A newly identified buccal interneuron initiates and modulates feeding motor programs in aplysia

Despite considerable progress in characterizing the feeding central pattern generator (CPG) in Aplysia, the full complement of neurons that generate feeding motor programs has not yet been identified. The distribution of neuropeptide-containing neurons in the buccal and cerebral ganglia can be used as a tool to identify additional elements of the feeding circuitry by providing distinctions between otherwise morphologically indistinct neurons. For example, our recent study revealed a unique and potentially interesting unpaired PRQFVamide (PRQFVa)-containing neuron in the buccal ganglion. In this study, we describe the morphological and electrophysiological characterization of this novel neuron, which we designate as B50. We found that activation of B50 is capable of producing organized rhythmic output of the feeding CPG. The motor programs elicited by B50 exhibit some similarities as well as differences to motor programs elicited by the command-like cerebral-to-buccal interneuron CBI-2. In addition to activating the feeding CPG, B50 may act as a program modulator.     

Central peptidergic neurons regulate gut motility in Aplysia

1. The small cardioactive peptides (SCPs) are potent modulatory neuropeptides in Aplysia. Buccal ganglia neurons B1 and B2 are the largest neurons that exhibit SCP-like immunoreactivity. High-pressure liquid chromatography (HPLC)-bioassay and in vivo radiolabeling procedures confirm that these neurons contain and synthesize very large quantities of SCPA and SCPB. 2. Both B1 and B2 innervate the gut. HPLC-bioassay measurements indicate that the SCPs are present throughout the anterior sections of the gut. SCP-like immunoreactivity was largely confined to fibers and varicosities in the gut, although occasional immunoreactive enteric neurons were also observed. The purpose of this study was to determine the physiological roles of B1 and B2 and to what extent these roles are mediated by release of the SCPs. 3. Low-frequency tonic stimulation of B1 led to an increase in peristaltic contractions in a relatively distal portion of the gut. This action could be mimicked by superfusion of the same portion of the gut with very low concentrations of the SCPs. 4. B2 produced discrete contractions of the anterior portions of the gut only when fired in bursts. These actions could not be reproduced by superfusion with the SCPs and may be mediated by ACh. 5. B1 and/or B2 are active during the swallowing cycle of each feeding movement, which suggests that these effects on the gut are likely to occur during feeding. Thus the SCPs play a major role in the central regulation of gut motility.     

Fast synaptic connections from CBIs to pattern-generating neurons in Aplysia: initiation and modification of motor programs

Consummatory feeding movements in Aplysia californica are organized by a central pattern generator (CPG) in the buccal ganglia. Buccal motor programs similar to those organized by the CPG are also initiated and controlled by the cerebro-buccal interneurons (CBIs), interneurons projecting from the cerebral to the buccal ganglia. To examine the mechanisms by which CBIs affect buccal motor programs, we have explored systematically the synaptic connections from three of the CBIs (CBI-1, CBI-2, CBI-3) to key buccal ganglia CPG neurons (B31/B32, B34, and B63). The CBIs were found to produce monosynaptic excitatory postsynaptic potentials (EPSPs) with both fast and slow components. In this report, we have characterized only the fast component. CBI-2 monosynaptically excites neurons B31/B32, B34, and B63, all of which can initiate motor programs when they are sufficiently stimulated. However, the ability of CBI-2 to initiate a program stems primarily from the excitation of B63. In B31/B32, the size of the EPSPs was relatively small and the threshold for excitation was very high. In addition, preventing firing in either B34 or B63 showed that only a block in B63 firing prevented CBI-2 from initiating programs in response to a brief stimulus. The connections from CBI-2 to the buccal ganglia neurons showed a prominent facilitation. The facilitation contributed to the ability of CBI-2 to initiate a BMP and also led to a change in the form of the BMP. The cholinergic blocker hexamethonium blocked the fast EPSPs induced by CBI-2 in buccal ganglia neurons and also blocked the EPSPs between a number of key CPG neurons within the buccal ganglia. CBI-2 and B63 were able to initiate motor patterns in hexamethonium, although the form of a motor pattern was changed, indicating that non-hexamethonium-sensitive receptors contribute to the ability of these cells to initiate bursts. By contrast to CBI-2, CBI-1 excited B63 but inhibited B34. CBI-3 excited B34 and not B63. The data indicate that CBI-1, -2, and -3 are components of a system that initiates and selects between buccal motor programs. Their behavioral function is likely to depend on which combination of CBIs and CPG elements are activated.     

Localization and identification of neurons with cholecystokinin and gastrin-like immunoreactivity in wholemounts of Aplysia ganglia

Immunohistochemical procedures were applied to wholemounts of the central nervous system and posterior intestine of the mollusc, Aplysia californica, to facilitate localization of cells that were immunoreactive to several antisera recognizing various epitopes of the peptides cholecystokinin and gastrin. Only antisera that recognized the carboxyl terminal sequence common to cholecystokinin and gastrin reacted with the Aplysia tissues tested. Intracellular electrophysiological studies of identified postsynaptic targets of immunoreactive neurons in the cerebral ganglia indicated that mammalian forms of gastrin 1-17, several cholecystokinin fragments, and the related peptide, amphibian caerulein, did not mimick the synaptic response mediated by the immunoreactive presynaptic neurons. Combinations of electrophysiological, immunohistochemical, and biochemical studies of several neurons in the buccal ganglia indicated that neurons B7 and B13 were immunoreactive to antisera against cholecystokinin and gastrin and that neuron B13 also contained a concentration of the neurotransmitter acetylcholine as high as in the identified cholinergic buccal neurons, B4 and B5. Several differences in the immunoreactivity of the various antisera were observed. Only one of the antisera was effective in staining neurons in the abdominal ganglia and another antiserum stained subsets of neurons that were immunoreactive to most of the other antisera recognizing the carboxyl terminus common to cholecystokinin and gastrin. The giant serotoninergic metacerebral neurons in Aplysia were not immunoreactive to the cholecystokinin/gastrin antisera even though it has been reported that the homologous neurons in a pulmonate mollusc contain cholecystokinin-like immunoreactivity. These studies demonstrated that there are many neurons with cholecystokinin/gastrin-like immunoreactivity in the Aplysia central and peripheral nervous system and suggested that the peptide may differ from vertebrate forms of cholecystokinin and gastrin. The identification of immunoreactive neurons with known postsynaptic target neurons and buccal neurons with acetylcholine co-localized with a cholecystokinin/gastrin-like peptide will facilitate elucidation of the functions of peptides in the nervous system since the Aplysia preparation is well known to be amenable to multidisciplinary studies.     

Crustacean cardioactive peptide (CCAP)-related molluscan peptides (M-CCAPs) are potential extrinsic modulators of the buccal feeding network in the pond snail Lymnaea stagnalis

We combine electrophysiological and immunocytochemical analyses in the snail Lymnaea stagnalis of M-CCAP1 and M-CCAP2, two molluscan peptides with structure similar to crustacean cardioactive peptide CCAP, originally isolated from the snail Helix pomatia. Both M-CCAP peptides (M-CCAP1 and M-CCAP2, 1 microM) had an excitatory effect, depolarizing all the identified neurons of the buccal feeding network (including motoneurons: B1, B2, B4 and modulatory interneurons SO, OC: 62 neurons in 33 preparations). Additionally, in 67% of preparations, rhythmic activity (fictive feeding) was recorded with a mean rate of 7 cycles/min. No significant difference in the proportion of preparations showing fictive feeding or mean feeding rate was found between M-CCAP1 and M-CCAP2. The extrinsic feeding modulator, the serotonergic CGC neuron, responds by increase of the spontaneous activity after M-CCAP application (9 of 18 preparations). Crustacean CCAP (1 microM) evokes a slight membrane depolarization in 3 out of 8 preparations but never evokes fictive feeding. Immunostaining revealed no cell bodies in the buccal ganglia, but a dense network of CCAP immunopositive fibers arborizing in the buccal neuropil. Many of these fibers originate from a symmetrical pair of CCAP-immunoreactive cerebro-buccal interneurons, which are the most likely candidates for extrinsic modulatory interneurons in the buccal feeding network. Our data are the first results suggesting that M-CCAP-peptides exist as effective modulators in mollusc.     

Glutamate is a fast excitatory transmitter at some buccal neuromuscular synapses in Aplysia.

Studies of the modulation of synaptic transmission in buccal muscle of Aplysia were limited because the conventional fast transmitter used by a number of large buccal motor neurons was unknown. Most of the identified buccal motor neurons are cholinergic because they synthesize acetylcholine (ACh) and their excitatory junction potentials (EJPs) are blocked by the cholinergic antagonist hexamethonium. However, three large identified motor neurons (B3, B6, and B38) do not synthesize ACh and their EJPs are not inhibited by hexamethonium. To identify the fast excitatory transmitter used by these noncholinergic motor neurons, we surveyed putative transmitters for their ability to evoke contractions. Of the noncholinergic transmitters tested, glutamate was the most effective at evoking contractions. The pharmacology of the putative glutamate receptor is different from previously characterized glutamate receptors in that glutamate agonists and antagonists previously used to classify glutamate receptors had little effect in this system. In addition, glutamate itself was the most effective agent tested at reducing EJPs evoked by the noncholinergic motor neurons presumably by desensitizing glutamate receptors. Finally, immunocytology using an antiserum raised to conjugated glutamate in parallel with intracellular fills indicated that the varicose axons of these motor neurons were glutamate-immunoreactive. Taken together, these results indicate that the fast transmitter used by the noncholinergic neurons is almost certainly glutamate itself. This information should help us understand the role of transmitters and cotransmitters in the generation of feeding behaviors in Aplysia.     

Modulation of fictive feeding by dopamine and serotonin in aplysia

The buccal ganglia of Aplysia contain a central pattern generator (CPG) that mediates rhythmic movements of the buccal apparatus during feeding. Activity in this CPG is believed to be regulated, in part, by extrinsic serotonergic inputs and by an intrinsic and extrinsic system of putative dopaminergic cells. The present study investigated the roles of dopamine (DA) and serotonin (5-HT) in regulating feeding movements of the buccal apparatus and properties of the underlying neural circuitry. Perfusing a semi-intact head preparation with DA (50 microM) or the metabolic precursor of catecholamines (L-3-4-dihydroxyphenylalanine, DOPA, 250 microM) induced feeding-like movements of the jaws and radula/odontophore. These DA-induced movements were similar to bites in intact animals. Perfusing with 5-HT (5 microM) also induced feeding-like movements, but the 5-HT-induced movements were similar to swallows. In preparations of isolated buccal ganglia, buccal motor programs (BMPs) that represented at least two different aspects of fictive feeding (i.e., ingestion and rejection) could be recorded. Bath application of DA (50 microM) increased the frequency of BMPs, in part, by increasing the number of ingestion-like BMPs. Bath application of 5-HT (5 microM) did not significantly increase the frequency of BMPs nor did it significantly increase the proportion of ingestion-like BMPs being expressed. Many of the cells and synaptic connections within the CPG appeared to be modulated by DA or 5-HT. For example, bath application of DA decreased the excitability of cells B4/5 and B34, which in turn may have contributed to the DA-induced increase in ingestion-like BMPs. In summary, bite-like movements were induced by DA in the semi-intact preparation, and neural correlates of these DA-induced effects were manifest as an increase in ingestion-like BMPs in the isolated ganglia. Swallow-like movements were induced by 5-HT in the semi-intact preparation. Neural correlates of these 5-HT-induced effects were not evident in isolated buccal ganglia, however.     

Esophageal mechanoreceptors in the feeding system of the pond snail, Lymnaea stagnalis

1. We identify esophageal mechanoreceptor (OM) neurons of Lymnaea with cell bodies in the buccal ganglia and axons that branch repeatedly to terminate in the esophageal wall. 2. The OM cells respond phasically to gut distension. Experiments with a high magnesium/low calcium solution suggest that the OM neurons are primary mechanoreceptors. 3. In the isolated CNS preparation, the OM cells receive little synaptic input during the feeding cycle. 4. The OM cells excite the motoneurons active in the rasp phase of the feeding cycle. 5. The OM cells inhibit each of the identified pattern-generating and modulatory interneurons in the buccal ganglia. Experiments with a saline rich in magnesium and calcium suggest that the connections are monosynaptic. 6. Stimulation of a single OM cell to fire at 5-15 Hz is sufficient to terminate the feeding rhythm in the isolated CNS preparation. 7. We conclude that these neurons play a role in terminating feeding behavior.     

Tight or loose coupling between components of the feeding neuromusculature of Aplysia?

Like other complex behaviors, the cyclical, rhythmic consummatory feeding behaviors of Aplysia-biting, swallowing, and rejection of unsuitable food-are produced by a complex neuromuscular system: the animal's buccal mass, with numerous pairs of antagonistic muscles, controlled by the firing of numerous motor neurons, all driven by the motor programs of a central pattern generator (CPG) in the buccal ganglia. In such a complex neuromuscular system, it has always been assumed that the activities of the various components must necessarily be tightly coupled and coordinated if successful functional behavior is to be produced. However, we have recently found that the CPG generates extremely variable motor programs from one cycle to the next, and so very variable motor neuron firing patterns and contractions of individual muscles. Here we show that this variability extends even to higher-level parameters of the operation of the neuromuscular system such as the coordination between entire antagonistic subsystems within the buccal neuromusculature. In motor programs elicited by stimulation of the esophageal nerve, we have studied the relationship between the contractions of the accessory radula closer (ARC) muscle, and the firing patterns of its motor neurons B15 and B16, with those of its antagonist, the radula opener (I7) muscle, and its motor neuron B48. There are two separate B15/B16-ARC subsystems, one on each side of the animal, and these are indeed very tightly coupled. Tight coupling can, therefore, be achieved in this neuromuscular system where required. Yet there is essentially no coupling at all between the contractions of the ARC muscles and those of the antagonistic radula opener muscle. We interpret this result in terms of a hypothesis that ascribes a higher-order benefit to such loose coupling in the neuromusculature. The variability, emerging in the successive feeding movements made by the animal, diversifies the range of movements and thereby implements a trial-and-error search through the space of movements that might be successful, an optimal strategy for the animal in an unknown, rapidly changing feeding environment.     

Transforming tonic firing into a rhythmic output in the Aplysia feeding system: presynaptic inhibition of a command-like neuron by a CpG element

Tonic stimuli can elicit rhythmic responses. The neural circuit underlying Aplysia californica consummatory feeding was used to examine how a maintained stimulus elicits repetitive, rhythmic movements. The command-like cerebral-buccal interneuron 2 (CBI-2) is excited by tonic food stimuli but initiates rhythmic consummatory responses by exciting only protraction-phase neurons, which then excite retraction-phase neurons after a delay. CBI-2 is inhibited during retraction, generally preventing it from exciting protraction-phase neurons during retraction. We have found that depolarizing CBI-2 during retraction overcomes the inhibition and causes CBI-2 to fire, potentially leading CBI-2 to excite protraction-phase neurons during retraction. However, CBI-2 synaptic outputs to protraction-phase neurons were blocked during retraction, thereby preventing excitation during retraction. The block was caused by presynaptic inhibition of CBI-2 by a key buccal ganglion retraction-phase interneuron, B64, which also causes postsynaptic inhibition of protraction-phase neurons. Pre- and postsynaptic inhibition could be separated. First, only presynaptic inhibition affected facilitation of excitatory postsynaptic potentials (EPSPs) from CBI-2 to its followers. Second, a newly identified neuron, B54, produced postsynaptic inhibition similar to that of B64 but did not cause presynaptic inhibition. Third, in some target neurons B64 produced only presynaptic but not postsynaptic inhibition. Blocking CBI-2 transmitter release in the buccal ganglia during retraction functions to prevent CBI-2 from driving protraction-phase neurons during retraction and regulates the facilitation of the CBI-2 induced EPSPs in protraction-phase neurons.     

Control of feeding in aplysia with ad libitum access to food: presence of food increases the intervals between feeding bouts

The patterning of feeding and the quantity eaten in Aplysia californica with ad libitum food access cannot be explained by the effects of three variables previously shown to control the patterning of consummatory feeding responses and the quantity eaten in animals hand-fed individual meals. Feeding in ad libitum conditions is regulated primarily by varying the time between feeding bouts rather than by modulating bout lengths or the efficacy of consummatory movements within a bout. Aplysia with steady-state food access are in a newly characterized feeding state in which they are relatively unresponsive to food. They eat very little (1-4% of the time), and the quantity eaten is unrelated to the quantity of food in the anterior gut. The steady state can be maintained by the presence of food, even if animals do not contact food. The chemosensory rhinophores signal the presence of food that maintains the steady state. Up to 24 h without food is needed for animals to recover from the inhibition of feeding by steady-state presence of food. Recovery from the steady state is partially governed by postingestion stimuli as shown by a faster recovery in animals that have not been in contact with food. Inhibition of feeding during the steady-state is mediated in part via humoral factors because bathing the cerebral and buccal ganglia in hemolymph from animals in the steady state inhibits the ability to elicit buccal motor programs via a cholinomimetic thought to simulate stimulation of the lips with food. After food deprivation that is sufficiently long so that the steady-state decays, animals eat a large meal the size and dynamics of which are consistent with regulation via the three variables previously identified. This large meal is modulated by pheromones secreted by conspecifics even in sexually immature Aplysia.     

Nitric oxide generation around buccal ganglia accompanying feeding behavior in the pond snail, Lymnaea stagnalis

Although there are many lines of evidence for both the presence of nitric oxide synthase (NOS) in the central nervous system (CNS) and the effects of NO on activating and modulating the feeding circuit in Lymnaea stagnalis, there has been no direct evidence that NO generation in the CNS accompanies feeding behavior. In the present study, we used a NO specific electrode to measure the increase in NO concentration around the buccal ganglia when the lips of semi-intact preparations of L. stagnalis were stimulated by sucrose. The NO concentration of the buccal ganglia was significantly increased by an application of sucrose to the lips. A NO scavenger and a NOS inhibitor suppressed this increase in NO concentration. A pair of putative NO-generative neurons in the buccal ganglia, the B2 cells, are active during the inter-feeding phase, and the bursting of the B2 cell elicited by sucrose application starts simultaneously with the feeding response. The rhythmic pulses of NO generation corresponded well with the rhythmic bursting of the B2 cells, which itself corresponds to the 'fictive feeding response'. The present data provide the first direct evidence that NO is generated in the buccal ganglia of L. stagnalis and is involved in a specific behavior such as feeding.     

Mechanisms underlying fictive feeding in aplysia: coupling between a large neuron with plateau potentials activity and a spiking neuron

The buccal ganglia of Aplysia contain a central pattern generator (CPG) that organizes the rhythmic movements of the radula and buccal mass during feeding. Many of the cellular and synaptic elements of this CPG have been identified and characterized. However, the roles that specific cellular and synaptic properties play in generating patterns of activity are not well understood. To examine these issues, the present study developed computational models of a portion of this CPG and used simulations to investigate processes underlying the initiation of patterned activity. Simulations were done with the SNNAP software package. The simulated network contained two neurons, B31/B32 and B63. The development of the model was guided and constrained by the available current-clamp data that describe the properties of these two protraction-phase interneurons B31/B32 and B63, which are coupled via electrical and chemical synapses. Several configurations of the model were examined. In one configuration, a fast excitatory postsynaptic potential (EPSP) from B63 to B31/B32 was implemented in combination with an endogenous plateau-like potential in B31/B32. In a second configuration, the excitatory synaptic connection from B63 to B31/B32 produced both fast and slow EPSPs in B31/B32 and the plateau-like potential was removed from B31/B32. Simulations indicated that the former configuration (i.e., electrical and fast chemical coupling in combination with a plateau-like potential) gave rise to a circuit that was robust to changes in parameter values and stochastic fluctuations, that closely mimicked empirical observations, and that was extremely sensitive to inputs controlling the onset of a burst. The coupling between the two simulated neurons served to amplify exogenous depolarizations via a positive feedback loop and the subthreshold activation of the plateau-like potential. Once a burst was initiated, the circuit produced the program in an all-or-none fashion. The slow kinetics of the simulated plateau-like potential played important roles in both initiating and maintaining the burst activity. Thus the present study identified cellular and network properties that contribute to the ability of the simulated network to integrate information over an extended period before a decision is made to initiate a burst of activity and suggests that similar mechanisms may operate in the buccal ganglia in initiating feeding movements.     

Control of feeding movements in the pteropod mollusc,Clione limacina

Summary (1) The buccal apparatus of the pteropod marine mollusc Clione limacina, isolated together with buccal ganglia, could perform rhythmic feeding movements. Movements of the radula and the hooks (which the Clione inserts into the body of its prey) as well as the electroneurogram of the radular nerve were recorded. Usually one could observe rhythmic radula movements alone, while the hooks were motionless. But sometimes the hooks also performed rhythmic movements which were more or less synchronous with those of the radula. The radula movement cycle consisted of the protraction and the retraction phases, which were occasionally followed by a quiescent phase. Corresponding to each radula movement was a burst of activity in the radular nerve, consisting of the protractor and the retractor components. (2) Isolated buccal ganglia were capable of feeding rhythm generation. Most of the buccal neurons exhibited rhythmic activity correlating with the activity in the radular nerve. According to the phase of activity in the feeding cycle, rhythmic neurons were divided into two groups — the protractor and the retractor ones. The neurons within each of the groups were electrically coupled with each other. The protractor and retractor neurons inhibited each other. (3) Protractor and retractor neurons were extracted from buccal ganglia by means of a microelectrode. Many isolated cells generated slow oscillations of membrane potential and bursts of spikes, the pattern of this activity being similar to that before isolation. (4) A model of the feeding rhythm generator is discussed. It consists of two (protractor and retractor) groups of neurons with mutual inhibitory connections, neurons of each group being endogenous bursters.     

Diverse synaptic connections between peptidergic radula mechanoafferent neurons and neurons in the feeding system of Aplysia

The buccal ganglion of Aplysia contains a heterogeneous population of peptidergic, radula mechanoafferent (RM) neurons. To investigate their function, two of the larger RM cells (B21, B22) were identified by morphological and electrophysiological criteria. Both are low-threshold, rapidly adapting, mechanoafferent neurons that responded to touch of the radula, the structure that grasps food during ingestive and egestive feeding movements. Sensory responses of the cells consisted of spike bursts at frequencies of 8-35 Hz. Each cell was found to make chemical, electrical, or combined synapses with other sensory neurons, motor neurons and interneurons involved in radula closure and/or protraction-retraction movements of the odontophore. Motor neurons receiving input included the following: B8a/b, B15, and B16, which innervate muscles contributing to radula closing; and B82, a newly identified neuron that innervates the anterodorsal region of the I1/I3 muscles of the buccal mass. B21 and B22 can affect buccal motor programs by way of their connections to interneurons such as B19 and B64. Fast, chemical, excitatory postsynaptic potentials (EPSPs) produced by RM neurons, such as B21, exhibited strong, frequency-dependent facilitation, a form of homosynaptic plasticity. Firing B21 also produced a slow EPSP in B15 that increased the excitability of the cell. Thus a sensory neuron mediating a behavioral response may have modulatory effects. The data suggest multiple functions for RM neurons including 1) triggering of phase transitions in rhythmic motor programs, 2) adjusting the force of radula closure, 3) switching from biting to swallowing or swallowing to rejection, and 4) enhancing food-induced arousal.     

Participation of GABA in establishing behavioral hierarchies in the terrestrial snail

GABA-immunoreactive fibers were observed in the neuropile of each ganglion of Helix lucorum, while GABA-immunoreactive neural somata were found only in the buccal, cerebral, and pedal ganglia. Bath application of 10(-5) M GABA to the preparation "buccal mass-buccal ganglia" elicited a sequence of radula movements characteristic of feeding behavior. Corresponding bursts of activity were recorded in the buccal nerves under GABA application and in the buccal neurons recorded optically. In preparations of isolated central nervous system, the bath applications of GABA (10(-5) to 10(-4) M) elicited no changes in synaptic input of the premotor interneurons involved in the withdrawal behavior. However, a significant decrease in amplitude of the synaptic input and in the number of spikes in responses elicited by the test nerve stimulation was observed in metacerebral serotonergic neurons involved in modulating the feeding behavior. GABA application inhibited the spontaneous spike activity in some pedal serotonergic neurons involved in the network underlying withdrawal responses and evoked bursting activity in the other neurons of this functional group. The effects of GABA application on mechanically isolated serotonergic neurons suggest that the primary effect of GABA is inhibition. Thus, our results give evidence of the putative role of GABA in activating the feeding behavior and in the synergistic suppression of serotonergic modulation of the withdrawal behavior and serotonergic modulation of feeding, which has corresponded to the observed behavioral suppression of withdrawal reactions during feeding.     

Control of feeding movements in the freshwater snail Planorbis corneus

(1) The buccal mass of the freshwater snail Planorbis corneus, dissected together with the buccal ganglia, performs rhythmic feeding movements. Radula movements and the electrical activity in various nerves of buccal ganglia were recorded in such a preparation. The cycle of radula movements consisted of three phases: quiescence (Q), protraction (P) and retraction (R). The activity in the radular nerve was observed mainly in the P-phase and that in the dorsobuccal nerve, largely in the R-phase. (2) Isolated buccal ganglia were capable of generating a feeding rhythm, the activity in buccal nerves being similar to that observed in the buccal mass-buccal ganglion preparation, i.e., a burst in the radular nerve preceded a burst in the dorsobuccal nerve. The activity of neurons in isolated buccal ganglia during generation of the feeding rhythm has been studied with intracellular microelectrodes. About 10% of ganglion neurons exhibited periodic activity related to the feeding rhythm ("rhythmic" neurons). (3) Rhythmic neurons have been divided into 7 groups according to the phase of their activity and to the characteristics of slow oscillations of the membrane potential during the feeding cycle. Group 1 neurons revealed a gradual increase of depolarization during the Q- and P-phases. In subgroup 1e neurons, spike discharges began in the Q-phase, while in subgroup 1d neurons activity started in the P-phase. During the R-phase, group 1 neurons were strongly hyperpolarized, and their discharges terminated. In group 2 neurons, small depolarization gradually increased during the Q- and P-phases. Then, in the R-phase, a large (20-50 mV) rectangular wave of depolarization arose with superimposed high-frequency oscillations. Group 3 neurons exhibited an excitatory postsynaptic potential (EPSP) in the P-phase and inhibitory postsynaptic potential (IPSP) in the R-phase. The neurons of group 4 revealed two EPSPs: a small one in the P-phase and a larger one in the R-phase. Group 5 neurons exhibited an EPSP in the P-phase, those of group 7 - an IPSP in the R-phase, and those of group 9 - IPSPs in the P- and R-phases. Neurons within each of the groups 1, 2 and 4 were electrically coupled, and in addition, there were also electrical connections between neurons of groups 2 and 4. (4) Data are presented showing that neurons of groups 1 and 2 are the main source of postsynaptic potentials in rhythmic neurons in the P-phase and in the R-phase of the cycle, respectively.     

Premotor neurons B51 and B52 in the buccal ganglia of Aplysia californica: synaptic connections, effects on ongoing motor rhythms, and peptide modulation

1. Two buccal ganglia interneurons, labeled here as B51 and B52, have been identified on the basis of morphological and physiological criteria. 2. These neurons have multipolar cell bodies. B51 extends a major neurite, which arborizes in the neuropil ipsilateral to the soma; extends into the buccal commissure, where it branches profusely; and projects an axon out the radular nerve (n1); other processes emanating from the soma arborize in the adjacent cell body layer. B52 arborizes ipsilateral to its cell body and sends a major process out of the ipsilateral hemiganglion into the sheath that attaches the buccal ganglia to the buccal mass proper. Here the B52 axon projects through a previously undescribed structure, which forms an arch over the buccal commissure that we designate the commissural arch. The extraganglionic B52 axon sends several branches into the connective tissue and then returns to the contralateral hemiganglion, where it again branches. 3. Each neuron exhibits a unique set of physiological properties. B51 frequently produces plateau potentials, which persist and are even enhanced in solutions where Ca2+ is replaced with Co2+. On the other hand, B52 shows a powerful posthyperpolarization rebound that contributes to its burst formation during spontaneous and nerve-elicited cyclic motor output. 4. B51 and B52 display distinctive rhythmic bursting on stimulation of the radular nerve or esophageal nerve. Their burst-firing tended to occur at certain phase relationships with respect to firing in other buccal premotor and motor neurons. 5. When firing frequency is measured as a function of intracellularly injected current, B51 shows a steplike increase in firing with increasing current, whereas B52 firing frequency is continuously graded. 6. B51 and B52 were found to make extensive synaptic connections within the buccal ganglia. B51 exhibited primarily excitatory electrical connections with known premotor and motor neurons, including an electrotonic synapse with its contralateral homologue. 7. In contrast, B52 made bilateral inhibitory synapses with nearly all of the premotor and motor neurons of the ventral motor cluster. Most of these connections appeared to be monosynaptic, producing synaptic potentials with short and fixed latencies that persisted when the ganglia were bathed in solutions containing elevated concentrations of Ca2+ and Mg2+. 8. Other synaptic potentials produced by B52 were more variable in size and latency; these included slow inhibition of the B4 and B5 neurons and excitation of an identifiable neuron that projected out the radular nerve.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ontogenesis of the snail, Helix aspersa: embryogenesis timetable and ontogenesis of GABA-like immunoreactive neurons in the central nervous system

Late stages of embryogenesis in the terrestrial snail Helix aspersa L. were studied and a developmental timetable was produced. The distribution of gamma-aminobutyric acid-like immunoreactive (GABA-ir) elements in the CNS of the snail was studied from embryos to adulthood in wholemounts. In adults, approximately 226 GABA-ir neurons were located in the buccal, cerebral and pedal ganglia. The population of GABA-ir cells included four pairs of buccal neurons, three neuronal clusters in the pedal ganglia, two clusters and six single neurons in the cerebral ganglia. GABA-ir fibers were observed in all ganglia and in some nerves. The first detected pair of GABA-ir cells in the embryos appeared in the buccal ganglia at about 63–64% of embryonic development. Five pairs of GABA-ir cell bodies were observed in the cerebral ganglia at about 64–65% of development. During the following 30% of development three more pairs of GABA-ir neurons were detected in the buccal ganglia and over fifteen cells were detected in each cerebral ganglion. At the stage of 70% of development, the first pair of GABA-ir neurons was found in the pedal ganglia. In the suboesophageal ganglion complex, GABA-ir fibers were first detected at about 90% of embryonic development. In the posthatching period, the quantity of GABA-ir neurons reached the adult status in four days in the cerebral ganglia, and in three weeks in the pedal ganglia. In juveniles, transient expression of GABA was found in the pedal ganglia (fourth cluster).     

Identified higher-order neurons controlling the feeding motor program of helisoma

A bilaterally symmetrical pair of neurons in the anterior region of the cerebral ganglia of the snail Helisoma trivolvis were found to have excitatory input to the feeding motor program contained in the buccal ganglia. Intracellular microelectrodes were used to stimulate and record from these cells while the motor output of the buccal ganglia was monitored with a combination of intracellular and extracellular recordings. Experimentally evoked tonic activity in an individual cerebral cell could initiate and maintain the patterned motor output from buccal ganglia, characteristic of the activity underlying buccal mass feeding movements. The rate of buccal motor output could be modified directly by varying the firing frequency of the cerebral cell. Cobaltous chloride backfills of cerebrobuccal connectives revealed that these higher-order neurons were the only large cells in the anterior portion of the ganglia to send processes into the connectives. Furthermore, they are the only cells in this region to fluoresce when processed with a sucrose-phosphate-glyoxylic acid solution, indicating that they contain an indoleamine, probably serotonin. Application of low concentrations of serotonin to isolated buccal ganglia or buccal ganglia-buccal mass preparations mimics the effects of the cerebral cells' activity on the buccal motor output, implying that serotonin is a putative transmitter for these cerebral cells.