Role of the giant serotonin-containing cell of the cerebral ganglion in the edible snail in organizing its food-acquiring behavior

By adding dopamine or serotonin to a bath with snail's isolated nervous system and by intracellular activation of giant cerebral serotonergic cells it was established in neurophysiological experiments that, in spite of activating effect of serotonin on buccal motorneurones, dopamine is the transmitter triggering feeding movements of the buccal mass and feeding pattern in buccal motorneurones. This conclusion is confirmed by behavioural experiments in which an experimental group was injected by neruotoxin 5,6-dihydroxytryptamine selectively impairing serotonergic neurones. The consumatory phase of feeding (triggered by dopamine) did not change in treated animals, while the appetitive phase was significantly impaired. It was noted that the giant metacerebral cell was activated during burst activity in buccal motoneurones. The conclusion is made that giant serotonergic cerebral cells only modulate but do not trigger the feeding behaviour in the snail Helix lucorum.     

Axonal branching pattern and coupling mechanisms of the cerebral giant neurones in the snail,lymnaea stagnalis

The axonal branching pattern of the two cerebral giant neurones (CGCs) of Lymnaea stagnalis was studied with intrasomatically applied horseradish peroxidase. The cells are symmetrical. Each CGC projects to the ipsilateral n. labialis medius and n. arteriae labialis, the subcerebral commissure, and to all ipsi- and contralateral buccal nerves. The contralateral buccal nerves are reached via the ipsilateral cerebro-buccal connective and the buccal commissure. The CGC fire action potentials 1:1 in a driver-follower relationship. Each cell is capable of both driving and following. The relationship depends on the membrane potentials of the somata. In driving CGC spikes are initiated in a cerebral spike trigger zone located near the soma. In following cells spikes are initiated in a distal zone located in the buccal ganglia. The buccal zone is only affected by the partner CGC. CGC are synchronized by three coupling mechanisms: mutual excitatory chemical synapses, electrotonic coupling, and common input. The chemical and electrotonic connections are located in the buccal ganglia. All spikes are relayed to the partner cell via the chemical synapses. The electrotonic coupling improves the efficiency of the chemical synapses. The dual connection selectively synchronizes the CGC-axonal spikes from each side of the buccal mass. Common excitatory input affects the cerebral spike trigger zones and can initiate simultaneous spikes in both cells. This results in bilateral synchrony of spikes in the CGC-axons in both the buccal and the lip nerves.     

Effect of serotonin on the activity of the neurons involved in the realization of the avoidance reflex of the snail

Bath application of 10(-5) mol/l of serotonin (5-HT) elicited a 50% increase of summary EPSPs recorded in command neurones for avoidance behaviour. No significant changes of rest potential and input resistance were seen in these cells. 5-HT evoked an increase of spontaneous level of firing in motoneurones involved in the same reflex, as well as an increase in the number of spikes which paralleled increase of EPSPs to the same stimulus in command neurones. In sensory cells, presynaptic to the command neurones, application of 5-HT evoked a significant increase of excitability and of input resistance. Monosynaptic EPSPs recorded in the command neurones showed a 40% increase after serotonin application. It is concluded that the major locus of plastic changes evoked by 5-HT application in the neuronal chain underlying avoidance reflex is the synaptic contact between sensory and command neurones.     

Serotonin regulates rhythmic whisking

Many rodents explore their environment by rhythmically palpating objects with their mystacial whiskers. These rhythmic whisker movements ("whisking"; 5-9 Hz) are thought to be regulated by an unknown brainstem central pattern generator (CPG). We tested the hypothesis that serotonin (5-HT) inputs to whisking facial motoneurons (wFMNs) are part of this CPG. In response to exogenous serotonin, wFMNs recorded in vitro fire rhythmically at whisking frequencies, and selective 5-HT2 or 5-HT3 receptor antagonists suppress this rhythmic firing. In vivo, stimulation of brainstem serotonergic raphe nuclei evokes whisker movements. Unilateral infusion of selective 5-HT2 or 5-HT3 receptor antagonists suppresses ipsilateral whisking and substantially alters the frequencies and symmetry of whisker movements. These findings suggest that serotonin is both necessary and sufficient to generate rhythmic whisker movements and that serotonergic premotoneurons are part of a whisking CPG.     

Radular mechanosensory neuron in the buccal ganglia of the terrestrial slug,Incilaria fruhstorferi

A radular mechanosensory neuron, RM, was identified in the buccal ganglia of Incilaria fruhstorferi. Fine neurites ramified bilaterally in the buccal ganglia, and main neurites entered the subradular epithelium via buccal nerve 3 (n3). When the radula was distorted by bending, RM produced an afferent spike which was preceded by an axonic spike recorded at n3. The response of RM to radular distortion was observed even in the absence of Ca²⁺, which drastically suppressed chemical synaptic interactions. Therefore, RM was concluded to be a primary radular mechanoreceptor.During rhythmic buccal motor activity induced by food or electrical stimulation of the cerebrobuccal connective, RM received excitatory input during the radular retraction phase. In the isolated buccal ganglia connected to the radula via n3s, the afferent spike, which had been evoked by electrical stimulation of the subradular epithelium, was broadened with the phasic excitatory input. Since the afferent spike was also broadened by current injection into the soma, depolarization due to the phasic input may have produced the spike broadening.Spike broadening was also observed during repetitive firing evoked by current injection. The amplitude of the excitatory postsynaptic potential in a follower neuron increased depending on the spike broadening of RM.Abbreviations CBC cerebrobuccal connective - EPSP excitatory postsynaptic potential - n1,n3 buccal nerves 1 and 3 - RBMA rhythmic buccal motor activity - RM radular mechanosensory neuron - SMT supramedian radular tensor neuron     

Activation by dopamine of patterned motor output from the buccal ganglia of Helisoma trivolvis

The buccal ganglia of the snail, Helisoma trivolvis, contain an intrinsic system of dopamine-containing neurons (Trimble, Barker, and Bullard, 1983). Dopamine, when bath applied to the isolated buccal ganglia, activates patterned motor output in a dose-dependent fashion. Haloperidol blocks the activating effect of dopamine, but the similar activation evoked by serotonin is not blocked by haloperidol. We suggest that there are two separate mechanisms for activating patterned motor output from the buccal ganglia. One is serotonergic, emanating from identified cerebral ganglion cells (Granzow and Kater, 1977), while the other is dopaminergic, involving neurons intrinsic to the buccal ganglia.     

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.     

Cerebral neurons underlying prey capture movements in the pteropod mollusc,Clione limacina

The pteropod mollusc Clione limacina is a highly specialized carnivore which feeds on shelled pteropods and uses, for their capture, three pairs of oral appendages, called buccal cones. Contact with the prey induces rapid eversion of buccal cones, which then become tentacle-like and grasp the shell of the prey. In the previous paper, a large group of electrically coupled, normally silent cells (A motoneurons) has been described in the cerebral ganglia of Clione. Activation of A neurons induces opening of oral skin folds and extrusion of the buccal cones. The present study continues the analysis of the electrical properties of A motoneurons.Brief intracellular stimulation of an A neuron can produce prolonged firing (afterdischarge), lasting up to 40 s, in the entire population of A neurons. Afterdischarge activity is based on an afterdepolarization evoked by an initial strong burst of A neuron spikes. The data suggest that this afterdepolarization represents excitatory synaptic input from unidentified neurons which in turn receive excitatory inputs from A neurons, thus organizing positive feedback. The main functional role of this positive feedback is the spread and synchronization of spike activity among all A neurons in the population. In addition, it serves to transform a brief excitatory input to A neurons into their prolonged and stable firing, which is required during certain phases of feeding behavior in Clione.     

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.     

SEROTONIN AND FREE AMINO ACID ANALYSIS OF GANGLIA AND ISOLATED NEURONES OF APLYSIA DACTYLOMELA

—The presence of serotonin and different amino acids was investigated in the ganglia and in isolated giant neurones of Aplysia dactylomela. With a few exceptions the pattern of substances was similar in all the ganglia. Of the many identified neurones studied only one giant neurone located in each cerebral ganglion was found to contain serotonin. GABA was detected in most extracts, including those of the serotonin-containing neurone, known cholinergic, and known neurosecretory neurones. Putrescine, recently detected in extracts of nervous tissue and isolated neurones of Helix, was not detected in Aplysia nervous tissue.     

The octopamine-containing buccal neurons are a new group of feeding interneurons in the pond snail Lymnaea stagnalis

In the pond snail, Lymnaea stagnalis, the paired buccal ganglia contain 3 octopamine-immunoreactive neurons, which have previously been shown to be part of the feeding network. All 3 OC cells are electrically coupled together and interact with all the known buccal feeding motoneurons, as well as with all the modulatory and central pattern generating interneurons in the buccal ganglia. N1 (protraction) phase neurons: Motoneurons firing in this phase of the feeding cycle receive either single excitatory (depolarising) synaptic inputs (B1, B6 neurons) or a biphasic response (hyperpolarisation followed by depolarisation) (B5, B7 motoneurons). Protraction phase feeding interneurons (SO, N1L, NIM) also receive this biphasic synaptic input after OC stimulation. All of protraction phase interneurons inhibit the OC neurons. N2 (retraction) phase neurons: These motoneurons (B2, B3, B9, B10) and N2 interneurons are hyperpolarised by OC stimulation. N2 interneurons have a variable (probably polysynaptic) effect on the activity of the OC neurons. N3 (swallowing) phase: OC neurons are strongly electrically coupled to both N3 phase (B4, B4cluster, B8) motoneurons and to the N3p interneurons. In case of the interneuronal connection (OC<->N3) the electrical synapse is supplemented by reciprocal chemical inhibition. However, the synaptic connections formed by the OC neurons or N3p interneurons to the other members of the feeding network are not identical. CGC: The cerebral, serotonergic CGC neurons excite the OC cells, but the OC neurons have no effect on the CGC activity. In addition to direct synaptic effects, the OC neurons also evoke long-lasting changes in the activity of feeding neurons. In a silent preparation, OC stimulation may start the feeding pattern, but when fictive feeding is already occurring, OC stimulation decreases the rate of the fictive feeding. Our results suggest that the octopaminergic OC neurons form a sub-population of N3 phase feeding interneurons, different from the previously identified N3p and N3t interneurons. The long-lasting effects of OC neurons suggest that they straddle the boundary between central pattern generator and modulatory neurons.     

Sensitivity of a peptide-activated neuron in Aplysia to serotonin and cyclic AMP-relevant agents

Serotonin depolarized Aplysia buccal motoneuron B16. The response could be obtained in high-magnesium/low-calcium medium, indicating a direct effect on B16 and supporting a putative monosynaptic input to B16 from the serotonergic metacerebral neurons. Similar depolarizing effects in high-magnesium/low-calcium medium were obtained in response to 8-bromo cyclic AMP, isobutylmethylxanthine, theophylline and forskolin. Tolbutamide, a putative inhibitor of cyclic AMP-dependent protein kinase, blocked or reversed responses of B16 to egg-laying hormone containing extracts and to serotonin. Serotonin and forskolin significantly increased the cyclic AMP content of buccal ganglia, whereas egg-laying hormone-containing extracts did not.     

Interaction of serotonin and nitric oxide (NO) in activation of the serotoninergic system in Helix

Serotonin and the NO donors were shown to induce excitation in all serotoninergic neurones under study and to activate synchronous synaptic input in the Helix brain. The serotonin effects may be blocked by 5,7-DOT and N-monomethyl-L-arginine. The 5,7-DOT blocked activation of the NO-induced synchronous bursts but did not affect their activating effect. The data obtained suggest that serotonin and NO equally regulate the serotoninergic system's function in the snail brain. The effects of serotonin and NO are interconnected and interdependent. A possible role of the NO as a second messenger during serotonin activation and as a serotonin co-transmitter in presynaptic neurones, is discussed.     

Feeding motor program in Limax. I. Neuromuscular correlates and control by chemosensory input

The feeding motor program(FMP) of the terrestrial slug Limax maximus was examined in vivo and in vitro. The feeding pattern of intact animals shows an initial increase in bite frequency followed by a plateau phase. Recordings obtained from semi-intact preparations of the lips, brain, and buccal mass established the correlation of activity in buccal ganglion nerve roots with the protraction-retraction bite cycle. A preparation of the lips, cerebral ganglia, and buccal ganglia was developed, such that, repetitive chemostimulation of the lips yields reproducible bouts of FMP. Sources of proprioceptive feedback from buccal muscles were demonstrated. The feasibility of computer scoring of the FMP is documented. The results demonstrate that aspects of in vivo feeding behavior are retained and identifiable in highly dissected, in vivo preparations.     

Rapid firing rates from mechanosensory neurons in copepod antennules

Small detection distances coupled with rapid movements require copepods to respond to stimuli with behavioral latencies on the order of milliseconds. Receiving adequate sensory information in such a short time necessitates extremely rapid firing rates of the efferent receptors. Here we show that copepod mechanoreceptors can fire at frequencies up to 5 kHz in response to fluid mechanical stimuli. Neural activity at these frequencies enables these animals to code for a range of fluid velocities thus providing important information regarding the nature of different fluid disturbances.     

Regulation of the plastic properties of an electro-excitable neuronal membrane by serotonin

The action of serotonin on plastic properties of electroexcitable membrane was studied in Helix lucorum parietal ganglia on neurones of two types: habituating (HC) and nonhabituating (NHC) to rhythmic intracellular stimulation by impulses of depolarizing current. Serotonin produced an effect of facilitation on HC (increase of responses to stimulation against the background of depolarization and rise of input resistance of the cell). Besides, serotonin completely blocked the ability of these cells to habituate to rhythmic stimulation. The obtained data testify that such action of serotonin may be based on suppression by it of C-dependent K-conductivity. Serotonin suppresses responses of NHC to stimulation and contributes to their habituation to rhythmic stimulation. Such action is due to serotonin suppression of Ca-conductivity, and, consequently, to elimination of the mechanism of action potential generation.     

Activation of command fibres to the stomatogastric ganglion by input from a gastric mill proprioceptor in the crab,Cancer pagurus †

In semi‐intact preparations of the crab Cancer pagurus the normal output from the stomatogastric ganglion (StG) was a regular pyloric cycle (Figure 4). Repeated stimulation of the posterior stomach nerve (psn) of the posterior gastric mill proprioceptor (PSR) often induced series of spontaneous gastric cycles. We were therefore able to describe the normal gastric cycle as recorded in the output nerves from StG and to identify most of the relevant motor neurones by reference to the muscles that they innervate (Figure 10). The gastric cycle output was variable (Figures 5, 6), although in many preparations one complex type of output predominated (Figure 7). The basic feature of the gastric cycle was an alternation of activity between the single cardio‐pyloric neurone (CP) and a complex variable burst in the lateral cardiac (LC), the gastro‐pyloric (GP), the gastric (GM), and other associated neurones. During this normally occurring complex gastric burst significant changes occurred in the pyloric cycle, notably an increase in activity of the pacemaker pyloric dilator (PD) group and an inhibition of the lateral pyloric (LP), inferior cardiac (IC) and ventricular dilator (VD) neurones (Figures 6, 7, 8, 9). These changes are probably associated with an opening of the cardio‐pyloric valve and food passage into the pyloric filter. The gastric output was related to the normally observed movements of the dorsal ossicles of the gastric mill and thus to the operation of the teeth of the mill (Figure 11). Increased input from the PSR is associated with the grinding action of the teeth which is caused by the complex gastric burst (Figure 12).

Stimulation of the psn during an ongoing regular pyloric output caused changes in the cycle which mimicked those occurring during the spontaneous gastric cycle (Figure 13; Table 1). Stimulation of the psn during ongoing gastric activity also affected the gastric units (Figure 14). The input pathway from the PSR is shown to be through the stomatogastric nerve (sgn), the connection between the commissural ganglia and the stomatogastric ganglion (Figure 15). The commissural ganglia are known to receive most of the sensory input from the foregut and PSR input is probably processed there. Recordings from the sgn show that psn stimulation activates a small number of centrally originating units, and that the activity of these units coincides with the pyloric output changes (Figures 15, 16). We therefore label the units command interneurones. Their effects could be mediated by direct connections to only the PD pacemaker neurones of the pyloric cycle. Control experiments showed that PSR input is not necessary for the pyloric output changes to occur during gastric output but that similar output changes can be evoked by input resulting from induced gastric movements (Figure 15(E)). We think that the pyloric cycle output changes are normally controlled by a number of mechanisms at different levels (Figure 17). We cannot easily explain the effects of PSR input on the gastric cycle neurones.

These findings are important because they allow us to study a specific input to the StG without disrupting its normal input‐output pathways to the central nervous system. Further experiments on the system designed to test the assumption that the sgn units are in fact responsible for the pyloric output changes, and to investigate the processing of the PSR input are outlined.     

Functional characteristics of an identified pair of neurones in the CNS of the pond snail (Lymnaea stagnalis L.).

The properties of 2 giant electrically coupled neurones (A10 and P1) identified in the visceral and right parietal ganglia of Lymnaea stagnalis were examined. The active and passive electrical parameters of the neurones, as well as the junction between them were measured. The main peripheral and interneuronal connections of the neurones were demonstrated using both electrophysiological and morphological methods. It is shown that the coupled cells are not neurones of the same function, but they are asymmetrical ones. This finding is supported by the following results: (1) the axonal pathways of neurones A10 and P1 are different; (2) there are significant differences in their afferent and efferent connections; (3) though the electrical junction between them is bidirectional, the junctional electrical characteristics prefer P1-A10 transmission. According to the electron microscopic results both neurones are possible neurosecretory cells. The differences demonstrated between the 2 giant neurones may have significance concerning their role in a special neuronal network.     

Locust flight behavior after hemisection of individual thoracic ganglia: evidence for hemiganglionic premotor centers

Summary The flight behavior of locusts with hemisected mesothoracic or metathoracic ganglia was observed in unrestrained animals and monitored electromyographically in tethered animals. Animals with hemisected mesothoracic ganglia were able to initiate and carry out free flight. Hemisection of the mesothoracic ganglion caused no significant changes in the pattern of flight muscle firing; both intra- and intersegmental coordination of flight muscle activity were retained (Figs. 3, 4). Additional transection of one meso-metathoracic connective altered the pattern of flight muscle firing but did not abolish rhythmic activity (Fig. 8). Deafferentation of the thoracic ganglia in animals with hemisected mesothoracic ganglia resulted in rhythmically coordinated motor activity (Fig. 5) which was indistinguishable from that shown by deafferented animals with all ganglia intact. Hemisection of the metathoracic ganglion resulted in an abnormal pattern of flight muscle firing. However, a basic rhythmicity of motor activity was still present (Fig. 6). The implications of these results for rhythm generation and motor coordination in the flight control system of the locust are discussed.     

The morphology and electrophysiology of the neurones of the paired pedal ganglia of Lymnaea stagnalis (L.)

1. A morphological and electrophysiological map of the identifiable neurones and neuronal clusters of the paired pedal ganglia has been prepared. 2. Neuronal morphology was investigated using the fluorescent dye, Lucifer Yellow CH, whilst electrophysiological properties were studied using conventional intracellular recording techniques and the phase plane technique. 3. The paired pedal ganglia are largely symmetrical and giant neurones usually have contralateral homologues. 4. Neuronal clusters are also paired, but minor asymmetries, both of identifiable neurones and neuronal clusters have been found to exist. 5. These asymmetries are thought to be related to asymmetries of body form. 6. Most of the individually identifiable neurones possess obligatory axon branches which are invariant from one preparation to the next, but variant branches also occur. 7. Within the neuronal clusters, morphology appears to be more variable. 8. Individually identifiable neurones and neuronal clusters were characterized electrophysiologically according to the criteria of action potential shape, spontaneous activity pattern, electrical coupling and common synaptic inputs. 9. Homologous pairs of neurones usually have similar electrophysiological properties, as do those within clusters. 10. A number of wide-acting synaptic inputs have been identified on neurones of the pedal, buccal, visceral and parietal ganglia.