Modulation of rhythmic motor activity by pyrokinin peptides

Pyrokinin (PK) peptides localize to the central and peripheral nervous systems of arthropods, but their actions in the CNS have yet to be studied in any species. Here, we identify PK peptide family members in the crab Cancer borealis and characterize their actions on the gastric mill (chewing) and pyloric (filtering) motor circuits in the stomatogastric ganglion (STG). We identified PK-like immunolabeling in the STG neuropil, in projection neuron inputs to this ganglion, and in the neuroendocrine pericardial organs. By combining MALDI mass spectrometry (MS) and ESI tandem MS techniques, we identified the amino acid sequences of two C. borealis pyrokinins (CabPK-I, CabPK-II). Both CabPKs contain the PK family-specific carboxy-terminal amino acid sequence (FXPRLamide). PK superfusion to the isolated STG had little influence on the pyloric rhythm but excited many gastric mill neurons and consistently activated the gastric mill rhythm. Both CabPKs had comparable actions in the STG and these actions were equivalent to those of Pevpyrokinin (shrimp) and Leucopyrokinin (cockroach). The PK-elicited gastric mill rhythm usually occurred without activation of the projection neuron MCN1. MCN1, which does not contain CabPKs, effectively drives the gastric mill rhythm and at such times is also a gastric mill central pattern generator (CPG) neuron. Because the PK-elicited gastric mill rhythm is independent of MCN1, the underlying core CPG of this rhythm is different from the one responsible for the MCN1-elicited rhythm. Thus neuromodulation, which commonly alters motor circuit output without changing the core CPG, can also change the composition of this core circuit.     

Long-lasting activation of rhythmic neuronal activity by a novel mechanosensory system in the crustacean stomatogastric nervous system

Sensory neurons enable neural circuits to generate behaviors appropriate for the current environmental situation. Here, we characterize the actions of a population (about 60) of bilaterally symmetric bipolar neurons identified within the inner wall of the cardiac gutter, a foregut structure in the crab Cancer borealis. These neurons, called the ventral cardiac neurons (VCNs), project their axons through the crab stomatogastric nervous system to influence neural circuits associated with feeding. Brief pressure application to the cardiac gutter transiently modulated the filtering motor pattern (pyloric rhythm) generated by the pyloric circuit within the stomatogastric ganglion (STG). This modulation included an increased speed of the pyloric rhythm and a concomitant decrease in the activity of the lateral pyloric neuron. Furthermore, 2 min of rhythmic pressure application to the cardiac gutter elicited a chewing motor pattern (gastric mill rhythm) generated by the gastric mill circuit in the STG that persisted for < or =30 min. These sensory actions on the pyloric and gastric mill circuits were mimicked by either ventral cardiac nerve or dorsal posterior esophageal nerve stimulation. VCN actions on the STG circuits required the activation of projection neurons in the commissural ganglia. A subset of the VCN actions on these projection neurons appeared to be direct and cholinergic. We propose that the VCN neurons are mechanoreceptors that are activated when food stored in the foregut applies an outward force, leading to the long-lasting activation of projection neurons required to initiate chewing and modify the filtering of chewed food.     

Gastric and pyloric motor pattern control by a modulatory projection neuron in the intact crab Cancer pagurus

Neuronal release of modulatory substances provides motor pattern generating circuits with a high degree of flexibility. In vitro studies have characterized the actions of modulatory projection neurons in great detail in the stomatogastric nervous system, a model system for neuromodulatory influences on central pattern generators. Less is known about the activities and actions of modulatory neurons in fully functional and richly modulated network settings, i.e., in intact animals. It is also unknown whether their activities contribute to the motor patterns in different behavioral conditions. Here, we show for the first time the activity and effects of the well-characterized modulatory projection neuron 1 (MCN1) in vivo and compare them to in vitro conditions. MCN1 was always spontaneously active, typically in a rhythmic fashion with its firing being interrupted by ascending inhibitions from the pyloric motor circuit. Its activity contributed to pyloric motor activity, because 1) the cycle period of the motor pattern correlated with MCN1 firing frequency and 2) stimulating MCN1 shortened the cycle period while 3) lesioning of the MCN1 axon reduced motor activity. In addition, gastric mill motor activity was elicited for the duration of the stimulation. Chemosensory stimulation of the antennae moved MCN1 away from baseline activity by increasing its firing frequency. Following this increase, a gastric mill rhythm was elicited and the pyloric cycle period decreased. Lesioning the MCN1 axon prevented these effects. Thus modulatory projection neurons such as MCN1 can control the motor output in vivo, and they participate in the processing of exteroceptive sensory information in behaviorally relevant conditions.     

Neurons that form multiple pattern generators: identification and multiple activity patterns of gastric/pyloric neurons in the crab stomatogastric system

1. The stomatogastric ganglion (STG) of decapod crustaceans has been characterized by its production of two motor patterns, the gastric mill rhythm and the pyloric rhythm. The period of the gastric rhythm is typically 5-10 s, whereas the period of the pyloric rhythm is approximately 1 s. 2. In the STG of the crab, Cancer borealis, we find routinely that many motor neurons are active in time with both the pyloric and gastric rhythms. We rigorously identified the motor neurons according to the muscles they innervate. Some neurons usually classified as members of the pyloric network can be active in time with the gastric rhythm. All of the gastric motor neurons except the dorsal gastric (DG) neuron can generate pyloric-timed firing patterns. 3. Two motor neurons innervate muscles found in several different regions of the stomach. The inferior cardiac (IC) neuron, usually considered part of the pyloric network, innervates cardiac sac, gastric mill, and pyloric muscles. The lateral posterior gastric (LPG) neurons innervate muscles of both the gastric mill and the pyloric chamber. 4. These data show that the gastric and pyloric networks in the crab are not separate groups of neurons that independently generate two different rhythmic behaviors. Rather, these neurons together provide a synaptically connected pool of neurons from which many different pattern-generating circuits can be assembled, under different physiological conditions.     

Peptidergic modulation of a multioscillator system in the lobster. I. Activation of the cardiac sac motor pattern by the neuropeptides proctolin and red pigment-concentrating hormone

1. The cardiac sac motor pattern consists of slow and irregular impulse bursts in the motor neurons [cardiac sac dilator 1 and 2 (CD1 and CD2)] that innervate the dilator muscles of the cardiac sac region of the crustacean foregut. 2. The effects of the peptides, proctolin and red pigment-concentrating hormone (RPCH), on the cardiac sac motor patterns produced by in vitro preparations of the combined stomatogastric nervous system [the stomatogastric ganglion (STG), the paired commissural ganglia (CGs), and the oesophageal ganglion (OG)] were studied. 3. Bath applications of either RPCH or proctolin activated the cardiac sac motor pattern when this motor pattern was not already active and increased the frequency of the cardiac sac motor pattern in slowly active preparations. 4. The somata of CD1 and CD2 are located in the esophageal and stomatogastric ganglia, respectively. Both neurons project to all four of the ganglia of the stomatogastric nervous system. RPCH elicited cardiac sac motor patterns when applied to any region of the stomatogastric nervous system, suggesting a distributed pattern generating network with multiple sites of modulation. 5. The anterior median (AM) neuron innervates the constrictor muscles of the cardiac sac. The AM usually functions as a part of the gastric mill pattern generator. However, when the cardiac sac is activated by RPCH applied to the stomatogastric ganglion, the AM neuron becomes active in antiphase with the cardiac sac dilator bursts. This converts the cardiac sac motor pattern from a one-phase rhythm to a two-phase rhythm. 6. These data show that a neuropeptide can cause a neuronal element to switch from being solely a component of one neuronal circuit to functioning in a second one as well. This example shows that peptidergic "reconfiguration" of neuronal networks can produce substantial changes in the behavior of associated neurons.     

Spectral analyses reveal the presence of adult-like activity in the embryonic stomatogastric motor patterns of the lobster, Homarus americanus

The stomatogastric nervous system (STNS) of the embryonic lobster is rhythmically active prior to hatching, before the network is needed for feeding. In the adult lobster, two rhythms are typically observed: the slow gastric mill rhythm and the more rapid pyloric rhythm. In the embryo, rhythmic activity in both embryonic gastric mill and pyloric neurons occurs at a similar frequency, which is slightly slower than the adult pyloric frequency. However, embryonic motor patterns are highly irregular, making traditional burst quantification difficult. Consequently, we used spectral analysis to analyze long stretches of simultaneous recordings from muscles innervated by gastric and pyloric neurons in the embryo. This analysis revealed that embryonic gastric mill neurons intermittently produced pauses and periods of slower activity not seen in the recordings of the output from embryonic pyloric neurons. The slow activity in the embryonic gastric mill neurons increased in response to the exogenous application of Cancer borealis tachykinin-related peptide 1a (CabTRP), a modulatory peptide that appears in the inputs to the stomatogastric ganglion (STG) late in larval development. These results suggest that the STG network can express adult-like rhythmic behavior before fully differentiated adult motor patterns are observed, and that the maturation of the neuromodulatory inputs is likely to play a role in the eventual establishment of the adult motor patterns.     

Central pattern generating neurons simultaneously express fast and slow rhythmic activities in the stomatogastric ganglion

Neuronal firing patterns can contain different temporal information. It has long been known that the fast pyloric and the slower gastric motor patterns in the stomatogastric ganglion of decapod crustaceans interact. However, the bidirectional influences between the pyloric rhythm and the gastric mill rhythm have not been quantified in detail from preparations that spontaneously express both patterns in vitro. We found regular and stable spontaneous gastric and pyloric activity in 71% of preparations of the isolated stomatogastric nervous system of the lobster, Homarus americanus. The gastric [cycle period: 10.96 +/- 2.67 (SD) s] and pyloric (cycle period: 1.35 +/- 0.18 s) patterns showed bidirectional interactions and coordination. Gastric neuron firing showed preferred phases within the reference frame of the pyloric cycle. The relative timing and burst parameters of the pyloric neurons systematically changed within the reference frame of the gastric cycle. The gastric rhythm showed a tendency to run at cycle periods that were integer multiples of the pyloric periods, but coupling and coordination between the two rhythms were variable. We used power spectra to quantify the gastric and pyloric contributions to the firing pattern of each individual neuron. This provided us with a way to analyze the firing pattern of each gastric and pyloric neuron type individually without reference to either gastric or pyloric phase. Possible functional consequences of these network interactions for motor output are discussed.     

RCPH modulation of a multi-oscillator network: effects on the pyloric network of the spiny lobster

The neuropeptide red pigment concentrating hormone (RPCH), which we have previously shown to activate the cardiac sac motor pattern and lead to a conjoint gastric mill-cardiac sac pattern in the spiny lobster Panulirus, also activates and modulates the pyloric pattern. Like the activity of gastric mill neurons in RPCH, the pattern of activity in the pyloric neurons is considerably more complex than that seen in control saline. This reflects the influence of the cardiac sac motor pattern, and particularly the upstream inferior ventricular (IV) neurons, on many of the pyloric neurons. RPCH intensifies this interaction by increasing the strength of the synaptic connections between the IV neurons and their targets in the stomatogastric ganglion. At the same time, RPCH enhances postinhibitory rebound in the lateral pyloric (LP) neuron. Taken together, these factors largely explain the complex pyloric pattern recorded in RPCH in Panulirus.     

甲壳类动物胃肠神经系统的数值分析

目的数值分析甲壳类动物胃肠神经系统神经元的电位发放。方法利用抑制神经系统的WLC(Winner less Competition)模型计算得到胃研磨和幽门神经系统的节律变化,数值说明龙虾胃研磨内的两侧牙齿和中间牙齿的运动出现切断食物、挤压食物和研磨食物的状态;结果幽门节律出现神经元AB(anterior burster)、PD(pyloric dilatator)和VD(ventricular dilator)之间的同步共振;胃研磨神经系统和幽门神经系统之间显示神经元LPG(lateral posterior gastricneuron)对神经元VD的发放传递等。结论数值结果再现了龙虾胃肠神经系统的实验现象。     

Gastric mill activity in the lobster. III. Effects of proctolin on the isolated central pattern generator

1. The response of the isolated gastric central pattern generator (CPG) to bath application of proctolin is characterized and compared with the previously analyzed behavioral response. 2. Proctolin had an excitatory effect on the ongoing spontaneous rhythm of "combined" preparations, in which the stomatogastric ganglion (STG) is connected to the esophageal and commissural ganglia by the stomatogastric nerve (STN). The effect started between 20 s and 5 min and was characterized by strongly increased burst durations as well as increased spike rates in all units except the two lateral posterior gastric (LPG) motoneurons. The effect was strongest in the dorsal gastric (DG) and lateral gastric (LG) motoneurons and was accompanied by a phase change of the DG burst. DG continued spiking throughout large parts of the burst of LG and of the gastric mill (GM) motoneurons, which are antagonists of DG. 3. The threshold concentration was approximately 10(-10) M, and the effects were dose dependent and reversible. 4. LG and DG were identified as target cells for the action of proctolin. In LG regenerative plateau properties were induced, as revealed by its long-lasting plateau potentials, sensitivity for triggering inputs, and the occurrence of oscillatory prepotentials. An induction of endogenous bursting in DG was concluded from preparations, in which DG was cycling alone or bursting with a much shorter period duration than other gastric neurons. Hyperpolarization of DG, which normally has no or weak driving power within the gastric network, demonstrated that under the influence of proctolin, firing of DG can accelerate the gastric rhythm from a 27- to a 9-s period duration. 5. Proctolin does not only have a modulatory influence on an ongoing rhythm, but it also can trigger gastric activity. This function was first concluded from proctolin-treated STGs, which, unlike normal preparations, continue bursting if inputs via the STN are blocked. Finally, triggering was demonstrated directly, since isolated STGs that were not oscillating started a gastric rhythm after 20-30 min of perfusion with proctolin. 6. The proctolin-induced changes of the CPG activity in isolated preparations are in agreement with the effect on gastric mill chewing in the intact animal, in which, depending on the dose, different modes of chewing could be elicited.     

Recruitment of crab gastric mill neurons into the pyloric motor pattern by mechanosensory afferent stimulation

1. The gastropyloric receptor (GPR) cells are stretch-sensitive muscle receptors in the crab stomatogastric nervous system that use both 5-hydroxytryptamine (serotonin) and acetylcholine as cotransmitters. Brief stimulation of these afferent neurons causes two gastric mill neurons to be recruited into the pyloric motor pattern. 2. The GPR cells evoke complex synaptic potentials in the lateral gastric (LG) and medial gastric (MG) motor neurons, two component neurons of the gastric mill central pattern generator. When the gastric mill is quiescent (as often happens in vivo), GPR stimulation transiently inhibits LG and MG. After this transient inhibition, these cells undergo a prolonged excitation during which they fire bursts of action potentials at a constant phase relation to the pyloric motor pattern. 3. To determine the causes for this effect, we examined the effects of GPR stimulation on these two cells and on the inferior cardiac motor neuron, which is electrically coupled to them. When GPR is stimulated, all three cells receive rapid biphasic synaptic potentials that are blocked by nicotinic antagonists, followed by a slow, prolonged depolarizing potential. 4. The slow, prolonged depolarizing potential is not blocked by nicotinic or muscarinic cholinergic antagonists but is mimicked and occluded by exogenously applied serotonin. 5. The prolonged excitation, mediated at least in part by serotonin, may be responsible for the recruitment of the gastric mill neurons into the pyloric motor pattern. Thus sensory input can directly exert prolonged modulatory effects that change the functional cellular composition of pattern-generating circuits.     

Serotonergic/cholinergic muscle receptor cells in the crab stomatogastric nervous system. II. Rapid nicotinic and prolonged modulatory effects on neurons in the stomatogastric ganglion

1. The gastropyloric receptor (GPR) cells, which are described in the preceding paper, are a set of proprioceptive cells in the crabs Cancer borealis and Cancer irroratus that contain serotonin (5-hydroxytryptamine, 5-HT) and choline acetyltransferase. These cells have a variety of synaptic effects on cells in the stomatogastric ganglion (STG). We used pharmacologic methods to distinguish the effects that were due to acetylcholine (ACh) from those that could be due to serotonin. 2. The GPR cells evoke excitatory postsynaptic potentials (EPSPs) in two gastric mill motor neurons [lateral and dorsal gastric (LG and DG)] in the stomatogastric ganglion. The EPSPs exhibit nicotinic pharmacology, indicating that they may be due to the release of ACh from the GPR cells. 3. A train of GPR action potentials induces plateau potential properties in the DG motor neuron. This plateau potential induction is not blocked by nicotinic or muscarinic antagonists, suggesting it might be due to serotonin released from the GPR cells. Bath-applied serotonin induces a tonic depolarization of DG with high-intensity spiking. 4. In the accompanying paper, it is shown that DG-evoked muscle contraction leads to the excitation of GPR2 through mechanical coupling of the muscles. Because GPR2 also excites DG, a positive feedback loop exists between GPR2 and DG. This reflex loop may be involved in the control of the medial tooth of the gastric mill. 5. GPR stimulation initiates or enhances rhythmic pyloric cycling. This is due at least in part to a direct enhancement of bursting in the pyloric dilator/anterior burster (PD/AB) pacemaker cell group and can outlast the period of GPR stimulation by up to 1 min. GPR-induced PD burst enhancement continues in the presence of nicotinic and muscarinic antagonists, indicating that the effect is probably not due to the release of ACh. Bath application of serotonin mimicks the neuromodulatory effect of GPR stimulation on the PD/AB group by inducing or enhancing bursting. 6. Thus the GPR cells elicit at least three different synaptic actions in the stomatogastric ganglion: 1) classical, fast nicotinic cholinergic EPSPs that may be important for reflex functions in the gastric mill; 2) noncholinergic, cycle-by-cycle plateau potential induction that might be critical for the timing and operation of the gastric mill, and 3) prolonged, noncholinergic burst enhancement in pyloric neurons that is mimicked by serotonin, lasts many cycles, and may act to assure that the pyloric central pattern generator (CPG) is activated and cycling strongly.     

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.     

Mechanisms of gastric rhythm generation in the isolated stomatogastric ganglion of spiny lobsters: bursting pacemaker potentials, synaptic interactions, and muscarinic modulation.

1. The gastric central pattern generator (CPG), located in the stomatogastric ganglion (STG) of the spiny lobster (Panulirus interruptus), is nonrhythmic when deprived of neuromodulatory inputs from anterior ganglia. Leaving these inputs intact in vitro can sustain a gastric rhythm but also introduces numerous, uncontrolled and largely unknown modulatory and synaptic influences that greatly complicate analysis of this CPG. 2. Here we induced gastric rhythms in the isolated STG, by superfusing a specific modulator, the muscarinic agonist, pilocarpine. Muscarinic agents sustain vigorous gastric rhythms in the isolated STG. Our aim was to analyze the pattern-generating functions of the restricted gastric circuit, free of complicating influences from other ganglia, and under specific (muscarinic) modulation. 3. We used combinations of multiple cell hyperpolarizations, photodeletions, and synaptic blockade by picrotoxin to assess the pattern-generating role of individual gastric neurons and to study the activity of subcircuits. 4. Four identified gastric neurons [lateral gastric (LG), dorsal gastric (DG), 2 electrically coupled lateral posterior gastric (2LPGs)] acted as pattern-generating cells. They showed bursting pacemaker potentials (BPPs), i.e., plateau (or driver) potentials that underlay bursts of axonal spikes and slow, interburst depolarizing potentials that underlay repetitive burst activity. LG and DG, at least, became conditional bursters, able to burst repetitively because of intrinsic oscillations. The other gastric neurons behaved mainly as follower cells and derived their rhythmic bursting from synaptic coupling to the pattern-generator cells and from their own intrinsic (but nonoscillatory) properties. 5. The pattern-generating neurons form a novel "kernel" circuit that works by the cooperative interaction of cellular properties and synaptic connectivity. 6. This study constitutes the first complete and fully consistent analysis of pattern generation in the gastric network of the isolated STG. These mechanisms pertain to muscarinic rhythms in particular but also, we suggest, to gastric rhythm generation and CPG function in general. We suggest that 1) rhythmicity normally depends on the induction of bursty membrane properties in at least some component neurons; 2) different subcircuits can produce rhythmic patterns and may be activated by different modulators; and 3) the gastric network shares several important "building blocks" with CPGs that have been analyzed in other systems. 7. Muscarinic inputs are implicated as an important gastric regulator. We compare these responses with the reported modulatory actions of the anterior pyloric modulator (AMP), an identified, putatively cholinergic input interneuron that may act via muscarinic mechanisms.     

Location of interganglionic neurons in the stomatogastric system of the spiny lobster

Summary The stomatogastric ganglion produces distinct and complex patterned output driving the mastication and filtration of food. It does so with a small number of neurons whose properties and interconnections have been extensively examined. The motor patterns are subject to modulation and integration by neurons of other ganglia. This paper reports a search for interneurons of the four interconnecting ganglia of this system, using cobalt chloride backfilling techniques. It was determined that only a small number of neurons may interconnect these ganglia: (1) In thestomatogastric ganglion there are two to three small neurons and six large neurons with neurites projecting anteriorly towards the other ganglia. (2) In the twocommissural ganglia there are one to three small neurons whose neurites project to the primary input nerve of the stomatogastric ganglion. (3) In theoesophageal ganglion there are three small neurons whose location would allow them to play a co-ordinating role in the output of all four ganglia.     

Synaptic perturbation and entrainment of gastric mill rhythm of the spiny lobster

The gastric mill rhythm of the lobster stomatogastric ganglion was perturbed with short trains of synaptic input from the inferior ventricular nerve (IVN) through fibers. The stimulus was delivered randomly for phase-response curve analysis or repetitively to examine entrainment. The responses depend on the phase of the stimulus in the endogenous rhythm. The stimulus may alter the internal coordination of the motor pattern. Stimuli that occur during a lateral gastric nerve-anterior lateral nerve-E-neuron (LG-GM-E) burst perturb the burst internally and produce a prolonged LG-GM-E burst, while those that occur during the silent interval between LG-GM-E bursts may evoke a triggered LG-GM-E burst. Spontaneous, prolonged, and triggered LG-GM-E bursts differ in their internal structure as well as the order of burst onsets and offsets. The intercalated triggered LG-GM-E burst delays the occurrence of the subsequent spontaneous LG-GM-E burst, thus strongly resetting the rhythm. These resetting effects have been formalized by phase-response curve analysis. Over limited constraints, cyclic IVN stimuli can entrain the rhythm. Repetitively delivered IVN stimuli have parametric effects on the rhythm that mask the predictive value of phase-response curve analysis for the determination of the phase relations during entrainment.     

Mechanisms underlying type I mGluR-induced activation of lobster gastric mill neurons

In addition to ionotropic effects, glutamate and acetylcholine have metabotropic modulatory effects on many neurons. Here we show that in the stomatogastric ganglion of the lobster, glutamate, one of the main ionotropic neurotransmitters, modulates the excitability of gastric mill neurons. The neurons in this well-studied system produce rhythmic output to a subset of lobster foregut muscles. Recently, metabotropic glutamate receptor (mGluR) agonists were suggested as modulators of the rhythmic output, in addition to the previously described muscarinic modulation by acetylcholine. However, the cellular mechanisms responsible for these effects on the pattern are not known. Using intracellular recording methods and calcium imaging, we show that glutamate has an excitatory effect on specific neurons in the stomatogastric ganglion, which is mediated by mGluRs. Responses to the application of mGluR type I agonists are transient oscillations in the system, probably arising from network interactions. We show that the excitatory effect is sensitive to phospholipase-C and IP(3) and is G-protein dependent. The G-protein dependency was demonstrated by GDPbetaS and GTPgammaS injection into identified neurons. The depolarizations and oscillations were accompanied by an increase of intracellular Ca(2+) levels and correlated Ca(2+) oscillations. By using cyclopiazonic acid, an endoreticular Ca(2+) uptake inhibitor, we show that some internal calcium release may augment the response, but is not crucial for its production. Interestingly, although Ca(2+) concentration increase is typically associated with the phosphoinositide pathway, in the lobster, the Ca(2+) concentration increase-either voltage dependent or independent-cannot account for the observed depolarization.     

Group I, II, and III mGluR compounds affect rhythm generation in the gastric circuit of the crustacean stomatogastric ganglion

We have studied the effects of group I, II, and III metabotropic glutamate receptor (mGluR) agonists on rhythm generation by the gastric circuit of the stomatogastric ganglion (STG) of the Caribbean spiny lobster Panulirus argus. All mGluR agonists and some antagonists we tested in this study had clear and distinct effects on gastric rhythm generation when superfused over combined oscillating or blocked silent STG preparations. A consistent difference between group I agonists and group II and III agonists was that group I agonists acted excitatory. The group I-specific agonists L-quisqualic acid and (S)-3,5-dihydroxyphenylglycine, as well as the nonspecific agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid accelerated ongoing rhythms and could induce gastric rhythms in silent preparations. The group II agonist (2S,1'S, 2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) and the group III agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) slowed down or completely blocked ongoing gastric rhythms and were without detectable effect on silent preparations. The action of L-CCG-I was blocked partially by the group-II-specific antagonist, (RS)-1-amino-5-phosphonoindan-1-carboxylic acid [(RS)APICA], and the group-III-specific antagonist (RS)-alpha-methyl-4-phosphonophenylglycine completely blocked the action of L-AP4. Besides its antagonistic action, the group-II-specific antagonist (RS)APICA had a remarkably strong apparent inverse agonist action when applied alone on oscillating preparations. The action of all drugs was dose dependent and reversible, although recovery was not always complete. In our experiments, the effects of none of the mGluR-specific agonists were antagonized or amplified by the N-methyl-D-aspartate (NMDA)-receptor-specific antagonist D(-)-2-amino-5-phosphonopentanoic acid, excluding the contamination of responses to mGluR agonists by nonspecific cross-reactivity with NMDA receptors. Picrotoxin did not prevent the inhibitory action of L-CCG-I and L-AP4. We conclude that mGluRs, probably similar to those belonging to groups I, II, and III described in mammals, may play a role as modulators of gastric circuit rhythm generation in vivo.     

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

Summary The 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.     

Episodic bouts of activity accompany recovery of rhythmic output by a neuromodulator- and activity-deprived adult neural network

The pyloric rhythm of the stomatogastric ganglion of the crab, Cancer borealis, slows or stops when descending modulatory inputs are acutely removed. However, the rhythm spontaneously resumes after one or more days in the absence of neuromodulatory input. We recorded continuously for days to characterize quantitatively this recovery process. Activity bouts lasting 40-900 s began several hours after removal of neuromodulatory input and were followed by stable rhythm recovery after 1-4 days. Bout duration was not related to the intervals (0.3-800 min) between bouts. During an individual bout, the frequency rapidly increased and then decreased more slowly. Photoablation of back-filled neuromodulatory terminals in the stomatogastric ganglion (STG) neuropil had no effect on activity bouts or recovery, suggesting that these processes are intrinsic to the STG neuronal network. After removal of neuromodulatory input, the phase relationships of the components of the triphasic pyloric rhythm were altered, and then over time the phase relationships moved toward their control values. Although at low pyloric rhythm frequency the phase relationships among pyloric network neurons depended on frequency, the changes in frequency during recovery did not completely account for the change in phase seen after rhythm recovery. We suggest that activity bouts represent underlying mechanisms controlling the restructuring of the pyloric network to allow resumption of an appropriate output after removal of neuromodulatory input.