Oscillatory command input to the motor pattern …post.queensu.ca/~locust/Publications/robertson...

J Comp Physiol (1981) 143:453 463 Journal of Comparative Physiology, A �9 Springer-Verlag 1981

Oscillatory Command Input to the Motor Pattern Generators of the Crustacean Stomatogastric Ganglion

I. The Pyloric Rhythm

Robert M. Robertson* and Maurice Moulins** Laboratoire de Neurobiologie Compar6e, CNRS et Universit6 de Bordeaux I, Place du DrPeyneau, F-33120 Arcachon, France

Accepted April 27, 1981

Summary. 1. In Homarus gammarus the central pattern generator of the pyloric rhythm (filtration to the midgut) is known to be located in the stomatogastric ganglion. The pyloric motor output is essentially generated by endogenous burster neurones (dilator neurones) which rhythmically inhibit follower neurones (constrictor neurones). However, in vitro recordings indicate that the pyloric motor output can be altered by an exogenous rhythmic input to the pyloric pattern generator (Fig. 2).

2. This phasic input, which excites the endogenous burster neurones, can be monitored by the subthreshold activity of an identified interneurone (F neurone) of the commissural ganglion (Fig. 3).

3. Blocking spike conduction between the stomatogastric ganglion and the commissural ganglion shows that this phasic input is generated, indepen- dently of the activity of the pyloric pattern generator, in the commissural ganglion (which contains a commissural pyloric oscillator, CPO) (Fig. 4).

4. There is a CPO in each commissural ganglion (Fig. 5).

5. It is shown that the pyloric motor output (monitored by the activity of the pyloric dilator neurones) exhibits several coordination modes with the CPO cycle (Fig. 6a, b) and that the pyloric bursts occur at preferred phases in the CPO period (Fig. 7a, b). This is an indication of a stable entrainment of the pyloric pattern generator by the CPO.

* Present address: Department of Physiology, The University of Alberta, Edmonton, Alberta, Canada T6G 2H7

** To whom correspondence should be addressed

Abbreviations: CG commissural ganglion; CPO commissural pyloric oscillator; STG stomatogastric ganglion; avn anterior ventricular nerve ; ion inferior oesophageal nerve ; ivn inferior ventricular nerve; lvn lateral ventricular nerve; mvn median ventricular nerve; son Superior oesophageal nerve; stn stomatogastric nerve; vlvn ventral lateral ventricular nerve; AB, PD, VD dilator neurones of the pyloric pattern generator; IC, LP, P Y constrictor neurones of the pyloric pattern generator

6. Dilator and constrictor motor neurones of the pyloric pattern generator can exibit different modes of coordination with the CPO (Fig. 6 c). This suggests that the CPO is able to modify not only quantitatively but also qualitatively the output of the pyloric pattern generator.

7. These results provide the first evidence that a pattern generator can be receiving separate phasic inputs containing timing cues able to participate in the generation of the motor output. In other words, our results suggest that a rhythmic motor behaviour can be organized by a hierarchy of linked oscillators (Fig. 9).

Introduction

Animal behaviour is built up by the sequential activation of different muscles which receive patterned motor discharges from the central nervous system. In deafferented animals and in isolated nervous systems, electrophysiological analysis of this patterned output has shown that it can be generated by a discrete group of neurones, even when these are isolated from the periphery and other regions of the central nervous system (Hagiwara 1961; Maynard 1972; Dellow and Lund 1971; Grillner and Zangger 1975; Wyman 1977). Such a discrete neuronal network has been called a 'pattern generator' and the concept has been particularly useful in the study of the neuronal control of rhythmical behaviour in vertebrates as well as in invertebrates (see Grillner 1977).

It is generally assumed that the expression of a pattern generator is under the control of command neurones or a command system and that these serve merely to turn on and/or maintain cycling of the pattern generator (Kupfermann and Weiss 1978). An

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454 R.M. Robertson and M. Moulins: Oscillatory Input to Motor Pattern Generators. I

i m p o r t a n t p r o p e r t y o f the re la t ionsh ip be tween com- m a n d input and pa t t e rned o u t p u t is tha t no specific t e m p o r a l pa t t e rn o f c o m m a n d d ischarge is necessary to elicit a perfect ly t imed ou tpu t f rom the pa t t e rn gene ra to r (Wi lson and W y m a n 1965; Stein 1977). Thus it is c o m m o n l y bel ieved, a l though no t d e m o n - s t ra ted (Gr i l lne r 1977), tha t pa t t e rn genera tors do no t receive i ndependen t phas ic inpu t con ta in ing t iming cues and able to pa r t i c ipa te in the gene ra t ion o f pa t t e rned ou tput .

In this p a p e r we d e m o n s t r a t e that , in the s t o m a t o - gastr ic ne rvous sys tem of d e c a p o d Crus tacea , phas ic act ivi ty with the f requency o f the py lor ic r h y t h m is genera ted comple t e ly wi thin each commissu ra l ganglion a l though the pa t t e rn genera to r for the py lor ic r h y t h m is k n o w n to be con ta ined in a d i s tan t gangl ion (the s t oma togas t r i c gangl ion) (Selvers ton et al. 1976). W e fur ther show tha t this commissu ra l ' p y l o r i c ' oscil- l a to r (CPO) projects to the py lor ic pa t t e rn genera tor , and affects its m o t o r ou tput . This p rovides the first evidence tha t a pa t t e rn gene ra to r can be inf luenced by separa te t iming in fo rmat ion .

The s toma togas t r i c ne rvous s y s t e m of d e c a p o d crus taceans con t ro l s the genera t ion o f a t least 4 different fo regut r hy thms and w o r k with these rhy thms has y ie lded results o f i m p o r t a n t genera l s ignif icance. In fact mos t o f the proper t ies , in te r re la t ionsh ips and con t ro l mechan i sms of rhy thmica l systems are exem- plif ied by this ' s i m p l e ' inver tebra te p r epa ra t i on . I t consists o f four gangl ia (the s tomatogas t r i c , the oesophagea l and the pa i red commissu ra l gangl ia) wi th

a to ta l o f a p p r o x i m a t e l y 1,400 neurones . The pa t t e rn genera to r for the oesophagea l r h y t h m (per is ta l t ic ; pe- r iod, 5-10 s) is as yet undesc r ibed bu t it is k n o w n to be loca ted in the oesophagea l and commissu ra l gangl ia and to be con t ro l l ed by a b i l a te ra l pa i r of osc i l la tors which p roduce a l t e rna ted series o f cycl ical act iv i ty (Moul ins and N a g y 1981). The gene ra to r of the ca rd i ac sac r h y t h m (s torage; per iod , 20-70 s) is s imi lar ly undesc r ibed bu t it is t hough t to have a m a j o r co -o rd ina t i ng role over to ta l fo regu t act ivi ty and to involve neurones in the oesophagea l gangl ion ( M o u - lins and Vedel 1977). I t is for the gastr ic ( t r i t u ra t ion ; per iod , 8-20 s) and pylor ic ( f i l t ra t ion; per iod , a r o u n d 1 s) rhy thms , which emana t e f rom the s toma togas t r i c gangl ion, tha t mos t i n f o r m a t i o n has accumula ted . The py lor ic pa t t e rn genera tor , which consists o f only 14 neurones , is p r o b a b l y one o f the bes t k n o w n pat - tern genera tors (Selvers ton et al. t976). W h e n the stoma togas t r i c gangl ion is d issected out connec ted with o ther ne rvous centres (Russel 1977) it is poss ib le to s tudy in vi t ro the con t ro l which can be exer ted by these centres on the py lor ic ou tput . The presen t w o r k was u n d e r t a k e n to f ind ou t if some independen t phas - ic inpu t can con t ro l the ac t iv i ty o f the py lor ic pa t t e rn

genera to r and affect the pylor ic output . A p re l imina ry r epo r t of this w o r k has been publ i shed elsewhere (Ro- be r t son and Mou l in s 1981).

Materials and Methods

All experiments were performed on aduIt Homarus gammarus which had been purchased locally and maintained in large tanks of circulating and aerated fresh sea water. Both males and females were used and there was no apparent difference in the results obtained with either. The foregut was removed from the animal and then the stomatogastric nervous system (Fig. l) dissected under saline (artificial sea water) and pinned in a Sylgard-lined Petri dish. The preparation was continuously perfused with cool, oxygen- ated saline. Prior to any recording the commissural and stomatogastric ganglia were desheathed by microdissection. To permit the blockage of conduction in certain nervous pathways, short lengths of selected connecting nerve trunks were desheathed and these portions were separately surrounded by walls of vaseline. The chambers thus formed were perfused either with saline or, for conduction blockage, with an isotonic (750 mM) sucrose solution (pH 7.4). In some experiments the stomatogastric ganglion (STG) was isolated from the other ganglia by blocking the stomatogastric nerve (stn). In other experiments three vaseline chambers (1, 2, 3 in Fig. 1) could be used (I) to isolate the three ganglia from each other (the STG and the 2 commissural ganglia, CG) (blockage in 1), (2) to keep one CG connected to the STG while the other CG is isolated (blockage in 2 and 3) or (3) to block only one of the superior oesophageaI nerve (son) and inferior oesophageal nerve (ion) (blockage in 2 or 3).

Extracellular activity was recorded by placing fine platinum wire electrodes against the nerve trunks and isolating them from the surrounding medium with vaseline. Intracellnlar recordings were taken from the cell bodies of neurones with fibre-filled glass microelectrodes pulled to a resistance of 15-30 MOhms and filled with 3 M KC1. Neurones in the STG were identified according to the muscles they innervate and their connections (Maynard 1972; Setverston et aI. 1976). To monitor extracellularly the output of the pyloric pattern generator, recordings were made from the lateral ventricular nerve (lvn) or the ventral lateral ventricular nerve (vlvn) in which the activity of the main dilator (PD) and constrictor (LP, PY) motor neurones can be identified. In Homarus recordings from the medial ventricular nerve vnvn) and the anterior ventricular nerve (avn) enabled the identification of VD and IC motor neurones respectively (Maynard and Dando 1974), which fire with the dilator neurones and the constrictor neurones. Finding the commissural interneurone which is described in this paper is difficult because each CG contains an estimated total of more than 685 neurones (Russell 1977), and there are no reliable morphological indications of their locations. However, once found, its axonal geometry, properties and connections enable it to be identified repeatedly in different preparations. Results were stored on 8 channel magnetic tape for subsequent filming and analysis. Figures 7 and 8 were prepared with the aid of a MINC II minicomputer (Digital) with a digitaliser (Calcomp) and a chart plotter (Tektronix). Some permanent visual records were taken during experiments using a 2 channel penre- corder (Brush) or a 8 channel electrostatic recorder (Gould ES 1000).

Results

The Pylor ic M o t o r Pat tern

The pylor ic m o t o r pa t t e rn can be recorded in vi t ro af ter i so la t ion o f the s toma togas t r i c ne rvous system

R.M. Rober tson and M. Moulins: Oscillatory Input to Motor Pattern

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Gvn Fig. 1. In vitro preparation of the stomatogastr ic nervous system. The pyloric pattern generator is localised in the stomatogastric ganglion (STG) (30 neurones) which is connected to the commissural gangli a (CG) by the single stomatogastric nerve (sin) and the paired superior and inferior oesophageal nerves (son and ion). Con- duction of action potentials between the STG and the CG can be blocked by perfusing isotonic sucrose solution in vaseline chambers (stippled rectangles) on the stn or on the other nerves (1, 2, 3). The pyloric output is recorded on the anterior ventricular nerve (avn), the medial ventricular nerve (mvn), the lateral ventricular nerve (lvn) and the ventral lateral ventricular nerve (vlvn). The CG are situated on the circumoesophageal connectives (cc), between the brain and the ventral cord; the stomatogastric nervous system is also connected to the brain via the inferior ventricular nerve (ivn). OG oesophageal ganglion

Generators. I 455

(Fig. 2 a). In this situation the output of the stomatogastric ganglion consists of an alternation between bursts of dilator motor neurones (PD) and bursts of constrictor motor neurones (LP, PY) (first trace in Fig. 2a). The IC and VD motor neurones, which control the cardiac valve, fire respectively with the pyloric con- strictors and the pyloric dilators (second and third trace in Fig. 2 a). It has been shown that this activity is underlain by the endogenous properties of three electrotonically coupled neurones (2 PD and AB) the membrane potentials of which exhibit regularly a wave of depolarisation with a burst of spikes inhibit- ing the constrictor neurones. All these neurones are located in the stomatogastric ganglion which is able to produce the pyloric output after isolation, at least after tonic stimulation of the only input nerve (the stomatogastric nerve). Thus it is clear that the pattern generator of the pyloric motor rhythm is located in the stomatogastric ganglion (Maynard 1972; Mayn- ard and Selverston 1975).

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Fig. 2a-e . The pyloric output, a It consists of a regular cycling of alternated bursts of dilator motor neurones (smaller spikes in the vlvn) and constrictor motor neurones (larger spikes in the vlvn). The IC motor neurone recorded in the avn and the VD motor neurone (larger spike in the rnvn) fire respectively with the pyloric constrictor and pytoric dilator neurones. According to Maynard and Dando (1974), the smaller spike recorded in the mvn belongs to the LC gastric motor neurone, b The pacemaker neurones (PD) receive phasic inputs which can induce a subthreshold depolarisation ( ~ ) or the strong depolarisations which underly bursts of spikes (circles). c IC bursts (as recorded in the avn) can occur when the pacemaker neurones (as recorded from the cell body of a PD neurone) are only slightly depolarised (single arrows) or not depolarised at all (double arrow). Scale: horizontal bars, 1 s; vertical bars, 10 mV

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R.M. Robertson and M. Moulins: Oscillatory Input to Motor Pattern Generators. I

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Fig. 3a-e. The commissural F cell. ~ The F ceil has an axon in the ivn; the oscilloscope sweep is triggered by the spike recorded in the cell body which was depolarised by injection of current, b Simultaneous intracellular recordings from F in the CG and AB (one of the pacemaker neurones of the pyloric pattern generator) in the STG shows that the 2 neurones exhibit time-locked epsps. e These epsps occur in bursts (as can be seen for the neurone F which is slightly hyperpolarised by injection of current to obtain larger synaptic events) which are time-locked with the pyloric output recorded from a PD neurone and from the lvn. In the third trace the two bursts of larger spikes belong to the gastric cycle. Scale: horizontal bars, 10 ms in 8, 500 ms in b, I s in e; vertical bars, 5 mV in a, 10 mV in b and e

to higher nervous centres (i.e. to the commissura l ganglia) (Fig. 1) the pyloric ou tpu t rhy thm is always apparent but it can be shown that, at this time, the higher nervous centres are no t only delivering tonic input bu t also phasic (rhythmic) input to at least some of the neurones o f the pyloric pat tern generator. Firstly the endogenous burster neurones (PD-AB) receive depolarising inputs expressed as bursts of epsps. These phasic inputs can induce only a subthreshold depolar isat ion or if sufficient the s trong depolar isat ion which underlies a burst o f spikes (Figs. 2b, 3b). Secondly at least some constr ic tor neurones as IC often burst at a f requency greater than the f requency of burst ing of the P D / A B group (which has always been considered as the pacemaker o f the pyloric pat tern generator) (Fig. 2c). In this case, in each cycle in which IC is burst ing wi thout the P D / A B neurones, the latter can exhibit an obvious depolar isat ion (single a r row in Fig. 2c) insufficient to give a burst of spikes. It is difficult to think that this depolar isat ion is sufficient to drive the constr ic tor neurones because sometimes these still cycle when PD does no t exhibit any depolar isat ion (double a r row in Fig. 2c). N o neurone in the s tomatogast r ic ganglion other than the P D / A B group has been shown to have endogenous oscillatory properties and thus it can be postulated that some phasic input, exoge-

nous to the s tomatogastr ic ganglion and the pyloric pat tern generator, is able to drive neurones of the pyloric cycle.

Identification o f the F Cell

To test this hypothesis the commissural ganglia were explored in an a t tempt to find a burst ing cell (or a g roup of burst ing cells) which could be at the origin of this input. A m o n g the 685 cells o f each ganglion one neurone, which is not at the origin of this input but which receives the same input, has been identified. This cell, with a soma diameter o f about 15 gm, is a follower of an oscil latory neurone or network and has been called the F cell (Rober t son and Moulins 1981). It can be identified by 3 characteristics:

(1) The F cell has an axon which runs to the brain in the inferior ventricular nerve (ivn) (Fig. 3 a) p robably via the ion (see Fig. 1).

(2) The F cell shows epsps which are t ime-locked with a constant latency to epsps exhibited by at least one of the endogenous burster neurones (AB) of the pyloric pat tern generator (Fig. 3 b). It is highly probable that F and AB are postsynapt ic to the same excitatory neurone.

(3) These epsps are always organised in bursts (i.e. the presynapt ic neurone is a burster neurone) and

R.M. Robertson and M. Moulins: Oscillatory Input to Motor Pattern Generators. I 457

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Fig. 4a-e. The activity of the phasic input is not generated by the pyloric pattern generator. The activity of the phasic input is monitored by recording from F in the commissural ganglion; the activity of the pyloric pattern generator is monitored by recording from a dilator neurone (VD in the mvn), a constrictor neurone (IC in the avn) and the viva. a Control. b Conduction is blocked in the stomatogastric nerve and the pyloric pattern generator stops cycling while the phasic input still cycles and activates F. c Recovery after unblocking the stomatogastric nerve. Scale: hoizontal bar, 1 s; vertical bars, 10 mV

the bursts are coordinated in some way with the pyloric output (Fig. 3c), This indicates that the pre- synaptic neurone gives a bursting input to the pyloric pattern generator. Thus F can be used to moni tor the activity of this phasic input.

Origin of the Phasic Input to the Pyloric Pattern Generator

By recording simultaneously from F and f rom neurones of the pyloric pattern generator (Fig. 4a) it is possible to show that the phasic input is not generated

by the pyloric pattern generator itself. This can be achieved by blocking conduction in the stomatogastric nerve with an isotonic sucrose solution. In this experimental situation the pyloric output of the stomatogastric ganglion usually stops (Fig. 4b) but the F cell in the commissural ganglion still receives bursts of epsps. Unblocking the stomatogastric nerve pro- duces complete recovery of the pyloric output which is again time-locked with the bursting activity received by the F cell (Fig. 4c). This not only shows that the phasic input is generated by a separate pyloric oscillator (not as a result of the activity of the pattern generator) but also that this pyloric oscillator is located


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Fig. 5a-c. There is a CPO in each commissural ganglion. In this experiment the pyloric output is recorded from the avn (in which the activity of the IC neurone can be identified) and from the vlvn (in which the activity of the PD neurones can be identified) and is marked by horizontal bars in a. The activity of the left CPO is monitored by the inputs to the left F cell. When the two CG are connected to the STG (a), the IC neurone can give bursts which are not correlated to F inputs (arrows) (first part of the record in a) or when the left F cell (and left CPO) is silent (second part of the record in a). This can be understood if it is assumed that the right CPO is responsible of these extra bursts. When the connection of the STG with the right CG are blocked (b) the left CPO regularly drives the IC and PD neurones (as was the right CPO in the second part or" a). Finally when the three ganglia are isolated (e) (by blocking with vaseline chamber 1; see Fig. 1) the pyloric output stops and the recorded left CPO is still cycling. Experimental situation indicated by an inset at the end of each recording. Scale: horizontal bars, 1 s; vertical bars 5 mV

anterior to the site o f conduc t ion block. Finally experiments where a commissural ganglion is com- pletely isolated (see Fig. 5c) show that the pyloric oscillator is located, as is the F cell, in the commissural ganglion. It will be called commissural pyloric oscillator (CPO).

B i l a t e r a l C P O s

It has no t been possible to penetrate s imultaneously bo th right and left F cells bu t there are s trong arguments to think that a CPO exists in each commissura l ganglion and drives the ipsilateral F cell.

In some experiments, for example the experiment of Fig. 5, the relationships between the input to the F cell (here in the left ganglion) and the ou tpu t o f the pyloric pat tern generator appear variable. The F input always precedes initiation of activity o f some pyloric neurones (Fig. 5a, first par t o f the record) but a neurone like IC (recorded in the avn) can give bursts impossible to correlate with any input to the recorded F cell (arrows in Fig. 5 a). These extra bursts

o f IC cannot be the result of the endogenous cycling of the pyloric pat tern generator because they can occur when the pacemaker neurones of the generator (PD/AB) are silent ( , in Fig. 5 a). I t can be assumed that these extra bursts o f IC are generated by the right CPO and that the lack o f coord ina t ion between the 2 CPO in this experiments is responsible for the irregularity o f the pyloric output . This is suggested by the fact that if the left CPO becomes silent, as attested by the complete failure of the F epsps (second par t o f Fig. 5a), the pyloric ou tpu t becomes regular. This can be experimentally demonst ra ted by blocking the right son and right ion (see Fig. 1) to isolate the right commissural ganglion and keep the s tomatogas- tric ganglion connected only to the left commissural ganglion. In this si tuation (Fig. 5 b), the pyloric output is regularly coordina ted with the C P O (moni tored by input to the ispilateral F cell) and identical with the output obtained when the left CPO was: silent (second par t o f Fig. 5a). This shows also that the 2 C P O are equivalent, i.e. have the same effects on the pyloric pat tern generator. Finally conduct ion


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Fig. 6a-e. Coordination modes between the activity of the CPO (monitored by F inputs) and the pyloric output (monitored by PD activity recorded from the vlvn and indicated by horizontal bars). In a a pyloric cycle occurs for each CPO cycle (coordination 1:1); in b a pyloric cycle occurs for two CPO cycles (coordination 2:1). In e a PD burst occurs for two CPO cycles (coordination 2:1) while an IC burst (recorded from the avn) occurs for each CPO cycle (coordination 1:1). Scale: horizontal bar, 1 s; vertical bars, 10 mV

block by using the vaseline chamber 1 (see Fig. 1) which isolates the three ganglia (the commissural and the stomatogastric ganglia) results in failure of the pyloric output (Fig. 5c). Nevertheless the left CPO still activates the left F cell and this confirms that in Fig. 5a (second part of the record) the pyloric output was probably induced by the right CPO.

Coordination of the Pyloric Pattern Generator with the CPO

It has been shown that an element of the CPO projects to AB (see Fig. 3b); this means that the group of the electrotonically coupled pacemaker neurones (PD/AB) of the pyloric generator receives an excitatory input from the CPO. Although not demonstrated it is highly probable that at least some constrictor neurones (LP, IC) are also receiving such rhythmic input from the CPO. This is attested by the fact that an excitatory burst received by F is always followed for example by an IC burst. IC seems to be much

more sensitive to CPO input than the PD/AB group (see e.g. Fig. 5b). Finally immediately following a burst of epsps in F there is always an alteration in the state of the pyloric cycle (see e.g. Fig. 5 a).

When a cycling system receives a rhythmic input it can be entrained. Each CPO when active is probably able to entrain the pyloric pattern generator but this is difficult to demonstrate because (1) it was impossible to modify experimentally the period of the CPO to study its effects on the pyloric generator and (2) the pattern generator receives input from 2 CPO the activities of which are not always coordinated, at least in our experimental conditions. Nevertheless an ex- amination of the spontaneous activity suggests that entrainment must exist.

Entrainment can be characterised by the facts that (1) the activities of the driven oscillator and of the driving oscillatory input are coordinated and (2) the driven oscillator is active at a preferred phase in the period of the driving oscillatory input (this preferred phase varying significantly with the period of the driv-


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Phase PD in F

Fig. 7a-d. Phase of pyloric output (monitored by the bursts of the PD/AB group) in the CPO period. The CPO period is measured from the onset of a burst of epsps in F to the onset of the next burst of epsps. The phase of the pyloric output is the time ratio between the onset of the burst of spikes of the PD/AB group (measured from the preceding onset of F epsps burst) and the corresponding CPO period. In a and b histograms there is clearly a preferred phase relationship of the pyloric output in the CPO period. The mean value of this phase relationship (0.47_+0.06 (SD) in a and 0.41 _+ 0.10 (SD) in b) varies with the mean value of the CPO period (1.80_+0.12 (SD) in a and 1.66+0.25 (SD) in b). In c histogram (which corresponds to the experiment of Fig. 5) the pyloric output did not exhibit a preferred phase relationship in the period of the recorded CPO (see Fig. 5a) but this preferred phase relationship appears when the STG is connected only to one CPO (d histogram), after blocking the conduction in the left ion and son (see Fig. 5b)

ing oscillator). Concerning the first point this is clearly what happened in many experiments in which the period of the pyloric output pattern is equal to the period of the CPO (Fig. 6a) or is double the CPO period (Fig. 6b). In such experiments an absolute coordination between the two oscillatory systems exists and this is respectively with 1 : 1 and 2:1 modes. Coordination between the CPO activity and the activity of single motor neurones of the pyloric pattern generator can also be observed and the coordination mode is not always the same for a given neurone as it is for the pacemaker neurones of the pattern generator. For example in Fig. 6c the coordination mode of the CPO activity with the IC neurone firing (recorded in the avn) is 1:1 while the coordination mode with PD firing (monitored in the vlvn) is 2:1.

Concerning the second point, when coordination occurs between the activity of the 2 oscillators there is always a preferred phase of activity for the pyloric output pattern (as monitored by PD activity) in the period of the CPO (Fig. 7a, b). The mean value of this phase relationship varies significantly with the mean value of the period of the CPO (compare

Fig. 7 a and b) and this is again evidence for entrainment. In some experiments as in Fig. 5a, the coordination between the two rhythms appears erratic and a preferred phase relationship for the activity of the pyloric output pattern in the period of the CPO cannot be demonstrated (Fig. 7c). Nevertheless it has been shown that in such experiments the activities of the two CPO are probably not coordinated (see Fig. 5a) and this can explain the results of Fig. 7c. If in the same experiment the influence of one CPO is removed by conduction block (see Fig. 5b), a preferred phase of activity for the pyloric output pattern in the period of the remaining CPO appears (Fig. 7d). It is also possible to test the phase in the CPO period of the activity of a single motor neurone. This was done for IC (Fig. 8) in the experiment of Fig. 5 and shows a strong preferred phase for the activity of IC. These results suggest again that some neurones (e.g. IC) are probably better entrained by the CPO than the pacemaker neurone of the pattern generator (compare Figs. 7d and 8b, concerning PD and IC respectively in the same experiment and same experimental conditions).


a)

5O

%Occ.

25

o

b) 50

% Occ.

25

N: 195

0-5 Phase IC in F

N: 250

1:0

65 b 1.0 Phase IC Jn F

Fig. 8a, b. Phase of the bursts of the IC motor neurone in the CPO period. The histograms correspond to the experiment of Fig. 5. The phase of IC bursts is measured in the same way as the phase of the PD/AB group in Fig. 7. In a the left and right CPO are connected to the STG while in b only the left CPO is connected to the STG (see inset). In b the motor neurone exhibits a preferred phase relationship in the left CPO period. In a the two CPO were active and the right CPO was probably responsible for the values above 0.3 which disappear in b after removing its influence

Discussion

The present results show that in each commissural ganglion there is one neurone which receives a bursting input correlated with the rhythmic motor output generated by the pyloric pattern generator in the stomatogastric ganglion. The bursting input to this F neurone can be recorded after total isolation of the commissural ganglion and it must be assumed that the commissural ganglion contains the oscillatory source of this input. This has been called the commissural pyloric oscillator (CPO). It seems to be the first example of an oscillator the activity of which is correlated with a well characterised behaviour and which does not directly drive a special set of motor neurones involved in this behaviour. In other words it seems to be the first example of rhythmic behaviour in which an oscillatory system is not an integral part of the pattern generator. This point will be discussed first. The present results also give evidence which shows that the CPO is able to modify the pyloric output generated by the pyloric pattern generator. Until now it has been assumed that the activity of a pattern generator is affected only by command neu-

rones (or command systems) which do not carry any timing cues. The demonstration of phasic command input to a pattern generator raises the question of its possible functional significance. This point will be discussed later.

The CPO Does Not Belong to the Pyloric Pattern Generator

It has been known for a long time (Maynard 1972; Maynard and Selverston 1975; Selverston et al. 1976) that the pytoric motor output can be recorded from an isolated stomatogastric ganglion. This means that the pyloric pattern generator, or at least a local centre of the pyloric pattern generator (Stein 1974), is located in the stomatogastric ganglion. Nevertheless, neurones firing with the pyloric output have already been described in the commissural ganglion of Panulirus (P neurones; Selverston et al. 1976) but their bursting firing is abolished when conduction is blocked in the stomatogastric nerve. It was possible to record from P neurones in Homarus and confirm that they belong to a long loop feedback system between the stomata- gastric ganglion and the commissural ganglion. A comparable long loop feedback between the spinal pattern generator and the higher nervous centres is known for the cat locomotory system (Orlovsky 1970; Arshavsky et al. 1972). It is clear neither F cell nor neurone(s) of the CPO cannot be compared with the commissural P neurones (and cannot belong to such a long loop system) because their bursting activity is not abolished when conduction between the stomatogastric and commissural ganglion is blocked.

Many complex behaviours are generated by dis- tributed pattern generators. For example locomotion in arthropods and mammals is generated by several local centres, each one driving one limb (Stein 1974, 1978). Each local centre has an oscillator able to produce a rhythmic output (Ikeda and Wiersma 1964; Grillner and Zangger 1974); it organises the temporal activation of the corresponding limb during locomotion. For such a complex behaviour the pattern generator consists of all the local centres connected to each other by coordinating neurones (Stein 1978). It could be argued that the CPO is only a local centre of the pyloric pattern generator which would possess another local centre in the stomatogastric ganglion. This can be disregarded because the CPO does not drive any commissural pyloric motor neurones. The anatomy of the pyloric muscles and their innervation are now well known (Maynard 1966; Maynard 1972; Maynard and Dando 1974; Selverston etal. 1976; Govind et al. 1976) and all the identified pyloric motor neurones (13) are located in the stomatogastric ganglion. The discovery of new pyloric motor neu-

462 R.M. Robertson and M. Moulins: OsciI1atory Input to Motor Pattern Generators. I

a)

Command Neurone 1 ( ~

Pattern Generator

PatternJd Output Pyloric

CPO

2 j~ -~ Pyloric

Pattern Generator

' Output

Fig. 9a, b. Comparison between the funtional status of command neurones (a) and the functional status of the CPO in the pyloric system of Homarus (b)

rones seems unlikely. Furthermore we never found any bursting activity whith the pyloric pattern in any of the output nerves of the commissural ganglion.

Finally, it can be argued that the CPO belong to a pattern generator responsible for another motor rhythm of the foregut. There are three other described foregut motor rhythms : the gastric rhythm (responsible of movements of the gastric teeth) (Selverston et al. 1976; Robertson and Moulins, in preparation); the oesophageal rhythm (Moulins and Nagy 1981) and the cardiac sac rhythm (Moulins and Vedel 1977). If the CPO was a local centre of one of these 3 pattern generators it must drive some corresponding motor neurones and its activity must exhibit some sort of coordination with activity of the appropriate pattern generator. As mentioned above no commissural motor neurone seems to follow the activity of the CPO. Moreover no coordination comparable with the one observed between the activity of the CPO and activity of the pyloric pattern generator was observed between the activity of the CPO and the activity of one of these pattern generators (which possess com- pletely different periods). Finally the CPO can be active for long periods when all the concerned pattern generators are silent. It is concluded that the CPO is an independent oscillatory system.

The CPO Can Probably Drive the Pyloric Motor Output

Because we were recording only from a follower cell of the CPO, it was not possible to modify its bursting frequency and to study directly its effects on the pyloric output. Nevertheless there are strong arguments to think that the CPO drives the pyloric output pattern :

(1) The pacemaker neurones (PD/AB) of the pyloric pattern generator receive an excitatory input

(burst of epsps) time-locked with the activity of the CPO ;

(2) at least some' follower' neurones of the pyloric pattern generator (e.g. IC) always fire after each burst of the CPO;

(3) in most experiments the timing of the activity of the pyloric pattern generator shows a strong correlation with the timing of the activity of the CPO. This correlation has the characteristics of an entrainment with absolute coordination;

(4) in the experiments in which this correlation does not appear clearly it can be shown that it is the result of the effects of two (left and right) uncoor- dinated CPO. In such an experiment if only one CPO remains connected to the stomatogastric ganglion an absolute coordination appears.

The pyloric pattern generator seems to be the first studied example of pattern generator receiving independent phasic input of central origin. This raised the question of the possible significance of such an input with regard to the pyloric output and it can be compared with the effects of a tonic input assumed to be carried by command neurones (see Fig. 9). The CPO projects probably directly to the pacemaker neurones of the pyloric pattern generator. It has been experimentally demonstrated that a rhythmic input is able to entrain these endogenous burster neurones (Ayers and Selverston 1979). Thus it must be assumed that when active the CPO is able to modify the period of the pyloric output, i.e., to speed up or slow pyloric cycling. This quantitative effect can also be obtained, at least experimentally, with a tonic input (Dando and Selverston 1972) which depolarises (or hyperpo- larises) the pacemaker neurones and alters the period of the pyloric output. Besides this quantitative effect, entrainment of the pacemaker neurones by the CPO will have a qualitative one, that is to say that the phase of bursting of the pacemaker neurones (and indirectly of the follower neurones) will be controlled. This means that such a phasic input not only deter- mines the period of the pyloric output but also its phase (time of occurrence) in another reference rhythm (for example another motor rhythm interesting another region of the foregut). This is impossible to obtain from a tonic input.

The CPO also probably projects on follower neurones of the pattern generator. At least the CPO seems to be responsible of the occurrence in the pyloric output of 'supernumerary extra bursts' of follower neurones. This is very clear for neurones such as IC when the coordination mode between the activity of the CPO and the pyloric output (monitored by PD bursts) is not 1:1 but 2:1 or 3:1 (see for example Fig. 2c and 5b). In such situations IC gives 1 or 2 'supernumerary extra bursts' (i.e. fires 2 or 3


times in each pyloric cycle). This can be explained by a very strong sensitivity of IC to the phasic activity of the CPO. Thus the firing pattern of follower neurones such as IC can be considered as the result of the combined effects of drive from both the CPO and from the timer neurones of the pattern generator (i.e. the PD/AB group). This shows that without dis- turbing the activity of the timer neurones of the pattern generator, a phasic input could be able to modify the output of the pattern generator. This seems to be a result of different sensitivities of the neurones of the pattern generator to this input.

In conclusion, the results support the proposal that the pyloric output rhythm is generated by the combined action of three oscillators (the PD/AB group and both CPO) and that the higher level oscillators (the CPO) are not part of the pyloric pattern generator but can act on it to alter the output rhythm both quantitatively and qualitatively. This is similar to the control system for the gastric rhythm (Robert- son and Moulins 1981) and will be discussed further in another paper (Robertson and Moulins in preparation). The existence of master high order oscillators driving motor pattern generators could be a general rule for the generation of rhythmic motor behaviours.

This work was supported by the DGRST (Grant 80.P.6049) and by a European Science Exchange Fellowship awarded to R.M.R. by the Royal Society and the CNRS. We thank Dr. F. Nagy for valuable suggestions and helpful criticism.

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