Evidence for homologous peptidergic neurons in the buccal ganglia of diverse nudibranch mollusks.

The buccal ganglia of seven nudibranches (Aeolidia papillosa, Armina californica, Dirona albolineata, D. picta, Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea) were examined to explore possible homologies between large cells that reacted with antibodies directed against small cardioactive peptide B (SCPB). The buccal ganglion of each species possessed a pair of large, dorsal-lateral, whitish neurons that contained an SCPB-like peptide. We refer to these neurons as the SLB (SCPB-immunoreactive Large Buccal) cells. In all species examined, the SLB cells project out the gastroesophageal nerves and appear to innervate the esophagus. In each species, an apparent rhythmic feeding motor program (FMP) was observed by intracellular recording from both SLB neurons and other neurons in isolated preparations of the buccal ganglia. SLB cells often fire at a high frequency, and usually burst in a specific phase relation to the FMP activity. Stimulation of SLB cells enhances expression of the feeding motor program, either by potentiating existing activity or eliciting the FMP in quiescent preparations. Finally, perfusion of isolated buccal ganglia with SCPB excites the SLB cells and activates FMPs. Thus, both the immunohistochemical and electrophysiological data suggest that the SLB cells within three suborders of the opisthobranchia (Dendronotacea, Arminacea, and Aeolidacea) are homologous. A comparison of our data with previously published studies indicates that SLB cell homologs may exist in other gastropods as well.     

Evidence for homologous peptidergic neurons in the buccal ganglia of diverse nudibranch mollusks

The buccal ganglia of seven nudibranches (Aeolidia papillosa, Armina californica, Dirona albolineata, D. picta, Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea) were examined to explore possible homologies between large cells that reacted with antibodies directed against small cardioactive peptide B (SCP_B). The buccal ganglion of each species possessed a pair of large, dorsal–lateral, whitish neurons that contained an SCP_B-like peptide. We refer to these neurons as the SLB (SCP_B-immunoreactive Large Buccal) cells. In all species examined, the SLB cells project out the gastroesophageal nerves and appear to innervate the esophagus. In each species, an apparent rhythmic feeding motor program (FMP) was observed by intracellular recording from both SLB neurons and other neurons in isolated preparations of the buccal ganglia. SLB cells often fire at a high frequency, and usually burst in a specific phase relation to the FMP activity. Stimulation of SLB cells enhances expression of the feeding motor program, either by potentiating existing activity or eliciting the FMP in quiescent preparations. Finally, perfusion of isolated buccal ganglia with SCP_B excites the SLB cells and activates FMPs. Thus, both the immunohistochemical and electrophysiological data suggest that the SLB cells within three suborders of the opistobranchia (Dendronotacea, Arminacea, and Aeolidacea) are homologous. A comparison of our data with previously published studies indicates that SLB cell homologs may exist in other gastropods as well.     

Acetylcholine activates cerebral interneurons and feeding motor program in Limax maximus

The cellular and network effects of acetylcholine (ACh) on the control system for feeding in Limax maximus were measured by intracellular recordings from feeding command-like interneurons and whole nerve recordings from buccal ganglion motor nerve roots that normally innervate the ingestive feeding muscles. The buccal ganglion motor nerve root discharge pattern that causes rhythmic feeding movements, termed the feeding motor program (FMP), was elicited either by attractive taste solutions applied to the lip chemoreceptors or by ACh applied to the cerebral ganglia. The ability of exogenous ACh applied to the cerebral ganglia to trigger FMP was blocked by the cholinergic antagonists curare and atropine. If the strength of the lip-applied taste stimulus was in the range of 1-2 times threshold, cerebral application of the cholinergic antagonists blocked or greatly decreased the ability of lip-applied taste solutions to trigger FMP (5 of 8 trials). The cerebral feeding interneurons, some of which activate FMP when stimulated intracellularly, are excited by small pulses of ACh applied directly to the cell body from an ACh-filled micropipette. A pulse of ACh that activates several of the feeding interneurons simultaneously triggers FMP. The data suggest that under certain stimulus conditions an obligatory set of cholinergic synapses onto the feedininterneurons must be activated for taste inputs to trigger ingestion. The determination of ACh's action within the feeding control system is necessary for understanding how enhanced cholinergic transmission leads to prolonged associative memory retention (Sahley, et al., 1986).     

Prey capture phase of feeding behavior in the pteropod mollusc,clione limacina: neuronal mechanisms

The prey capture phase of feeding behavior in the pteropod mollusc Clione limacina consists of an explosive extrusion of buccal cones, specialized structures which are used to catch the prey, and acceleration of swimming with frequent turning and looping produced by tail bend. A system of neurons which control different components of prey capture behavior in Clione has been identified in the cerebral ganglia. Cerebral B and L neurons produce retraction of buccal cones and tightening of the lips over them — their spontaneous spike activities maintain buccal cones in the withdrawn position. Cerebral A neurons inhibit B and L cells and produce opening of the lips and extrusion of buccal cones. A pair of cerebral interneurons C-BM activates cerebral A neurons and synchronously initiates the feeding motor program in the buccal ganglia. Cerebral T neurons initiate acceleration of swimming and produce tail bending which underlies turning and looping during the prey capture. Both tactile and chemical inputs from the prey produce activation of cerebral A and T neurons. This reaction appears to be specific, since objects other than alive Limacina or Limacina juice do not initiate activities of A and T neurons.     

The Role of Small Cardioactive Peptide, SCPB, in the Regulatory Responses of Terrestrial Slugs

The feeding motor program in Limax maximus is the neural correlateof feeding and consists of a discrete pattern of cyclical efferentactivity generated by the buccal ganglia in response to stimulationof chemosensory pathways. The small cardioactive peptide, SCP_B(10^–6 to 10^–9 M), increases the responsiveness ofthe FMP and the endogenous activity of specific feeding motoneuronssuch as the fast salivary burster. Stimulation of buccal neuron,B1, which contains SCP_B-like immunoreactive substance, similarlyincreases the activity of feeding motoneurons. Furthermore,both exogenous SCP_B and stimulation of Bl increase the contractileforce of the heart. Thus it appears that the peptidergic neuronBl is a multifunctional interneuron that is involved in thecontrol of both peripheral and central targets.     

Distribution and anatomy of GABA-like immunoreactive neurons in the central and peripheral nervous system of the snail Helix pomatia

Gamma-aminobutyric acid (GABA)-like immunoreactive neurons were studied in the central and peripheral nervous system of Helix pomatia by applying immunocytochemistry on whole-mount preparations and serial paraffin sections. GABA-immunoreactive cell bodies were found in the buccal, cerebral and pedal ganglia, but only GABA-immunoreactive fibers were found in the viscero-parietal-pleural ganglion complex. The majority of GABA-immunoreactive cell bodies were located in the pedal ganglia but a few could be found in the buccal ganglia. Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes run into the connectives and commissures of the ganglia, indicating an important central integrative role of GABA-immunoreactive neurons. GABA may also have a peripheral role, since GABA-immunoreactive fibers could be demonstrated in peripheral nerves and the lips. Glutamate injection did not change the number or distribution of GABA-immunoreactive neurons, but induced GABA immunoreactivity in elements of the connective tissue ensheathing the muscle cells and fibers of the buccal musculature. This shows that GABA may be present in different non-neural tissues as a product of general metabolic pathways.     

The molluscan neuropeptide, SCPB, increases the responsiveness of the feeding motor program of Limax maximus

Small cardioactive peptide B (SCPB) has an excitatory effect on both buccal neurons and musculature in numerous molluscan species. The present study reports the effects of SCPB on the activity of specified buccal neurons and the expression of the feeding motor program of the terrestrial slug, Limax maximus. Superfusion of an isolated CNS preparation with 10(-6)M SCPB results in a 3-4-fold increase in the burst frequency of the fast salivary burster neuron (FSB), while having no effect on the activity of another endogenous burster, the bilateral salivary neuron (BSN). The response of the FSB to SCPB is dose dependent, with a threshold concentration of 2 X 10(-8)M. The response of the FSB to SCPB showed no indication of desensitization, even after long-term exposure (20 min). The feeding motor program (FMP) in Limax is a discrete pattern of cyclical motor activity that can be initiated by lip nerve stimulation. In the presence of SCPB a previously subthreshold stimulus can initiate the full FMP. The pattern of the FMP, once initiated, appears unaffected by SCPB. Thus it is the responsiveness of the initiation process that is enhanced by SCPB. Histochemical studies revealed a number of buccal neuron somata and fibers that stain for SCPB-like immunoreactive material (SLIM).     

Octopamine-containing (OC) interneurons enhance central pattern generator activity in sucrose-induced feeding in the snail <Emphasis Type="Italic">Lymnaea</Emphasis>

In the pond snail Lymnaea stagnalis octopamine-containing (OC) interneurons trigger and reconfigure the feeding pattern in isolated CNS by excitation of the central pattern generator. In semi-intact (lip–mouth—CNS) preparations, this central pattern generator is activated by chemosensory inputs. We now test if sucrose application to the lips activates the OC neurons independently of the rest of the feeding central pattern generator, or if the OC interneuron is activated by inputs from the feeding network. In 66% of experiments, sucrose stimulated feeding rhythms and OC interneurons received regular synaptic inputs. Only rarely (14%) did the OC interneuron fire action potentials, proving that firing of OC interneurons is not necessary for the sucrose-induced feeding. Prestimulation of OC neurons increased the intensity and duration of the feeding rhythm evoked by subsequent sucrose presentations. One micromolar octopamine in the CNS bath mimicked the effect of OC interneuron stimulation, enhancing the feeding response when sucrose is applied to the lips. We conclude that the modulatory OC neurons are not independently excited by chemosensory inputs to the lips, but rather from the buccal central pattern generator network. However, when OC neurons fire, they release modulatory octopamine, which provides a positive feedback to the network to enhance the sucrose-activated central pattern generator rhythm.     

Cerebral interneurons controlling fictive feeding in Limax maximus

Summary Initiation and modulation of fictive feeding by cerebral to buccal interneurons (CBs) was examined in an isolated CNS preparation of Limax maximus. Three CBs which are phasically active during fictive feeding, CB₁, CB₃ and CB₄, will reliably trigger bouts of fictive feeding when activated alone or in pairs. Another phasic CB, CB_EC, is not effective for triggering feeding. One CB which is tonically active during fictive feeding, CB_ST, drives fictive feeding in 50% of preparations when activated alone and enhances triggering of feeding when co-activated with phasic CBs. The metacerebral giant cell (MGC) was found to be capable of triggering fictive feeding in preparations with an intact subcerebral commissure. The MGC was especially effective at increasing the effectiveness of other CBs for initiation of feeding. Short high-frequency bursts of phasic CB or MGC action potentials are capable of resetting ongoing fictive feeding. Resetting effects of CB action potentials are relatively independent of the phase of the bite-cycle in which they are activated. CB₄ phase-advances the bite-cycle while the other phasic CBs phase-delay the bite cycle. Moderate frequency stimulation of CB₄ speeds up the bite rate while moderate frequency stimulation of CB₃ slows biting. All CBs, except the tonic CB, CB_DL, increase the intensity of buccal motor neuron bursting during feeding. The excitatory effects of phasic CBs and the tonic CB, CB_EPSP, on fictive feeding persist for many seconds after the offset of stimulation. CBs form both monosynaptic excitatory and monosynaptic inhibitory connections with different BG motor neurons.Abbreviations BG buccal ganglion - BR buccal root - CB cerebral-buccal interneuron - CBC cerebral-buccal connective - CPG central pattern generator - FB fast burster neuron - FMP feeding motor program - IBI interbite interval - MGC metacerebral giant cell     

Responses of cerebral GABA-containing CBM neuron to taste stimulation with seaweed extracts in Aplysia kurodai

Aplysia kurodai distributed along Japan feeds well on Ulva pertusa but rejects Gelidium amansii with distinctive patterned movements of the jaws and radula. On the ventral side of the cerebral M cluster, four cell bodies of higher order neurons that send axons to the buccal ganglia are distributed (CBM neurons). We have previously shown that the dopaminergic CBM1 modulates basic feeding circuits in the buccal ganglia for rejection by firing at higher frequency after application of the aversive taste of seaweed such as Gelidium amansii. In the present experiments immunohistochemical techniques showed that the CBM3 exhibited gamma-aminobutyric acid (GABA)-like immunoreactivity. The CBM3 may be equivalent to the CBI-3 involved in changing the motor programs from rejection to ingestion in Aplysia californica. The responses of the CBM3 to taste stimulation of the lips with seaweed extracts were investigated by the use of calcium imaging. The calcium-sensitive dye, Calcium Green-1, was iontophoretically introduced into a cell body of the CBM3 using a microelectrode. Application of Ulva pertusa or Gelidium amansii extract induced different changes in fluorescence in the CBM3 cell body, indicating that taste of Ulva pertusa initially induced longer-lasting continuous spike responses at slightly higher frequency compared with that of Gelidium amansii. Considering a role of the CBM3 in the pattern selection, these results suggest that elongation of the initial firing response may be a major factor for the CBM3 to switch the buccal motor programs from rejection to ingestion after application of different tastes of seaweeds in Aplysia kurodai.     

Properties of a symmetric pair of serotonin-containing neurones in the cerebral ganglia of Planorbis.

1. There is a bilaterally symmetric pair of large serotonin-containing neurones in the cerebral ganglia of Planorbis corneus. 2. In some animals these neurones are connected by a non-rectifying electrotonic synapse, and fire in synchrony even at prolonged high frequency. In other animals the neurones are not coupled, and fire independently except when driven by common input. Occasionally the coupling is weak. 3. Both coupled and non-coupled serotonin neurones have processes in the major nerve trunks of both buccal ganglia. 4. Synapses are made with many neurones in the buccal ganglia. The serotonin neurones can initiate firing in several motoneurones and thus produce movements of the buccal mass. 5. During spontaneous feeding cycles the input and firing pattern of the serotonin neurones do not bear any obvious relation to the movements of the buccal mass. 6. The data suggest that the serotonin neurones are modulatory cells, altering the level of excitability of buccal ganglion neurones.     

Sprouting and functional regeneration of an identified serotonergic neuron following axotomy

An identified serotonergic neuron (C1) in the cerebral ganglion of Helisoma trivolvis sprouts following axotomy and rapidly (seven to eight days) regenerates to recover its regulation of feeding motor output from neurons of the buccal ganglia. The morphologies of normal and regenerated neurons C1 were compared. Intracellular injection of the fluorescent dye, Lucifer Yellow, into neuron C1 was compared with serotonin immunofluorescent staining of the cerebral and buccal ganglia. The two techniques revealed different and complimentary representations of the morphology of neuron C1. Lucifer Yellow provided optimal staining of the soma, major axon branches, and dendritic arborization. Immunocytochemical staining revealed terminal axon branches on distant targets and showed an extensive plexus of fine fibers in the sheaths of ganglia and nerve trunks. In addition to C1, serotonin-like immunoreactivity was localized in approximately 30 other neurons in each of the paired cerebral ganglia. Only cerebral neurons C1 had axons projecting to the buccal ganglia. No neuronal somata in the buccal ganglia displayed serotonin-like immunoreactivity. Observations of regenerating neurons C1 demonstrated: Actively growing neurites, both in situ and in cell culture, displayed serotonin-like immunoreactivity; severed distal axons of C1 retained serotonin-like immunoreactivity for up to 28 days; axotomized neurons C1 regenerated to restore functional control over the feeding motor program.     

Chemosensory stimulation of the oral cavity of the snail Helisoma

Food extracts, perfused through the oral cavity of the snailHelisoma trivolvis, lead to synaptic activation of identifiedbuccal ganglia motor neurons. Both retractor and protractormotor neurons displayed cyclic bursts of firing characteristicof that observed during expression of the central feeding motorprogram(CFM). The possibility that leakage of food extracts from theoral cavity had a pharmacological effect on buccal neurons wasconsidered. Direct application of the extracts to the exposedganglionic surface did not evoke similar neuronal activity.Oral perfusion with a behaviorally aversive compound inhibitedboth the activity evoked by acceptable taste solutions and "spontaneously"generated activity in some preparations. It is concluded thatoral chemosensory receptors in the snail exert both an excitatoryand inhibitory influence on buccal motor neurons. The significanceof these results for cellular neurophysiological investigationof the synaptic events underlying the central processing ofafferent chemosensory information is discussed.     

Electrophysiological study of serotoninergic neuron Cl in the pteropod mollusk Clione

Functional characteristics of cerebral serotoninergic neuron Cl, axons of which terminate at the buccal ganglia [7], were investigated in the pteropod molluskClione. Stimulating neuron Cl induced activation of the feeding rhythm generator located in the buccal ganglia — an effect arising after a long latency and persisting for some tens of seconds once stimulation had ended. Neuron Cl receives feedback from buccal ganglion cells and this brings about periodic modulation in ganglia activity during the generation of feeding rhythm. Activity of neuron Cl is correlated with operation of the locomotor rhythm generator located in the pedal ganglia. The firing rate of Cl neurons increased upon activation of the locomotor generator (whether spontaneous or induced by stimulating certain command neurons). The correlation found between workings of the locomotor generator and activity of Cl neurons is thought to be one of the manifestations of feeding synergy involving simultaneous activation of the locomotor and buccal apparatus.Institute for Research on Information Transmission, Academy of Sciences of the USSR, Moscow. M. V. Lomonosov State University, Moscow. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 18–25, January–February, 1991.     

Phase-dependent coordination of two motor programs in the buccal ganglion of a pteropod mollusk

Rhythmic activities of two feeding structures of the pteropod mollusk Clione limacina, redula and hooks, controlled by the neural networks in the buccal ganglia must be coordinated in order to produce a meaningful feeding response. Optical recording from the buccal ganglia, which allows the simultaneous activities of numerous neurons to be traced, revealed that such coordination exists in a phase-dependent manner. Instead of recording four theoretically possible phases of neuronal rhythmic activity, we always recorded only two phases, even after the electrical stimulation of the cerebro-buccal connective, which triggers both radula and hook rhythmic movements in the preparation.     

Cerebral-buccal pathways in Aplysia californica: synaptic connections, cooperative interneuronal effects and feedback during buccal motor programs

Ingestion of seaweed by Aplysia is in part mediated by cerebral-buccal interneurons that drive rhythmic motor output from the buccal ganglia and in some cases cerebral-buccal interneurons act as members of the feeding central pattern generator. Here we document cooperative interactions between cerebral-buccal interneuron 2 and cerebral-buccal interneuron 12, characterize synaptic input to cerebral-buccal interneuron 2 and cerebral-buccal interneuron 12 from buccal peripheral nerve 2,3, describe a synaptic connection between cerebral-buccal interneuron 1 and buccal neuron B34, further characterize connections made by cerebral-buccal interneurons 2 and -12 with B34 and B61/62, and describe a novel, inhibitory connection made by cerebral-buccal interneuron 2 with a buccal neuron. When cerebral-buccal interneurons 2 and 12 were driven synchronously at low frequencies, ingestion-like buccal motor programs were elicited, and if either was driven alone, indirect synaptic input was recruited in the other cerebral-buccal interneuron. Stimulation of BN2,3 recruited both ingestion and rejection-like motor programs without firing in cerebral-buccal interneurons 2 or 12. During motor programs elicited by cerebral-buccal interneurons 2 or 12, high-voltage stimulation of BN2,3 inhibited firing in both cerebral-buccal interneurons. Our results suggest that cerebral-buccal interneurons 2 and 12 use cooperative interactions to modulate buccal motor programs, yet firing in cerebral-buccal interneurons 2 or 12 is not necessary for recruiting motor programs by buccal peripheral nerve BN2,3, even in preparations with intact cerebral-buccal pathways.     

Modulation of the feeding system by a radular mechanosensory neuron in the terrestrial slug,Incilaria fruhstorferi

We investigated the modulatory role of a radular mechanoreceptor (RM) in the feeding system of Incilaria. RM spiking induced by current injection evoked several cycles of rhythmic buccal motor activity in quiescent preparations, and this effect was also observed in preparations lacking the cerebral ganglia. The evoked rhythmic activity included sequential activation of the inframedian radular tensor, the supramedian radular tensor, and the buccal sphincter muscles in that order.In addition to the generation of rhythmic motor activity, RM spiking enhanced tonic activities in buccal nerve 1 as well as in the cerebrobuccal connective, showing a wide excitatory effect on buccal neurons. The excitatory effect was further examined in the supramedian radular tensor motoneuron. RM spiking evoked biphasic depolarization in the tensor motoneuron consisting of fast excitatory postsynaptic potentials and prolonged depolarization lasting after termination of RM spiking. These depolarizations also occurred in high divalent cation saline, suggesting that they were both monosynaptic.When RM spiking was evoked in the fictive rasp phase during food-induced buccal motor rhythm, the activity of the supramedian radular tensor muscle showed the greatest enhancement of the three muscles tested, while the rate of ongoing rhythmic motor activity showed no increase.Abbreviations CPG central pattern generator - EPSP excitatory postsynaptic potential - RBMA rhythmic buccal motor activity - RM radular mechanosensory neuron - SMT supramedian radular tensor neuron     

GABA regulates the buccal motor output of Helisoma by two pharmacologically distinct actions

GABA was tested for its effects on patterned motor activity (PMA) underlying feeding. Using buccal motoneuron B19 to monitor PMA through intracellular recordings, GABA was found to exert effects at two levels. First, GABA stimulated rhythmic patterned activity resembling fictive feeding, which is under the control of the buccal CPG. In addition, GABA produced a direct inhibition of neuron B19. Both effects were observed when the buccal ganglia were studied in isolation from the rest of the central nervous system, suggesting local interactions with GABA receptors of buccal neurons. Furthermore, these two actions of GABA were found to be pharmacologically distinguishable. The direct hyperpolarization of neuron B19 was mimicked by muscimol, but not baclofen, and involved an increased chloride conductance, which was blocked by picrotoxin.Baclofen duplicated CPG activation by GABA. Picrotoxin had no effect on GABA- or baclofen-induced PMA.These results demonstrate that the Helisoma buccal ganglia have two GABA receptor types which resemble, pharmacologically, mammalian GABA_A and GABA_B receptors, and that GABA plays a key role in feeding patterned motor activity in Helisoma.Abbreviations CPG central pattern generator - GABA gammaamino butyric acid - HPLC high performance liquid chromatography - IPSP inhibitory postsynaptic potential - PMA patterned motor activity - SLRT supralateral radular tensor muscle     

Distribution of serotonin-containing neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of serotonin (5HT)-containing neurons in the central nervous system of the snail Helix pomatia has been determined in whole-mount preparations by use of immunocytochemical and in vivo 5,6-dihydroxy-tryptamine labelling. 5HT-immunoreactive neuronal somata occur in all but the buccal and pleural ganglia. Immunoreactive fibres are present throughout the central nervous system. The 5HT-immunoreactive neuronal somata characteristically appear in groups, located mainly in the cerebral, pedal, visceral and right parietal ganglia. The majority of 5HT-immunoreactive neurons is located in the pedal ganglia. Additionally a dense network of 5HT-immunoreactive varicose fibres is found in the neural sheath of the central nervous system including all the nerves and ganglia. The number and distribution of 5HT-immunoreactive neurons correlates with that demonstrated by 5,6-dihydroxytryptamine labelling method.