Maurice Moulins

The pyloric central pattern generator in Crustacea: a set of conditional neuronal oscillators

SummaryIn the stomatogastric nervous system of the Cape lobster,Jasus lalandii, all the neurons comprising the pyloric central pattern generator are cellular oscillators. This was shown by isolating in situ pyloric neurons from all other elements in the pyloric network. These neuronal oscillators are conditional and require modulatory inputs from anterior centers in order to express their oscillatory capabilities.1.Pyloric neurons were isolated from their counterparts in the network by photoinactivation of most of their presynaptic neurons (Miller and Selverston 1979, 1982a), and completed by pharmacological blockade of the remaining presynaptic influences. Under these conditions, all pyloric neurons display spontaneous bursting and underlying oscillations of their membrane potential.2.Both period and amplitude of oscillation in all isolated pyloric neurons vary when their membrane potential is modified by current injection. Furthermore, in isolated constrictor neurons (LP, PY and IC) the duration of the rhythmic regenerative depolarizations is also dependent on membrane potential.3.After isolation in situ, each type of pyloric neuron manifests a particular oscillatory behavior. The isolated interneuron AB has the fastest oscillation period with the shortest depolarizing phase. These oscillations closely resemble AB oscillations in the intact network. At the other extreme are the constrictor neurons (LP, PY and IC) which, when isolated, display spontaneous oscillations rather different from their oscillations in the intact network. Oscillations of isolated pyloric constrictor neurons are rhythmical plateaus whose duration and frequency are fairly irregular.4.When axonal conduction is blocked in the single input nerve to the stomatogastric ganglion, none of the isolated pyloric neurons is able to oscillate whether at rest or when their membrane potential is modified by current injection. Under these conditions, electrical stimulation of the input nerve on the ganglionic side of the block restores oscillations in isolated pyloric neurons.5.In conclusion, the pyloric network can be considered as a set of conditional oscillatory neurons, one of which, the interneuron AB, has a pacemaker function.

Expression of the crustacean pyloric pattern generator in the intact animal

Summary1.InJasus lalandii the activity of the central pattern generator of the pyloric motor output (the pyloric CPG) has been studied in intact free animals by electromyographic recording of the pyloric muscles (Fig. 2).2.Two patterns of activity (pattern 1 and pattern 2) can be observed. Each pattern corresponds to a specific behavioural situation: pattern 1 is only observed in unfed lobsters and feeding invariably produces a switch from pattern 1 to pattern 2 (Fig. 3).3.The two pyloric patterns differ in the period of the cycle (Fig. 4), the burst duration of the constrictors (Fig. 5), the latency of firing of the constrictors in the cycle (Fig. 6) and the phase of the constrictors in the cycle (Fig. 7). Correlation between period and constrictor latency is weak in pattern 1 and strong in pattern 2 (Fig. 8).4.The two pyloric patterns can be recorded from in vitro preparations only when the stomatogastric ganglion (i.e. the CPG) is connected to the more rostral commissural ganglia (Fig. 9).5.At least in pattern 1, cycling of the constrictors can occur when the dilators (known as pacemakers in the CPG) are silent (Fig. 10).6.For these reasons, we concluded that a commissural oscillator could be participating in the organization of the pyloric output. However, the synaptic relationships which exist between the motor neurones (i.e. the CPG) cannot be neglected (Fig. 11) and this suggests that, in the intact animal, the motor output is organized by the cooperation of a premotor oscillator and a motor oscillator.

Myogenic oscillatory activity in the pyloric rhythmic motor system of Crustacea

Summary1.The dorsal dilator muscle of the pylorus of the shrimpPalaemon (Fig. 1) is innervated, as in lobsters, by two electrically coupled excitatory motorneurons, the pyloric dilator (PD) neurons located in the stomatogastric ganglion (Fig. 2). The PD motorneurons are conditional oscillators and, when bursting, they rhythmically drive the pyloric dilator muscle (Fig. 3).2.When isolated the pyloric dilator muscle can undergo spontaneous rhythmic contractions with associated electrical membrane events. This spontaneous rhythmic activity is not of neural origin since: (1) it cannot be correlated with any injury discharge in the cut motor nerve; (2) it remains unaffected by bath application of tetrodotoxin to suppress neuronal spiking (Fig. 4).3.Electrical stimulation of the motor nerve or membrane depolarization with injected current shows that an otherwise quiescent pyloric dilator muscle can express either a non-oscillatory state (Fig. 5A, C, E) or an oscillatory state (Fig. 5 B, D, F). It is always possible to switch from the non-oscillatory. state to the oscillatory state by bath application of dopamine (Fig. 6); it is concluded that the muscle is a conditional oscillator.4.In its oscillatory state the pyloric dilator muscle displays properties characteristic of endogenous oscillators. These include: (1) phasic response to tonic stimulation; (2) voltage-dependence of the cycle frequency of the rhythmic activity (Fig. 7); (3) ability of cycling to be reset by a brief stimulus (Fig. 8); (4) ability to be entrained by repetitive stimuli (Fig. 9).5.The pyloric dilator neuromuscular system ofPalaemon thus appears to consist of a conditional motorneuronal oscillator (PD) and a conditional muscle oscillator; the second can be entrained by the first (Fig. 10). The functional implications of such a neuromuscular control system are tested (Fig. 11) and discussed.

Identification of all GABA-immunoreactive neurons projecting to the lobster stomatogastric ganglion

SummaryThe stomatogastric ganglion of lobsters (Homarus or Jasus) contains a large number of gamma-aminobutyric acid-immunoreactive processes originating from ten fibres in the single input nerve, the stomatogastric nerve. The cell bodies and axonal pathways of these ten fibres have been identified using gamma-aminobutyric acid immunohistochemistry in combination with Lucifer Yellow staining (double labelling) and nickel chloride backfilling (selective gamma-aminobutyric acid immunoinhibition).It is shown that eight gamma-aminobutyric acid-immunoreactive neurons project to the stomatogastric ganglion: gamma-aminobutyric acid neurons 1 and 2, found posterior to the oesophageal ganglion, entering the stomatogastric nerve via the oesophageal nerve as well as sending an axonal branch into each superior oesophageal nerve; gamma-aminobutyric acid neurons 3 and 4, found anterior to the oesophageal ganglion, each sending an axonal branch into each inferior oesophageal nerve to reach the stomatogastric nerve via the commissural ganglion and the superior oesophageal nerve; and gamma-aminobutyric acid neurons 5 and 6, found in each commissural ganglion, projecting into the stomatogastric nerve via the inferior oesophageal nerve, the oesophageal ganglion and the oesophageal nerve.These gamma-aminobutyric acid-immunoreactive neurons were also characterized by electrophysiological methods coupled with Lucifer Yellow labelling, and their picrotoxin-sensitive effects on several stomatogastric ganglion neurons were demonstrated.The present results provide a firm basis for further studies concerning the physiological significance of one class of neurochemically-defined input neurons to stomatogastric ganglion networks.

Tetrodotoxin-sensitive dendritic spiking and control of axonal firing in a lobster mechanoreceptor neurone.

1. A primary mechanosensory neurone, the anterior gastric receptor (AGR) associated with gastric mill muscle in the lobster foregut was examined in vitro with extra‐ and intra‐cellular recording techniques to understand processes of dendritic integration and dendro‐axonal communication. 2. AGR has a ‘T’‐shaped geometry; its two long (> 3 mm) primary dendrites project distally to spatially separate, stretch sensitive terminals and converge centrally onto a common apical neurite that leads to a bipolar soma and single axon. 3. The receptors bilateral dendrites are independently capable of generating action potentials. These appear to be Na+ dependent since they are blocked by tetrodotoxin, but not by Co2+ or a lack of Ca2+ in the bath saline. 4. Both dendrites are autogenically active, although impulses in the dendrite with the higher intrinsic excitability may cross over and activate the trigger zone on the contralateral side. Moreover, spikes arising on either dendrite do not actively invade the soma, but are conveyed as decremented potentials to a third trigger zone on the initial axon segment. 5. Focal applications of TTX (tetrodotoxin) demonstrated the existence and allowed precise definition of a central membrane compartment of AGR that appears to lack in functional Na+ channels. This inexcitable region includes the soma, the apical neurite and the central branch point of the two dendrites. A failure to observe collision block of bilateral dendritic potentials as they traverse the neurite supported this conclusion. 6. Horseradish peroxidase injections and staining revealed two morphological features of the apical neurite that differed markedly from other regions of the cell. In addition to a relatively large diameter, the neurites plasma membrane is heavily convoluted and coiled to form a lamellar transverse profile. This latter feature may itself contribute to membrane inexcitability while the former is consistent with an elevated space constant for electrotonic conduction. 7. It is concluded that the inhomogeneous distribution of membrane excitability in AGR enhances the integrative capability of the receptors dendrites, permitting mechanical input at diverse loci to be encoded and processed prior to transformation into axonal discharge.

Modulation and dynamic specification of motor rhythm-generating circuits in crustacea

The operation of central pattern generators (CPGs), oscillatory neural circuits responsible for rhythmic motor behavior, is now known to depend both on the synaptic interactions between constituent neurons and their intrinsic membrane properties (oscillatory, plateauing, etc). Moreover, these synaptic and cellular properties are not invariant, but are subject to a wide range of neuromodulatory influences that, by modifying the bioelectrical character of individual neurons and/or the strength of their synapses, are able to adapt the output of a given CPG circuit to the changing needs of the animal. Despite this ability to produce different functional configurations, however, the assumption remains of a CPG as a predefined assemblage of interconnected neurons dedicated to a particular behavior and functionally distinguishable from other circuits responsible for other tasks. However, our recent studies on the stomatogastric nervous system (STNS) of crustacea have begun to question this concept of the CPG as a discrete and identifiable entity within the central nervous system. Here we review evidence showing that under neuromodulatory instruction, individual neurons can participate in different oscillatory motor circuits and hence more than one rhythmic behaviour, and even more profoundly, preexisting networks can be dismantled to specify dynamically a new circuit for an entirely different behaviour. This de novo network construction is achieved again by neuromodulatory-induced alterations in the oscillatory and synaptic properties of individual target neurons. On this basis, therefore, a functional CPG network must be seen in a more dynamic context than previously thought since it may exist only in a particular behavioural situation dictated by modulatory influences.

Oscillatory command input to the motor pattern generators of the crustacean stomatogastric ganglion

Summary1.InHomarus 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, independently 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. 6 a, b) and that the pyloric bursts occur at preferred phases in the CPO period (Fig. 7 a, b). This is an indication of a stable entrainment of the pyloric pattern generator by the CPO.6.Dilator and constrictor motor neurones of the pyloric pattern generator can exibit different modes of coordination with the CPO (Fig. 6c). 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).

CONTROL OF RHYTHMIC BEHAVIOUR BY A HIERARCHY OF LINKED OSCILLATORS IN CRUSTACEA

In Homarus, the central pattern generators for the rhythmic motor activities of the gastric teeth and the pyloric chamber are located in the stomatogastric ganglion. It is shown that independent gastric and pyloric oscillators are also contained in higher nervous centres (the commissural ganglia) and provide a phasic rhythmic input to the stomatogastric pattern generators. This demonstrates that rhythmic behaviour can be organized by a hierarchy of linked oscillators each capable of producing the rhythm.

Conditional dendritic oscillators in a lobster mechanoreceptor neurone.

1. Intra‐ and extracellular recordings were made from in vitro preparations of the lobster (Homarus gammarus) stomatogastric nervous system to study the nature and origin of pacemaker‐like activity in a primary mechanoreceptor neurone, the anterior gastric receptor (AGR), whose two bilateral stretch‐sensitive dendrites ramify in the tendon of powerstroke muscle GM1 of the gastric mill system. 2. Although the AGR is known to be autoactive, we report here that in 20% of our preparations, rather than autogenic tonic discharge, the receptor fired spontaneously in discrete bursts comprising three to ten action potentials and repeating at cycle frequencies of 0.5‐2.5 Hz in the absence of mechanical stimulation. Intrasomatic recordings revealed that such rhythmic bursting was driven by slow oscillations in membrane potential, the frequency of which was voltage sensitive and dependent upon the level of stretch applied to the receptor terminals of the AGR. 3. Autoactive bursting of the AGR originated from an endogenous oscillatory mechanism in the sensory dendrites themselves, since (i) during both steady, repetitive firing and bursting, somatic and axonal impulses were always preceded 1:1 by dendritic action potentials, (ii) hyperpolarizing the AGR cell body to block triggering of axonal impulses revealed attenuated somatic spikes that continued to originate from the two peripheral dendrites, (iii) the timing of burst firing could be phase reset by brief electrical stimulation of either dendrite, and (iv) spontaneous bursting continued to be expressed by an AGR dendrite after physical isolation from the GM1 muscle and the stomatogastric nervous system. 4. Although a given AGR in vitro could switch spontaneously from dendritic bursting to tonic firing and vice versa, exogenous application of micromolar (or less) concentrations of the neuropeptide F1 (TNRNFLRFamide) to the dendritic membrane could rapidly and reversibly switch the receptor firing pattern from repetitive firing to the bursting mode. Exposure of the somatic and axonal membrane of the AGR to F1 was without effect, as were applications of other neuroactive substances such as serotonin, octopamine and proctolin. 5. We conclude that, as for many oscillatory neurones of the central nervous system, the intrinsic activity pattern of this peripheral sensory neurone may be dynamically conferred by extrinsic modulatory influences, presumably according to computational demands. Moreover, the ability of the AGR to behave as an endogenous burster imparts considerable integrative complexity since, in this activity mode, sensory coding not only occurs through the frequency modulation of on‐going dendritic bursts but also via changes in the duration of individual bursts and their inherent spike frequencies.

Extrinsic Inputs and Flexibility in the Motor Output of the Lobster Pyloric Neural Network

In recent years, our understanding of motor behavior in terms of single-cell activity has been primarily concerned with determining the functional structure of central pattern generators (CPGs) (Selverston, 1980). To analyze such a structure, i.e., to identify the neuronal components and determine the mutual interactions between these components, “naked” CPGs (i.e., completely deafferented CPGs) must be used. Nevertheless, the patterned activity that can be recorded from such an isolated CPG is relatively stereotyped, and until now, little was known about the mechanisms by which such a network could exhibit flexibility in its output in the intact animal. The goal of this chapter is to show how extrinsic inputs to a well-known CPG (the pyloric network of Crustacea, see Chapter 3, this volume) can continuously control the expression of the intrinsic properties of the neurons and thereby continuously “rewire” the network.