humoral induction of pyloric rhythmic output in … · (pd) motor neurones, cl muscles by the...

J. exp. Biol. 163, 209-230 (1992) 2 0 9Printed in Great Britain © The Company of Biologists Limited 1992

HUMORAL INDUCTION OF PYLORIC RHYTHMIC OUTPUTIN LOBSTER STOMATOGASTRIC GANGLION: IN VIVO AND

IN VITRO STUDIES

BY ELIZABETH REZER AND MAURICE MOULINS

Laboratoire de Neurobiologie et Physiologie Comparees,Universite de Bordeaux I, CNRS, Place Peyneau, 33120 Arcachon, France

Accepted 29 October 1991

Summary

In the lobster Jasus lalandii, 14 neurones of the stomatogastric ganglion (STG)are organized in a network that produces rhythmic pyloric outputs. In vitroexperiments have shown that the STG neurones receive, via the stomatogastricnerve (stn), neuromodulatory inputs that influence the expression of the burstingproperties of the neurones and the ability of the network to produce its rhythmicoutput.

In contrast to these in vitro observations, in vivo transection of the stn does notabolish the pyloric rhythm. Rhythmic output can be recorded by electromyogra-phy immediately after stn transection and for up to 2 years afterwards. We haveshown that, under these experimental conditions, the STG appears to be isolatedfrom any neuronal input that might account for the maintenance of the rhythmicoutput. Experiments carried out in the 2 days after stn transection showed that anin vitro preparation of the isolated STG was unable to produce any rhythmicoutput, but blood serum added to the system could restore the pyloric output.

These results suggest strongly that the pyloric network receives neural andhumoral modulatory influences in parallel and that each type of influence alone isable to maintain the bursting capability of the pyloric neurones.

Introduction

Central pattern generators (CPGs) are networks of neurones that generate thetiming and phasing cues for rhythmic movements (Delcomyn, 1980; Grillner,1977). The neuronal characteristics and the synaptic connectivity that underlie theendogenous rhythmicity of these networks have been investigated in severalpreparations, but relatively complete analysis has been achieved in only a fewsimpler invertebrate circuits (Roberts and Roberts, 1983; Selverston and Moulins,1985; Getting, 1988). Moreover, recent studies have shown that these circuits arethe targets of modulatory influences, with long-lasting effects shaping the finaloutput (Weiss et al. 1981; Harris-Warrick, 1988). These modulatory influences can

Key words: lobster stomatogastric system, central pattern generator, neuronal oscillators,electromyography, Jasus lalandii.

210 E. REZER AND M. MOULINS

act directly through neuronal pathways or indirectly through the action ofcirculating hormones. However, it is difficult to find an experimental situation inwhich it can be demonstrated that a given neuronal circuit can be the target ofneuronal and humoral influences acting in parallel. In the present work, we haveused one of the best known invertebrate CPGs, the crustacean pyloric CPG(Selverston and Moulins, 1987) and have designed experimental procedures thatstrongly suggest that this CPG is subjected to both neuronal and humoralmodulatory influences under physiological conditions.

The pyloric CPG is a small neuronal network (14 neurones) in the stomatogas-tric ganglion (STG) that produces a triphasic rhythmic output. The pyloric outputcan be recorded from in vitro preparations of the stomatogastric nervous system(Maynard, 1972). Using such preparations, it has been demonstrated that theexistence of the rhythmic pattern is dependent upon the intrinsic burstingproperties of the component neurones (see Miller, 1987). However, all theneurones of the pyloric CPG are conditional oscillators, requiring neuromodula-tory inputs from anterior centres in order to express their oscillatory capabilities invitro (Bal etal. 1988). In other words, neuromodulatory influences are necessaryto initiate and maintain the rhythmic output (Moulins and Cournil, 1982; Nagy andMiller, 1987). Consequently, the presence or absence of rhythmic output is a goodindication of whether or not the pyloric CPG is receiving permissive modulatoryinfluences at any time.

In vitro, neuromodulatory inputs to the pyloric CPG can be removed by cuttingor blocking impulse conduction in the single input nerve to the STG, thestomatogastric nerve (stn). This results in an inactivation of pyloric output.However, in such preparations the pyloric network is devoid of other modulatoryfactors, such as those carried by the blood in the intact animal. In this study, wehave taken a complementary approach: we cut the input nerve of an otherwiseintact animal so that the stomatogastric ganglion no longer received neuronalmodulatory influences but remained exposed to putative blood-borne influences.Electromyographic recordings (Rezer and Moulins, 1980, 1983) from suchpreparations have enabled us to test the ability of the pyloric CPG to produce arhythmic output when exposed only to humoral influences. Our data suggest that,under physiological conditions, the pyloric CPG is the target of both neuronal andhumoral modulatory influences able independently to induce the oscillatoryproperties of the neurones that generate the rhythmic output of this network.

Materials and methodsExperiments were performed on 46 (36 operated, 10 intacts) adult male and

female specimens of the lobster Jasus lalandii (Milne Edwards), obtained from alocal market and kept in large tanks of running sea water. The animals were fedwith mussels twice a week.

In vivo electromyographic recordings (EMG)The recording electrodes (Teflon-insulated silver wire, 130 ,um in diameter) were

Humoral induction of rhythmic motor pattern 211

implanted in the muscles through small-diameter holes made in the cephalothorax.Electrodes were then attached to the surface of the carapace with tissue glue(Histoacryl glue from Ligatures Peters) and dental cement. Animals were replacedin a small tank of sea water and the free ends of the wires were connected toamplifiers (Grass P5 a.c. preamplifier). Data were recorded onto a Gould ES1000electrostatic recorder and simultaneously stored on a Schlumberger tape recorder.This method allowed us to record simultaneously the activity of several pyloricmuscles. After experimentation, animals were killed, and the muscles whoseactivity had been recorded were identified using a marking technique describedpreviously (Rezer and Moulins, 1980). Muscles were classified according toMaynard and Dando (1974).

In most experiments, we recorded from one pyloric dilator muscle (cpvla,b),here referred to as a dilator muscle (D), and two pyloric constrictor muscles (piand p8-pl2), here referred to as the anterior constrictor (Cl) and posteriorconstrictor (C2) (Fig. 1A). D muscles were innervated by the two pyloric dilator(PD) motor neurones, Cl muscles by the single latero-pyloric (LP) motor neuroneand C2 muscles by the eight pyloric (PY) motor neurones (Maynard and Dando,1974). These motor neurones form a pattern-generating network, the pyloricCPG, in the stomatogastric ganglion (see Selverston and Moulins, 1987) (Fig. IB).

Simultaneous myographic and movement recordings

In several experiments, it was necessary to record simultaneously both themyographic activity and the corresponding movement. This was achieved by usingthe same wire both as an electromyographic recording electrode and as amovement transducer. A high-frequency electrical field (40xl03 Hz) was gener-ated (with a Tektronix FG 501 generator) between two steel sheets placed againstthe walls of the aquarium. An electromyographic (EMG) recording electrode wasimplanted in a muscle and the displacement of the tip of this electrode in theelectrical field (resulting from movement of the muscle) was recorded at the sametime as the electrical activity of the muscle. The two signals (EMG and movement)were separated using low- and high-pass filters. The EMG has a frequency of lessthan 300 Hz, so we used a low-pass (below 300 Hz) filter placed in the first EMGamplifier stage to extract the EMG signal from the movement signal. Themovement was obtained by demodulating the high-frequency carrier. We used acapacitative isolation unit to prevent polarization effects. In these experiments theanimals had to be restrained.

To block the neuromuscular junction of the D muscle (and suppress itsmovements), d-tubocurarine (Sigma) was injected into the animal. Enoughrf-tubocurarine was injected to achieve a final concentration of 10~4molP1 in thehaemolymph, assuming that the total haemolymph volume was 20 % of the weightof the animal (Phillips etal. 1980). The solution to be injected was made up at aconcentration that gave a final injection volume of no more than 1.5 ml, and wasinjected into the body cavity near the D muscle.


STGB

CG

CG

vlvn

STG

PDn

STGdlvn.

Ivri

lcm

C2

Fig. 1. The pyloric neuromuscular system of Jasus lalandii. (A) Schematic drawing ofthe stomatogastric nervous system in situ. The complete stomatogastric nervous systemconsists of the stomatogastric ganglion (STG), the oesophageal ganglia (OG), thepaired commissural ganglia (CG) and the nerves that connect them; B, brain. Thesingle input nerve to the STG is the stomatogastric nerve (stn). The pyloric dilator (D)and constrictor (Cl and C2) muscles are innervated by the lateral ventricular nerve(Ivn). The arrow indicates where the stn is cut in the operated animals. The marks onthe peri-oesophageal connective indicate the portion of the connective kept with the invitro preparation, avn, anterior ventricular nerve; mvn, medial ventricular nerve.(B) Innervation of the pyloric muscles and circuit diagram of the pyloric network (theblack circles represent inhibitory synapses). (C) In vitro preparation of the stomatogas-tric nervous system. The system was dissected from the foregut and pinned in aSylgard-lined Petri dish. One Vaseline pool was built around the desheathed STG. Thecircle around the stn indicates where it was cut. Pin electrodes were used to record themotor activity of the pyloric dilator neurone (PD) on the PD nerve (PDn) and LP andPY on the ventral and dorsal branches of the latero-ventricular nerve (vlvn, dlvn).(D) Electromyographic activity of the D, Cl and C2 muscles. Such a recording givesdirect access to the suprathreshold activity of the motor neurones (PD, LP and PY)that constitute the pyloric network in the STG (see B).


In vitro and ex vivo neurographic recordings

The stomatogastric nervous system was dissected from the foregut and pinnedout under saline (400mmoir1 NaCl, lOmmolP1 KC1, lOmmolT1 CaCl2,52mmoir 1 MgCl2, 28mmoll~1 Na2SO4, Smmoll"1 NaHCO3, O.emmoir1

NaBr) in a Sylgard-lined Petri dish (combined preparation of Selverston et al.1976) (see Fig. 1C).

We called this combined preparation dissected from an intact animal an in vitropreparation: the STG was still attached to the anterior centres. We called thestomatogastric nervous system dissected from an operated animal an ex vivopreparation. In this case, as the stn had been cut in vivo, the preparation wasreduced to a short piece of the stn attached to the STG and the output nerves (seeFig. 1B,C).

Extracellular recordings and stimulation of the various nerves of the preparationwere achieved via platinum electrodes according to electrophysiological tech-niques previously reported (Moulins and Nagy, 1981).

A Vaseline pool built around the stomatogastric ganglion was used to superfuseblood serum. Blood of Jasus lalandii or Cancerpagurus was taken from the base ofthe fourth leg just before experimentation and serum was obtained by centrifugingit for 2min at 2000 revs min"1. Serum tests were carried out on ex vivopreparations, i.e. stomatogastric systems isolated from animals previously oper-ated upon.

Operated animals

Animals were anaesthetized by gradual cooling; they were left for 2h at 10°C,then 5h at 5°C. They were then kept on ice throughout the operation. A smallpiece of carapace was carefully removed from the cephalothorax with sterilizedinstruments to allow access to the stn. This piece of carapace was placed in a sterilePetri dish during the operation. Under a binocular microscope, the epidermis wasremoved and the stn was cut anterior to its emergence from the aorta. The naturaltension of the nerve was sufficient to cause retraction of the cut ends. Theepidermis was then glued back in place with Histoacryl glue. This piece ofepidermis is essential for the generation of the new carapace necessary for thesurvival of the animal at the next moult. The carapace piece was then put back inplace and fixed to the rest of the carapace with dental cement. Afterwards, animalswere placed in individual tanks of running sea water. More than 90% of theoperated animals survived for 3 weeks or longer and some survived for as long as 2years. All the animals were kept with fresh mussels in their aquarium. The dayafter the operation, the animals appeared to have recovered completely andEMGs could be recorded from the pyloric muscles. Spontaneous feeding began10-20 days after the operation (see Table 1). EMG recordings were carried out on36 animals at different times after the operation. Some animals were used for tworecording sessions, giving a total of 42 in vivo experiments (see Table 1). For theseanimals we used wires of different colour, cut and left in place after the first


recording session. The muscles were identified at the end of the second recordingsession. In a few animals, we recorded the pyloric activity with the same electrodesin place before and after the operation (see Fig. 5).

Results

In vivo transection of stn does not abolish the rhythmic pyloric output

Electromyographic recordings from intact Jasus lalandii show that the pyloricmotor activity consists of a regular triphasic rhythmic pattern with an activation ineach cycle of the dilator muscles (D), the anterior constrictor muscles (Cl) and theposterior constrictor muscles (C2) (Fig. ID) (Rezer and Moulins, 1983).

It has been shown, using in vitro recordings from the isolated stomatogastricnervous system of Panulirus, that this activity originates from a network of 14neurones in the STG (Maynard, 1972; Maynard and Selverston, 1975). Similarresults have been obtained subsequently for Jasus lalandii (Nagy and Dickinson,1983). This network has been extensively studied (for a review, see Selverston andMoulins, 1987) and it has been demonstrated that its triphasic rhythmic outputarises both from the synaptic connectivity between pyloric neurones (see Fig. IB)and from the ability of these neurones to produce bursting pacemaker potentials(BPP) (Miller and Selverston, 1982).

In the Cape lobster Jasus lalandii, Bal et al. (1988) have clearly demonstratedthat all the neurones of the pyloric CPG are conditional oscillators, i.e. theirabilities to produce BPPs are conditional and can be expressed only underextrinsic modulatory input to the pyloric network. In an in vitro preparation of thestomatogastric nervous system, the suppression of impulse traffic in the stncompletely abolishes the ability of pyloric neurones to produce BPPs and, thereby,the ability of the network to produce rhythmic output (Moulins and Cournil, 1982;Nagy and Miller, 1987) (Fig. 2A).

The related experiment performed in vivo, however, does not give the sameresults as above (compare Fig. 2A and 2B). For all the operated animals tested(see Table 1), EMG recordings from pyloric muscles show a rhythmic pattern ofactivity similar to the pyloric pattern recorded in the non-operated animals(Fig. 2B). This triphasic pattern has been observed in animals just after 5m

Fig. 2. Comparison between the effects of deafferentation on the activity of the STGin vitro (A) and in vivo (B). (A) In the control preparation, rhythmic pyloric activitywas recorded from an isolated stomatogastric nervous system. The three types ofmotor neurones fired in successive bursts: PD in the PD nerve (PDn), LP and PY in thevlvn. The neurone that fired with PD in the stn is the anterior burster. Thirty-fiveminutes after stn section, pyloric activity had completely disappeared. (B) In thecontrol preparation (intact animal), rhythmic pyloric activity was recorded byelectromyography. The D muscles (innervated by PD), Cl muscles (innervated by LP)and C2 muscles (innervated by PY) were successively active. After cutting the stn(3-day-old operated animal), the D, Cl and C2 muscles were still rhythmically active,indicating that the PD, LP and PY motor neurones were still bursting.

Humoral induction of rhythmic motor pattern

In vitro Control

PDn

vlvn

sin cut

215

Jiliiiiiil mtmmmmmm

I ' I " > I

B In viv

Cl

C2

" M M * *

stn cut animal

' I Mil ll , | M |

Intact animal

I s

•17 riiifpn

Is

y M » « * » | ^

I s

Fig. 2


Table 1. Rhythmic pyloric pattern tested in vivo in operated animals

Time afterstn section

0-24 h24-48h3-8 days9-15 days1 month2 months4-6 months2 years

N

108544353

Percentagefeeding

000

50100100100100

Pyloric patternin vivo

++++++++

N represents the number of tests performed on the animals at different times after the stn hadbeen cut. These 42 tests were performed on 36 animals (six animals were tested twice).

+ indicates the presence of a rhythmic pyloric pattern.All the animals had free access to fresh mussels in their aquaria; feeding was monitored daily.

transection, as well as in animals 5 months and even 2 years after stn transection(Fig. 3 and Table 1).

We isolated the stomatogastric system from operated animals and recorded thepyloric output in the resulting ex vivo preparation. Prior to each ex vivo recordingexperiment, we had tested for the presence of rhythmic pyloric output in vivo:electromyographic recordings showed that all the animals tested in this way had arhythmic pyloric motor output. Nevertheless, when these stomatogastric systemswere rapidly transferred to Petri dishes, they appeared to be unable to producerhythmic pyloric output. In recordings from the pyloric motor nerves in nine exvivo preparations dissected 1 day after stn transection and in seven preparationsdissected 2 days after stn transection, no rhythmic activity was seen.

The discrepancy between the results obtained in vivo and ex vivo can beexplained by one of two hypotheses: (1) although all the nervous inputs to the STG(and the pyloric network) are generally thought to travel in the stomatogastricnerve, some inputs may use other routes and, thereby, may be responsible for thecontinuation of a pyloric output in the operated animal; (2) there is a blood-bornefactor (normally acting in parallel with nervous influences) that is not present inthe in vitro preparations and that is present in sufficient quantity in the operatedanimal to maintain the ability of the neurones to generate BPPs and thus therhythmic pyloric output.

The following results strongly support this second possibility.

Fig. 3. Pyloric patterns recorded in operated animals at different times after stntransection. These electromyographic recordings, obtained from five animals at 2h,24 h, 8 days, 5 months (142 days) and 2 years after stn transection, show that the pyloricrhythmic pattern was present just after the operation and had not disappeared even 2years later.

Humoral induction of rhythmic motor pattern 2172h

"I

C2

24 h

D *II • I I

I s

ci mm

8 days

I s

Cl •***»

C2k»m*^

5 months

Cl

C2

Is

2 years

D

Cl

Is

~jK "• '" ' • ' • ^ ' jP n i 1 ' - 1 - 1 ' P i n p i u " I . I j ^ ,• ,j|p p

Fig. 3

Is


Startle pyloric responses disappear in operated animals

The first test that can be used to determine whether the pyloric network of anoperated animal is effectively isolated from sensory inputs is to compare theresponses of the network to different sensory stimuli in the intact animal withthose, if any, in the operated one.

Fig. 4 compares the effects of mechanical (Fig. 4A,C) and chemical (Fig. 4B,D)stimuli applied to an intact animal and to an operated animal. The mechanicalstimulus used here was a non-specific tapping on the dorsal carapace. Tappingproduced a general startle reaction and a transient modification (primarilyacceleration) of the pyloric rhythm (Fig. 4A). Although highly evident, thisresponse exhibited habituation in the intact animal, disappearing almost com-

A Intact animal

C Operated animal

D

C2

StimulusD

Is

Is

Fig. 4. Effects of a mechanical (A,C) and a chemical (B,D) stimulus on intact andoperated animals. (A,B) In an intact animal, a mechanical stimulus (A) applied to thedorsal carapace was always associated with an immediate brief modification of thepyloric motor pattern: the frequency of the rhythm increased for several cycles, and theburst duration of the constrictor in these cycles decreased. The activities of D (blacktriangle) and Cl (black circle) were recorded simultaneously with a single electrode.The stimulus time is marked by a bar under the record. A chemical stimulus (B)(mussel juice introduced into the aquarium) was also always associated with similartransient modifications of the pyloric rhythm. (C,D) In an operated animal, the samestimuli (mechanical in C and chemical in D) were never associated with any change inthe pyloric rhythm. Stimulus time is marked by a bar.


pletely after only five repetitive trials. By contrast, the same stimuli never had aneffect on the existing pyloric output of operated animals (compare Fig. 4A and4C).

The chemical stimulus used was more specific; juice extracted from freshmussels was introduced into the aquarium. In the intact animal, this alwaysinduced a modification of the pyloric rhythm that lasted for several cycles(Fig. 4B). However, the same stimulus applied to an operated animal nevercaused a modification of the pyloric rhythm (Fig. 4D). Each of these stimuli wasapplied to 10 operated animals, and we never recorded a startle response.Moreover, we have been unable to find any sensory stimulus that inducesobservable effects on the pyloric motor output in operated animals.

The pyloric pattern is not modified at the onset of feeding in operated animals

Feeding is a specific stimulus that alters the activity of the pyloric CPG in intactanimals (Rezer and Moulins, 1983). In animals unfed for more than 8 days, thepyloric motor output is highly irregular: the pyloric period is long (mean greaterthan 2s) and varies from one cycle to another (left-hand part of Fig. 5Ai). Afterfeeding, the pyloric motor output is very regular, with a shorter (less than 2 s) andalmost constant period; this pattern continues without changing for more than 12 hafter feeding (right-hand part of Fig. 5Ai). The output of the pyloric CPG switchesfrom one pattern to the other immediately upon feeding (middle part of Fig. 5Ai).In continuous recordings from six operated animals, feeding was never associatedwith any change in the pyloric pattern (Fig. 5Bi). The pyloric period remainedvariable and did not decrease after feeding. Thus, the pyloric period had a clearlybimodal distribution when measured in the intact animal during a time intervalthat included feeding (Fig. 5Aii). When measured in the operated animal underthe same conditions, the distribution of the pyloric period was unimodal(Fig. 5Bii).

Although the origin of the modification of the pyloric output in an intact animalis not known, it is clear that its abrupt occurrence at feeding is the mark of extrinsicnervous influences impinging on the pyloric network. This again indicates that thestomatogastric ganglion in the operated animal was completely isolated: its pyloricoutput was no longer correlated with the behavioural context.

Phasic proprioceptive feedback cannot account for pyloric rhythm generation inoperated animals

It has been demonstrated using in vitro preparations that the pyloric networkconnected to the more rostral ganglia is able to produce organized rhythmic outputwithout proprioceptive feedback (Maynard, 1972; Selverston et al. 1976). How-ever, although proprioceptive inputs are not necessary to produce this output, itremains possible that, under our experimental conditions, they are sufficient togenerate the rhythmic pattern.

This hypothesis has been tested with respect to the proprioceptive feedbackassociated with D muscle movement by (1) pharmacological suppression of the


Ai

Intact animal Time (min)

0 2 4 6 8 10 121 1-

14~i 1 1 r

Aii Intact animal

20

Is

Bi

Operated animal Time (min)0 2 4 6 8 10 121 1 1 1 r— "i

14T r~

N=302

Bii Operated animal

50

r

-

3D period

Time (s)

Fig. 5. Evolution of the pyloric period at feeding time in intact and operated animals.The evolution of the pyloric period (measured in seconds between the onsets ofsuccessive dilator bursts) has been plotted over 14 min during which the animal fed.The electromyographic recording presented under the graph corresponds to thefeeding time. In the intact animal (Ai), the pyloric output changed abruptly uponfeeding: the pyloric period, which was long and variable, became short and stable.(Aii) Distribution histograms of D period as a percentage of the total cycle periodbefore and after the feeding. D period has a bimodal distribution; for the first mode(1), corresponding to the pattern recorded after feeding, the mean is 0.96±0.079s; forthe second mode (2), corresponding to the pattern recorded before feeding, the meanis 2.97+0.79s (N= 700) (see text). In the operated animal (B), the pyloric output is notmodified at feeding. In Bi, the electromyographic recording is interrupted for 1.30 min,so as to include recordings both before and after feeding. (Bii) Distribution histogramof D period before and after feeding; here the D period has a unimodal distribution(3), with a mean of 5.26±2.81s (N=700).

16

movement and (2) experimental perturbation of existing movement. Suchperturbation or suppression should modify any proprioceptive information thatmay derive directly from contraction of the muscle itself and drive pyloric output.

In intact animals, it has been possible to record simultaneously the EMGactivity of the pyloric muscles and the movements they produce via the tip of thesame electrode inserted into the muscle (see Materials and methods). This isshown in the experiments of Fig. 6A, in which movements and electrical activity ofthe D muscle were recorded simultaneously. It is known that the dilator muscle is


innervated by two cholinergic motor neurones (Marder, 1987). This property hasbeen used to suppress the movements of the D muscle pharmacologically. This wasachieved by injecting rf-tubocurarine to a final concentration of about 10~4 mol P 1

into the haemolymph. Fig. 6B shows that, at this concentration, contractions ofthe D muscle disappeared.

If the pyloric pattern recorded from an operated animal was due in some way toproprioceptive feedback, suppression of the feedback coming from the D muscle(by suppression of D muscle contractions) would be expected to stop this patternor at least to alter it considerably. The experiment shown in Fig. 6C,D indicatesthat this is not the case. Injection of d-tubocurarine into an operated animal (at thesame concentration as in Fig. 6B) does not abolish or modify the pattern: acomparison of Fig. 6C and Fig. 6D shows only a slight increase in the periodattributable to direct central effects of the drug on the pyloric network. It shouldbe noted that, although we have shown that d-tubocurarine blocks the neuro-muscular junction (Fig. 6B), electrical activity was still recorded by the electrodeimplanted in the D muscle. This activity must be of motor neuronal origin, andwhat we term EMG in such recordings is likely to be primarily a direct monitor ofmotor neurone terminal activity (neurography).

Another way to test the possible role of proprioceptive feedback fromcontracting muscles in generating the pyloric output is to perturb existingmovement and to look for effects on the motor pattern. To do this, we implantedtwo electrodes into the D muscle of operated animals; one electrode was used forelectromyographic and movement recordings, the other was used to stimulate themuscle electrically. As shown in Fig. 7, a brief stimulation of the muscle wasapplied at the beginning (Fig. 7A), in the middle (Fig. 7B) and at the end(Fig. 7C) of the cycle. None of these perturbations had any effect on the pyloricperiod (Fig. 7D,E). The same perturbations imposed by electrical stimulation toeither Cl or C2 muscles are also without effects on the pyloric period (not shown).Together with the above pharmacological data, this strongly suggests thatproprioceptive inputs cannot be responsible for the organization of the rhythmicoutput recorded in operated animals.

Blood-borne influences may be involved in the generation of rhythmic pyloricoutput in the operated animals

The preceding results indicate that the pyloric rhythmic pattern recorded in anoperated animal is not due to neuronal inputs to the STG, suggesting that blood-borne influences alone are able to maintain the pyloric pattern, possibly byinducing oscillatory properties in the pyloric neurones.

This hypothesis was tested in ex vivo preparations of the stomatogastric nervoussystem removed from the animal 24 or 48 h after stn transection. The presence ofrhythmic pyloric output was first verified by in vivo EMG recordings (Fig. 8A); thestomatogastric system was then removed from the animal, and activity wasrecorded on the motor nerves. Under these conditions, none of the nervoussystems tested (N=16) exhibited any rhythmic pyloric activity (Fig. 8B). To show


A Intact animal: control

D+Cl

C2

Movement

B Intact animal: d-tubocurarine

^ * M * S W H * ^ ^

^/VW-A/W>VAw'•*^/*/^^^

C Operated animal: control

Is

Is

D Operated animal: d-tubocurarine

Fig. 6

Js


Fig. 6. Injection of d-tubocurarine abolished dilator contraction (A,B) but did notabolish the rhythmic pyloric output of an operated animal (C,D). (A,B) In the control(A), the electrode implanted in the D muscles was used to monitor D contractions.Note that this electrode recorded simultaneously the activity in D (black triangle) andCl (black circle). Injection of rf-tubocurarine into the animal (to a final concentrationof 10~4 mol 1~' in the haemolymph) stopped the movement (B). (C,D) Injection of thesame concentration of d-tubocurarine into an operated animal did not abolish (or evendisturb) the pyloric pattern (compare D with C).

that humoral factors might be responsible for the activity recorded in vivo, weattempted to restore pyloric cycling by superfusing the STG with blood serumfrom Jasus lalandii. In only two of nine such ex vivo preparations was rhythmicactivity restored. One possible explanation for this low success rate is that theblood may have coagulated during application. Centrifugation of the bloodgenerally does not prevent clotting (Durliat, 1985) and, although such clotting canbe prevented with strontium chloride (Durliat and Vranckx, 1981; Vella andTripp, 1983), this salt strongly affects the pyloric rhythm and thus could not beused. Instead, we tried blood from the crab Cancer pagurus, since clotting of crabblood is effectively suppressed by centrifugation. Bathing the STG of an ex vivopreparation in saline plus crab serum (at least 70% v/v) restored the pyloricrhythm (Fig. 8B) in five of seven preparations tested. Interestingly, the bloodserum used in the five preparations in which rhythmic activity was successfullyrestored was taken from the animal after 20:00 h local time, whereas the serumthat gave the two negative results was taken from the animal earlier in the day.These results indicate that blood serum was able to induce rhythmic pyloricactivity in an isolated STG.

Discussion

It is now well established that, in several crustacean species, a completelyisolated STG in vitro is unable to produce any rhythmic pyloric output. This hasbeen demonstrated in Homarus gammarus (Moulins and Cournil, 1982), in Jasuslalandii (Dickinson and Nagy, 1983, and present paper) and in Panulirusinterruptus (Russell and Hartline, 1978; Nagy and Miller, 1987). Under suchconditions, the pyloric neurones lose their regenerative bursting properties; theseproperties are expressed only in the presence of permissive neuromodulatoryinputs from the rostral ganglia (Bal et al. 1988).

In the present paper we have shown that, in vivo, an isolated STG remains ableto produce the rhythmic pyloric pattern. Under these conditions (1) non-specificsensory stimuli that modify the pyloric pattern in the intact animal are withouteffect after stn transection; (2) the change in the pyloric pattern that occurs uponfeeding in the intact animal does not occur in the operated animal; (3) thesuppression or perturbation of putative proprioceptive feedback is without effecton the pyloric activity.

From these results we conclude that the rhythmic pattern observed in the

E. REZER AND M. MOULINS

- l

0.5

P - plr— p 2 . _ p 3 ^ pS

• • h fIS

Stimulation

Movementv = ii v = p p

pO ' pO

Fig. 7. Perturbation of the dilator contractions had no effect on the pyloric period ofan operated animal. (A-C) The same electrode was used to record simultaneously theelectrical activity of the D muscle and the associated movement. A second electrodewas implanted in the same muscle and was used for stimulation. An electric shock wasdelivered randomly to the D muscle during the pyloric period; stimulations wereseparated by at least six cycles. The electric shock induced a strong modification of themovement, but no modification of the pyloric period, regardless of the phase [early(A), medium (B) or late (C)] at which the stimulus occurs in the pyloric cycle. (D,E)The observation that the pyloric period does not vary when the dilator muscle isstimulated is shown graphically in D. The formulae used to calculate the points plottedon the abscissa (x) and on the ordinate (y) are shown in E; pi, p2, p3 are pyloricperiods without stimulation, pS is the stimulated pyloric period, and IS is the latency ofthe stimulation in the pyloric period. In the graph, each point represents the mean andstandard deviation (vertical bar) of points falling within the corresponding bin. It isclear that there was no variation of the pyloric period when the D muscle wasstimulated. This has been confirmed by an analysis of variance, which shows that thereare no significant differences among the bins (P=0.3804). Moreover, there is nosignificant difference (with a confidence level of 99%) between the mean pyloricperiod of the cycles just before the stimulation (1.549±0.134s), the cycles in whichstimulation occurred (1.549±0.151s) and the cycles just after the stimulation(1.534±0.146s).


A In vivo

B Ex vivo

dlvn

vlvn

Is

Ringcr+bloocl serum

Fig. 8Is

Fig. 8. Haemolymph serum can restore rhythmic pyloric output to a silent ex vivopreparation. (A) EMG pyloric activity of a 48 h operated animal (in vivo). (B) TheSTG (and output nerves) was transferred to a Petri dish (ex vivo) where it was unableto produce any rhythm in the presence of saline alone. Addition of crab blood serum tothe saline was sufficient to restore the pyloric output.


operated animal is organized by the STG alone, i.e. with no extrinsic neuronalinfluences. The STG of an operated animal becomes electrically silent whenremoved from the animal and studied in vitro. However, it is possible to restorethe pyloric rhythmic activity of such an ex vivo preparation by applying bloodserum to the ganglion. We conclude that humoral influences are sufficient tomaintain the bursting properties of the pyloric neurones.

Does cutting the stn suppress all STG neuromodulatory inputs?

The stn is the single connection between the STG and the higher centres (seeMaynard and Dando, 1974). There is considerable immunohistochemical andpharmacological evidence to suggest that a large population of modulatory fibrestravels to the STG from rostral centres via the 5m; these fibres are able to influencethe activity of networks in the STG (Marder, 1987). Among these fibres are thosesuch as APM (the anterior pyloric modulator of Jasus lalandii, Nagy et al. 1981),which have a permissive influence on the oscillatory properties of the pyloricmotor neurones. The firing of APM restores cycle activity in a quiescent pyloricnetwork (Nagy and Dickinson, 1983) by inducing the oscillatory properties of theneurones (Bal etal. 1988). Such permissive influences are suppressed by cuttingthe stn. The results obtained in vitro, i.e. cessation of the rhythmic pyloric outputafter stn conduction has been blocked or after the stn has been cut, are readilyexplained by such modulation. In contrast, the results obtained in vivo, i.e.continuation of the rhythmic pyloric output after stn transection, are more difficultto understand.

One possible explanation is that the sectioned input fibres of the stn remain ablespontaneously to liberate modulatory transmitters in the STG, thus ensuring thatthe bursting properties of the neurones continue to be expressed. It is well known,especially in Crustacea, that axons can remain alive for a long time aftertransection (Bittner, 1988). Moreover, this has been specifically noted for the STGinput fibres. Based on the morphology of the ganglion more than 200 days after stntransection, Royer (1987) concluded that at this time there were still some inputfibres that had not degenerated. Nevertheless, it has not been demonstrated thatthese axons are still functional. If we are to explain our results with this hypothesis,we must assume that the modulatory sectioned axons are still able to synthesizeand liberate transmitter 2 years after axon transection. We must also explain theobservation that in vitro, stn blockade or transection resulted in the cessation ofthe pyloric pattern in less than 15 min.

In the stomatogastric system, modulatory neurones that do not use the stn toproject to the STG have been identified in the crabs Cancer borealis and Cancerirroratus. These gastropyloric receptors (GPRs) (Katz etal. 1989) are primarysensory afferent cells that are active in phase with the movements of the gastricmill and that have direct prolonged neuromodulatory effects on neurones of thepyloric and gastric CPGs of the crab STG (Katz and Harris-Warrick, 1989). Theyproject to the STG via the lateral ventricular nerve (Ivn), which can be consideredas the output nerve of the STG. Cutting the stn does not suppress the STG input


from GPRs and it could be argued that the modulatory activity of the GPR cellscan ensure that the pyloric rhythm continues in operated animals. However, it hasnot been demonstrated that firing of the GPR cells is sufficient to cause rhythmicactivity in the pyloric system. In addition, no gastric muscles were active in any ofthe animals whose stn had been cut less than 1 month before recording. Since theGPR cells are activated by gastric movements, it seems probable that they weresilent in the operated animals. Thus, it is unlikely that they are involved in thegeneration of the rhythmic pyloric activity recorded in vivo for at least the firstmonth after stn transection.

It remains a possibility that movements of the pyloric region might be able toactivate the GPR cells, which in turn could induce cycling of the pyloric neurones.Although such a feedback cannot be totally rejected, it must be mentioned thatperturbation and suppression of the dilator phase in the pyloric cycle (Figs 6 and 7)are without effect on the activity of the CPG.

Can the pyloric rhythm of an operated animal be the result of phasicproprioceptive feedback?

The work of Maynard (1972) clearly demonstrated that sensory inputs are notnecessary for the genesis of a rhythmic pyloric pattern. Nevertheless, thesesensory inputs exist, and their activities modify the existing pyloric rhythmicpattern (Dando et al. 1974). These inputs (except those of the GPR) have indirectaccess to the STG, travelling via the stn (see Moulins and Nagy, 1985; Simmers andMoulins, 1988). Cutting the stn suppresses their influence on the neurones of theSTG. After stn transection, pyloric output appears to be produced without anyrelationship to the behavioural context. For example, the stimuli that normallyresult in a startle response in the intact animal are without effect on the pyloricrhythm of an operated animal. This loss of responsiveness is clearly demonstratedby the fact that feeding itself, which produces significant changes in the intactanimal, does not produce any 'adaptative' modification of the pyloric output.

It can also be argued that proprioceptive feedback can introduce timing cuesthat will be sufficient to maintain a rhythmic pattern of behaviour in the pyloricneurones. Putative candidates for this role are proprioceptors associated with thepyloric muscles and travelling in a nerve other than the stn. However, our resultshave shown that suppression of the dilator movements (i.e. the suppression ofpossible feedback) does not perturb the pyloric pattern of an operated animal.Similarly, altering the activity of putative proprioceptors (by electrically disturbingthe activity of the pyloric muscles) does not alter the pyloric output.

In the operated animal the bursting properties of the STG neurones are under thecontrol of humoral agents

It could also be argued that deafferentation of the STG results in a profoundmodification of the membrane properties of the pyloric neurones. In the presentcase, the conductances involved in the oscillatory properties of the neuroneswould have to be modified in such a way that these properties became non-


conditional (i.e. can be expressed in the absence of extrinsic influences). Althoughsuch modifications after denervation have yet to be described, experimental workis in progress to test this hypothesis. However, it seems unlikely that such amodification could be achieved 2 h after stn transection (see Fig. 3). Furthermore,this hypothesis fails to explain why the STG of an operated animal is unable toproduce any rhythmic output when tested in vitro.

The best explanation for our results is that, under our experimental conditions(in the operated animal), the bursting properties of the pyloric neurones areinduced and maintained by blood-borne modulatory influences. This has beenpostulated previously, based on pharmacological and histochemical results (Beltzet al. 1984). This hypothesis is supported by the observation that serum is able torestore the ability of an in vitro isolated STG to produce a rhythmic pyloric output.Previous experiments performed in vitro had shown that neuronal modulatoryinfluences from higher centres are sufficient to induce the bursting properties ofthe pyloric neurones (Russell and Hartline, 1978; Miller and Selverston, 1982;Moulins and Cournil, 1982; Dickinson and Nagy, 1983). The data presented hereshow that humoral influences are also sufficient to induce the bursting propertiesof the pyloric neurones. We can consider the pyloric system as a CPG made fromconditional oscillators that receive modulatory inputs from two sources, oneneuronal and one humoral. Either is sufficient to induce rhythmic activity; neitheris necessary.

From results obtained in vitro by bath application, it is clear that numerousamines and peptides could be responsible for the induction of the pyloric cycling(Marder, 1987), and in the intact animal the STG is probably exposed to a largenumber of active molecules. However, only a few of these substances have beendemonstrated to be circulating hormones in Crustacea. Among them we mustmention the red pigment concentrating hormone-like peptide, which is known tobe a strong activator of the pyloric CPG (Nusbaum and Marder, 1988). Anotherputative candidate for a humoral inducing factor is the cholecystokinin-likepeptide that has been identified in the haemolymph of Panulirus and is known toenhance the oscillatory activity of the pyloric neurones, but is without effect on thefrequency of the rhythm (Turrigiano and Selverston, 1989). Interestingly, inPanulirus its level increases after feeding (Turrigiano and Selverston, 1990) but ifthe same is true in Jasus lalandii this does not induce any obvious modification ofthe pyloric pattern (see Fig. 5B).

This in vivo preparation will be useful in analyzing the effects of humoralmodulatory influences on the pyloric network and in analyzing how these humoralfactors interact with the neuronal modulatory influences that also impinge on thisnetwork. Moreover, our preparation will now allow us to compare in vivo theactivity of a well-known CPG subjected only to humoral influences with theactivity of the same CPG subjected to both neuronal and humoral influences.

This work was supported by a Grant from the Human Frontier Science


Programme. The apparatus used to record simultaneously, with a single electrode,muscle electrical activity and movement, was built by Pierre Ciret.

ReferencesBAL, T., NAGY, F. AND MOULINS, M. (1988). The pyloric central pattern generator in crustacean:

a set of conditional neuronal oscillators. J. comp. Physiol. 163, 715-727.BELTZ, B., EISEN, J., FLAMM, R., HARRIS-WARRICK, R. M., HOOPER, S. L. AND MARDER, E.

(1984). Serotonergic innervation and modulation of the stomatogastric ganglion of threedecapod crustaceans (Panulirus interruptus, Homarus americanus and Cancer irroratus).J. exp, Biol. 109, 35-54.

BITTNER, G. D. (1988). Long term survival of severed distal axonal stumps in vertebrates andinvertebrates. Am. Zool. 28, 1165-1179.

DANDO, M. R., CHANUSSOT, B. AND NAGY, F. (1974). Activation of command fibres to thestomatogastric ganglion by input from a gastric mill proprioceptor in the crab, Cancerpagurus. Mar. Behav. Physiol. 2, 197-228.

DELCOMYN, F. (1980). Neural basis of rhythmic behavior in animals. Science 210, 492-498.DICKINSON, P. S. AND NAGY, F. (1983). Control of a central pattern generator by an identified

modulatory interneurone in Crustacea. II. Induction and modification of plateau properties inpyloric neurones. J. exp. Biol. 105, 59-82.

DURLIAT, M. (1985). Clotting processes in crustacean Decapoda. Biol. Rev. 60, 473-498.DURLIAT, M. AND VRANCKX, R. (1981). Action of various anticoagulants on hemolymphs of

lobsters and spiny lobsters. Biol. Bull. Mar. biol. Lab., Woods Hole 160, 55-68.GETTING, P. A. (1988). Comparative analysis of invertebrate central pattern generators. In

Neural Control of Rhythmic Movements (ed. A. H. Cohen, S. Rossignol and S. Grillner), pp.101-127. New York: John Wiley and Sons, Inc.

GRILLNER, S. (1977). On the neural control of movement. A comparison of different basicrhythmic behaviors. In Function and Formation of Neural Systems (ed. G. S. Stent), pp.197-224. Berlin: Dahlem Konferenzen.

HARRIS-WARRICK, R. M. (1988). Chemical modulation of central pattern generators. In NeuralControl of Rythmic Movements (ed. A. H. Cohen, S. Rossignol and S. Grillner), pp. 285-331.New York: John Wiley and Sons, Inc.

KATZ, P. S., EIGG, M. H. AND HARRIS-WARRICK, R. M. (1989). Serotonergic/cholinergic musclereceptor cells in the crab stomatogastric nervous system. I. Identification and characterizationof the gastropyloric receptor cells. J. Neurophysiol. 62, 558-570.

KATZ, P. S. AND HARRIS-WARRICK, R. M. (1989). Serotonergic/cholinergic muscle receptor cellsin the crab stomatogastric nervous system. II. Rapid nicotinic and prolonged modulatoryeffects on neurones in the stomatogastric ganglion. /. Neurophysiol. 62, 571-581.

MARDER, E. (1987). Neurotransmitters and neuromodulators. In The Crustacean StomatogastricSystem: A Model for the Study of Central Nervous Systems (ed. A. I. Selverston andM. Moulins), pp. 263-300. Heidelberg: Springer Verlag.

MAYNARD, D. M. (1972). Simpler networks. Ann. N.Y. Acad. Sci. 193, 59-72.MAYNARD, D. M. AND DANDO, M. R. (1974). The structure of the stomatogastric neuromuscular

system in Callinectes sapidus, Homarus americanus and Panulirus argus. Phil. Trans. R. Soc.Lond. B 68, 161-220.

MAYNARD, D. M. AND SELVERSTON, A. I. (1975). Organization of the stomatogastric ganglion ofthe spiny lobster. IV. The pyloric system. J. comp. Physiol. 100, 161-182.

MILLER, J. P. (1987). Pyloric mechanisms. In The Crustacean Stomatogastric System: A Modelfor the Study of Central Nervous System (ed. A. I. Selverston and M. Moulins), pp. 109-136.Heidelberg: Springer Verlag.

MILLER, J. P. AND SELVERSTON, A. I. (1982). Mechanisms underlying pattern generation inlobster stomatogastric ganglion as determined by selective inactivation of identifed neurones.II. Oscillatory properties of the pyloric neurones. /. Neurophysiol. 48, 1378-1391.

MOULINS, M. AND COURNIL, I. (1982). All-or-none control of the bursting properties of thepacemaker neurones of the lobster pyloric pattern generator. J. Neurobiol. 13, 447-458.

MOULINS, M. AND NAGY, F. (1981). Participation of an unpaired motor neurone in the bilaterally


organized oesophageal rhythm in the lobsters Jasus lalandii and Palinurus vulgaris. J. exp.Biol. 90, 205-230.

MOULINS, M. AND NAGY, F. (1985). Extrinsic inputs and flexibility in the motor output of thelobster pyloric neural network. In Model Neural Networks and Behavior (ed. A. I.Selverston), pp. 49-68. New York: Plenum Publishing Corp.

NAGY, F. AND DICKINSON, P. S. (1983). Control of a central pattern generator by an identifiedmodulatory interneuron in Crustacea. I. Modulation of the pyloric motor output. J. exp. Biol.105, 33-58.

NAGY, F., DICKINSON, P. S. AND MOULINS, M. (1981). Modulatory effects of a single neurone onthe activity of the pyloric pattern generator in Crustacea. Neurosci. Lett. 23, 167-173.

NAGY, F. AND MILLER, J. P. (1987). Pyloric pattern generation in Panulirus interruptus isterminated by blockade of activity throught the stomatogastric nerve. In The CrustaceanStomatogastric System: A Model for the Study of Central Nervous System (ed. A. I. Selverstonand M. Moulins), pp. 136-139. Heidelberg: Springer Verlag.

NUSBAUM, M. P. AND MARDER, E. (1988). A neuronal role for a crustacean red pigmentconcentrating hormone-like peptide: neuromodulation of the pyloric rhythm in the crab,Cancer borealis. J. exp. Biol. 135, 165-181.

PHILLIPS, B. F., COBB, J. S. AND GEORGE, R. W. (1980). General biology. In The Biology andManagement of Lobsters (ed. J. S. Cobb and B. F. Phillips), pp. 2-82. New York: AcademicPress.

REZER, E. AND MOULINS, M. (1980). Modalit6s d'expression du gdndrateur du rythme pyloriquechez les Crustaces: analyse 61ectromyographique. C.R. hebd. Seanc. Acad. Sci. Paris 291,353-356.

REZER, E. AND MOULINS, M. (1983). Expression of the crustacean pyloric pattern generator inthe intact animal. J. comp. Physiol. 153, 17-28.

ROBERTS, A. AND ROBERTS, B. L. (eds) (1983). Neural origin of rhythmic movements. Symp.Soc. exp. Biol. 37. Cambridge: Cambridge University Press.

ROYER, S. M. (1987). Chronic effects of de-afferentation on the stomatogastric ganglion ofPanulirus. In The Crustacean Stomatogastric System: A Model for the Study of CentralNervous System (ed. A. I. Selverston and M. Moulins), pp. 251-257. Heidelberg: SpringerVerlag.

RUSSELL, D. F. AND HARTLINE, D. K. (1978). Bursting neural networks: a reexamination.Science 200, 453-456.

SELVERSTON, A. I. AND MOULINS, M. (1985). Oscillatory neural networks. A. Rev. Physiol. 47,29-48.

SELVERSTON, A. I. AND MOULINS, M. (eds) (1987). The Crustacean Stomatogastric System: AModel for the Study of Central Nervous System. Heidelberg: Springer Verlag.

SELVERSTON, A. I., RUSSEL, D. F., MILLER, J. P. AND KING, D. G. (1976). The stomatogastricnervous system: structure and function of a small neural network. Prog. Neurobiol. 1,215-289.

SIMMERS, A. J. AND MOULINS, M. (1988). A disynaptic sensorimotor pathway in the lobsterstomatogastric system. J. Neurophysiol. 59, 740-756.

TURRIGIANO, G. G. AND SELVERSTON, A. I. (1989). Cholecystokinin-like peptide is a modulatorof a crustacean central pattern generator. J. Neurosci. 9, 2486-2501.

TURRIGIANO, G. G. AND SELVERSTON, A. I. (1990). A cholecystokinin-like hormone activates afeeding-related neural circuit in lobster. Nature 344, 866-868.

VELLA, F. A. AND TRIPP, M. R. (1983). Strontium chloride inhibition of clotting in blue crabhemolymph. /. Invert. Pathol. 42, 400.

WEISS, K. R., KOCH, V. T., KOESTER, J., MANDELBAUM, D. E. AND KUPFERMANN, I. (1981).Neural and molecular mechanisms of food-induced arousal in Aplysia californica. InNeurobiology of Invertebrates, vol. 23 (ed. J. Salanki), pp. 305-344. Budapest: Pergamon.

humoral induction of pyloric rhythmic output in … · (pd) motor neurones, cl muscles by the...

Documents