig. 7,4 and 5). Also the simultaneous application of scopolamine (50 PM) and the nicotinic antagonist mecamylamine in concentrations as high as 50 PM did not interrupt
the proctolin-induced
activity (n = 2, not shown). Therefore the expression of a proctolin-induced
swimmeret motor pattern does not depend on cholinergic neurotransmission. However, during application of atropine or scopolamine the proctolin-induced
burst frequency increased from
1.26&0.13Hzto
1.51 &0.17Hz(n=4;P<O.Q5,pairedt
test). After washing the frequency decreased to the initial
level [ 1.3 t 0.15 Hz]. Application of mecamylamine
in
concentrations 550 PM had no effect on the burst frequency (n = 3, not shown).
DISCUSSION
Initiation of the motor pattern and modulation
frequency
of burst
Our results demonstrate that cholinergic agonists can activate the swimmeret motor pattern and modulate the burst
frequency in a dose-dependent manner (Figs. l-5 ). In the
swimmeret system, activation of the motor pattern by the
muscarinic agonist oxotremorine
has been reported recently (Chrachri and Neil 1993). We confirmed and extended these findings with the use of a different muscarinic
agonist (Figs. 1 and 5 ). Muscarinic induction of patterned
activity has also been found in other arthropod locomotor
systems including crayfish thoracic ganglia (Chrachri and
Clarac 1990), lobster stomatogastric ganglion ( Elson and
Selverston 1992; Marder and Eisen 1984), and locust thoracic ganglia (Ryckebusch and Laurent 199 1) , indicating a
widespread role for muscarinic receptors in activation of
motor systems throughout the arthropods.
An important finding in the present study is that nicotinic receptors also seem to be involved in the frequency
modulation of the motor pattern. Nicotine enhanced the
burst frequency more effectively than pilocarpine (Fig.
SC). Carbachol, which affects both muscarinic and nicotinic receptors, both activated the motor pattern and modulated its burst frequency as effectively as nicotine (Figs. 2B
and 5C). With maximum
burst frequencies > 3 Hz (Fig.
5C), nicotine and carbachol came close to the frequency
range observed in intact crayfish (unpublished results) and
the burst frequencies obtained by stimulation of axons in
the connectives between the first and second abdominal
ganglia in the nerve cord of the lobster (Davis and Kennedy
1972). The motor neuron bursts in different ganglia occurred at constant phase at different frequencies, a prerequisite for the maintenance of coordinated swimmeret beating
(Fig. 6).
Relation
ofmuscarinic
and nicotinic receptor activation
Pilocarpine elicited and maintained a stable motor pattern in a wide range of concentrations (Figs. 1 and 5 B). In
contrast, the modulatory effect of nicotine was limited to a
narrow concentration range (Figs. 2A and 5 B). At higher
concentrations nicotine disrupted the rhythm, and it produced only tonic activity in resting preparations (see RESULTS). A similar dualism of muscarinic and nicotinic effects has been reported for the pyloric rhythm in the foregut
of crabs (Marder and Meyrand 1989). Marder and Meyrand ( 1989) explain this by the different mode of action of
muscarinic and nicotinic agonists: pilocarpine activates
slow, voltage-dependent conductances (Freschi and Livengood 1989 ) , whereas nicotine activates “classical” conductances that depolarize the membrane irrespective of the
state of individual neurons and thus tends to disrupt rhythmic activity, especially at high concentrations. This explanation might also apply to the results reported here. However,
the application of agonists to an in vitro preparation only
poorly reflects the spatial and temporal patterning of nervous activity in vivo. It seems probable that ACh released
by neurons in the crayfish swimmeret system acts on muscarinic and nicotinic receptors and thus can initiate the motor
program and modulate its frequency.
Intracellular recordings showed that different swimmeret
motor neurons can respond differently to similar doses of
nicotine (Fig. 3, A and B). Given the small numbers of
neurons tested, we cannot say whether the motor neuron
pool can be divided into distinct groups with a different
sensitivity to nicotine. However, the example in Fig. 3 B,
which shows strong depolarization and changes in the number of spikes per cycle, suggests that this motor neuron
could receive cholinergic inputs other than from the central
pattern-generating circuit. In motor neurons like this, cholinergic sensory feedback or descending pathways (Davis
1968, 1969) could exert correctional action that would still
be phase-locked with the ongoing motor pattern.
Although we cannot rule out the idea that some of the
observed effects of cholinergic drugs might reflect the influence of sensory feedback, the activation of resting preparations by muscarinic drugs is not what would be expected of
sensory afferents providing information to central circuits. -+
CHOLINERGIC
MODULATION
Sensory afferents are commonly modulated by the pattern-renerating network itself to provide phase-locked feedback
kithout disruption of the ongoing activity (Clarac et al.
1992) and central modulation of sensory afferents does occur in the crayfish swimmeret system (Paul 1989). Moreover, eserine increased the burst frequency in active preparations, probably by enhancing cholinergic transmission
through inhibition of AChE (Fig. 4). Because there was no
sensory feedback present in isolated nerve cords and the
terminals of sensory afferents were not active, this also suggests that the observed effects of cholinergic drugs reflect
the activity of central cholinergic interneurons.
Relation ofcholinergic
motor pattern
and proctolinergic
excitation of the
Previously, application of the peptide proctolin was the
only pharmacological way to activate the swimmeret motor
pattern in vitro (Mulloney et al. 1987 ) . At least three pairs
of descending interneurons, probably originating in the subesophageal ganglion, were shown to be immunoreactive
for
proctolin, and proctolin is released when some of these
axons are stimulated ( Acevedo 1990). Thus the application
of proctolin in vitro is likely to mimic the activation of the
abdominal motor system by higher centers. Neither muscarinic nor nicotinic antagonists blocked the proctolin-induced activity (Fig. 7, and see RESULTS).
This suggests that
proctolinergic and cholinergic activation of the swimmeret
motor system mimic the effects of two different pathways
capable of activating the system. Studies on cardiac motor
neurons in lobster showed that proctolin and muscarinic
agonists elicit quite similar sodium inward currents, although they act on different receptors (Freschi 1989; Freschi and Livengood 1989). Therefore it is conceivable that
proctolinergic and cholinergic pathways might to some extent converge on common targets in the neural circuitry
that produces the swimmeret motor pattern.
Although cholinergic neurotransmission is not necessary
for the expression of the proctolin-induced
motor pattern,
it seems to be involved since the AChE-inhibitor
eserine
increased the burst frequency in proctolin-activated
preparations (Fig. 4). Eserine had no effect on resting preparations (see REsuLTs), so its effects on active preparations
imply the activity of cholinergic neurons in the pattern-generating or coordinating circuits (Mulloney et al. 1993; Murchison et al. 1993 ) , whose effects were enhanced by inhibiting AChE.
A second inference of cholinergic activity in the swimmeret circuits follows from the observations that muscarinic antagonists increased the proctolin-induced
burst frequency (Fig. 7) and that the effects of simultaneous application of proctolin and pilocarpine were not additive;
rather, proctolin slightly decreased the burst frequency
caused by pilocarpine alone (Fig. 5, B and C) . It is conceivable that in the proctolin-activated
state the application of
muscarinic antagonists abolishes hidden cholinergic transmission and thus increases the burst frequency. The nicotinic antagonist mecamylamine
had no detectable effect on
the frequency of the proctolin-induced
pattern (see RESULTS),
which suggests that nicotinic receptors either play a
negligible role during low levels of activitv or that the appli-
OF
MOTOR
ACTIVITY
2397
cation of nicotine activated elements in the circuitry that
are not active in the presence of proctolin alone.
Because phasic sensory feedback was absent from the
deafferented preparations, we think that the muscarinic
pharmacology reflects the presence of cholinergic interneurons in the swimmeret system that can initiate and modulate the expression of the swimmeret motor pattern. Because our knowledge about central cholinergic neurons in
arthropods is scarce, a future investigation of the physiology of cholinergic neurotransmission
will be of broad interest.
We thank D. Murchison,
C. Sherff, and K. Sigvardt for reviewing
earlier
drafts of this manuscript
and W. Hall for help with the illustrations.
This work was supported
by National
Science Foundation
Grants BNS
87-19397
and IBN 92-22470
to B. Mulloney.
Address for reprint
requests:
B. Mulloney,
2320 Storer Hall, UCD,
Davis CA 956 16.
Received
5 March
1993; accepted
in final
form
13 July
1993.
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