Cellular reactions to elaboration of a backward conditioned connection in Helix lucorum

After 10-15 food stimuli paired with electrical shock in semi-intact snail preparation, responses to strong tactile stimuli identified feeding behaviour neurones were studied. Inhibition evoked by tactile stimulation in these cells before learning procedure disappeared and in some cases noxious stimulus evoked synaptic activation corresponding to feeding reactions in the intact animal. Changes in second-order sensory neurones pre-synaptic to the command neurones of avoidance behaviour are suggested to be the mechanism of forward conditioned connection as well as the mechanism of backward conditioned connection.     

Neuronal correlates of changes in the defensive behavior of Helix lucorum L. during ontogeny

In experiments on the isolated nervous system, responses of the same command neurones of avoidance behaviour to electrical stimulation of the intestinal nerve were compared in adult snails (Helix pomatia) and in snails 1-20 days old. Dynamics of spike reactions of the command neurones to rhythmic nerve stimulation differed in adult and newborn snails. In the neurones of young snails, no sensitization, common in the adult animals, was observed. Study of excitatory input in the command neurones during rhythmic nerve stimulation suggests independence of habituation of synaptic input amplitude and spike response sensitization, because dynamics of habituation coincided in the young and adult snails while spike response underwent sensitization only in adult animals.     

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.     

Differences in the responses of identified neurons to chemical stimuli in satiated and hungry edible snails

Reactions of command neurones for avoidance behaviour to food were investigated in hungry and satiated snails in "CNS-chemoreceptor" preparations, in which hemolymph was washed out by saline. Neuronal responses in hungry animal preparations differed significantly from responses in satiated animals preparations. Perfusion of hungry animal preparations with hemolymph of satiated animals changed significantly the responses of command neurones for avoidance behaviour. These responses resembled the reactions of the same neurones to food after aversive conditioning to food of hungry snails. The role of humoral factor in learning is discussed.     

Sensitization and habituation of command neurons during a defensive reflex in grape snails

All investigated neurones were classified into functional groups depending on their participation in unconditioned avoidance reflex of pneumostome closure. Habituation was produced by tactile stimuli applied with a frequency of 0.1 c/s. Spike responses of neurons participating in processing sensory information habituated gradually, while reactions of command neurones of avoidance behaviour became sensitized to the second or third stimulus of the series. Behavioral reaction was sensitized in parallel with command neurones in spite of habituation in other types of nerve cells. A conclusion is drawn that behavioral habituation is due to waning of responses in all the participating neurones, but the independnet process of sensitization is due to sensitization of command neurones. Probable mechanisms of neuronal sensitization are discussed.     

Dynamic defensive and feeding reactions to the acquisition of sensitization in edible snails]

In snails changes of defensive and alimentary behaviour and of reactions of command and modulator neurones of these kinds of behaviour were studied during elaboration of sensitization. After a single action of 50% chinine solution on mollusc's head, a short-term (during 50-70 min) and long-term (hours or days) facilitation was found of animals defensive reactions and responses of command neurones of defensive behaviour to tactile and chemical sensory stimuli. Alimentary behaviour of the snails and reactions of modulator neurones of alimentary behaviour to carrot juice presentation were inhibited in sensitized animals. Dynamics differences of defensive responses to tactile and chemical stimuli in the same sensitized animals were observed. Short-term responses facilitation under sensitization correlated chiefly with depolarization of the membrane potential of defensive behaviour neurones and with an increase of plasmatic membrane excitability. Prolonged facilitation of responses was due mainly to a change of effectiveness of synaptic transmission. The described model of sensitization elaboration can be a base for studying of molecular-cellular mechanisms, underlying the learning.     

Effect of serotonin and noradrenaline on the magnitude of the response of the command neurons for defensive behavior in Helix lucorum L

Changes in synaptic responses of identified command neurones of avoidance behaviour to the electric nerve stimulation were investigated in the isolated nervous system of the snail during bath application of serotonin or noradrenaline. Serotonin (10(-5) M) elicited an increase of summary EPSP amplitude in the cells without changes of input resistance and resting potential. Noradrenaline (10(-5) M) application evoked an increase of EPSP amplitude, accompanied by an increase of the input resistance. Mechanisms of serotonin and noradrenaline influence on synaptic responses are discussed.     

The neurochemical basis of recurrent inhibition in the reflex arch of the defensive reaction]

Mechanisms of habituation in the network of identified neurones were investigated in isolated preparation of central nervous system in the snail Helix. It has been found that intracellularly induced spike discharge in premotor command neurones decreases synaptic responses to repeated nerve stimulation in all recorded command neurones. Application of the neuropeptide FMRFamide elicits similar changes in the network. Taking into account that the investigated command neurones contain FMRFamide, as was shown immunochemically, it is possible to assume the existence of recurrent inhibition in the network underlying avoidance reactions. This recurrent inhibition causes habituation of the network output in the cases when the repeated stimuli do not evoke sensitization via activation of serotonergic cells.     

The role of dopamine and serotonin in modulating the defensive behavior of the edible snail

Dopamine application in concentration of 10(-5)-10(-6) M into saline around the snail CNS leads to decrease of excitability of LPa7 neurone which is presynaptic in relation to defensive behaviour command neurones, and to decrease of amplitude of monosynaptic excitatory postsynaptic potential (EPSP) in the command neurones elicited by intracellular stimulation of LPa7 neurone. Besides, the dopamine causes a decrease of summated EPSP amplitude in the studied neurones in response to intestinal nerve stimulation (70% in average), a change of rest potential towards hyperpolarization for 6-8 mV, a reduction of the command neurones input resistance (20% in average). The described influences can lead to a general increase of the threshold of defensive system reaction to stimulation. Dopamine action on the defensive behaviour command neurones is significantly weakened in serotonine presence. Against the dopamine background, the efficiency of serotonine influence on the value of EPSP in command neurones in response to testing stimulus is reduced. According to the obtained data, a conclusion is made that interrelation of dopamine and serotonine concentrations can be a base for formation of behaviour choice in snail.     

Postsynaptic mechanism of withdrawal reflex sensitization in the snail

Repeated tactile stimulation of the skin evoked sensitization and subsequent habituation of a behavioral avoidance reaction of pneumostome closure in the land snail, Helix lucorum L. Spike responses in putative command neurons for avoidance behavior increased and subsequently decreased parallel to behavioral events. Decrease of spike responses in command neurons was due to decreased synaptic input evoked by repeated tactile stimulation. Intracellular investigation of the increase in the spike response in command neurons for pneumostome closure, which underlies behavioral sensitization, revealed a synpatically-evoked, long-lasting depolarization. No conductance changes during this depolarization were found, but neuronal excitability was altered.     

The effect of a vasopressin analog on the reaction of the command neurons in the defensive behavior of the edible snail during nerve stimulation

On the level of snail command neurones of the defensive behaviour an increase was revealed of the amplitude of summary EPSP recorded in command neurones in response to nerve stimulation, an increase of probability of appearance of action potential in the reaction and, under certain conditions, a significant decrease of habituation speed at rhythmic (0.1 Hz) nerve stimulation against the background of peptide. The latter effect was found during comparison of groups of neurones--control neurones and those to which the peptide was presented before the first series of stimulation in the test, i. e. without preliminary elaboration of habituation. The decrease of the speed of habituation concerned both the amplitude of summary EPSP and the probability of action potential appearance in the reaction. All these changes against the background of vasopressin analogue may be the basis of the increase of spike reactions of command neurones of the defensive behaviour and thus the basis of the initiation or the increase of behavioural defensive reactions. The obtained effects were not protracted and took place only in the presence of the peptide in the extracellular medium.     

Feeding clusters and olfaction in the mangrove snail Terebralia palustris (Linnaeus) (Potamididae: Gastropoda)

Large numbers of the snail Terebralia palustris (Linnaeus) (Potamididae; Gastropoda) are frequently observed feeding in a cluster on a single fallen mangrove leaf, yet none are present on leaves nearby. Consequently, we investigated the food-finding ability of T. palustris in a Kenyan mangrove forest using field experiments. We estimated the attractive effect of different cues and analysed the paths (video-recorded) of snails when approaching a food-related odour. This intertidal snail can potentially use both air-borne and water-borne odours to locate food. T. palustris is attracted to conspecifics feeding on leaves, while intact leaves as well as non-feeding snails are not attractive. Moreover, the guiding stimulus appears to be compounds released when the leaves are damaged.T. palustris also seems able to discriminate between different food items; it is more strongly attracted to green mangrove leaves than senescent or fallen ones or mangrove propagules, probably because green leaves release a greater amount of attractive cues.Feeding snails thus recruit more snails to feed on the same leaf. The ecological implications of this behaviour are discussed: a large number of snails on the same leaf counteracts the ability of crabs to remove the leaf being eaten by the snails.     

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.     

Selectivity of stimulus induced responses in cultured hippocampal networks on microelectrode arrays

Sensory information can be encoded using the average firing rate and spike occurrence times in neuronal network responses to external stimuli. Decoding or retrieving stimulus characteristics from the response pattern generally implies that the corresponding neural network has a selective response to various input signals. The role of various spiking activity characteristics (e.g., spike rate and precise spike timing) for basic information processing was widely investigated on the level of neural populations but gave inconsistent evidence for particular mechanisms. Multisite electrophysiology of cultured neural networks grown on microelectrode arrays is a recently developed tool and currently an active research area. In this study, we analyzed the stimulus responses represented by network-wide bursts evoked from various spatial locations (electrodes). We found that the response characteristics, such as the burst initiation time and the spike rate, can be used to retrieve information about the stimulus location. The best selectivity in the response spiking pattern could be found for a small subpopulation of neurones (electrodes) at relatively short post-stimulus intervals. Such intervals were unique for each culture due to the non-uniform organization of the functional connectivity in the network during spontaneous development.     

Selective involvement of non-histone chromatin proteins in the reproduction of averse reaction to food in snail

Participation of the brain-specific non-histone chromatin proteins Np-3.5 in the reproduction of an averse habit to certain kind of food was studied in experiments onHelix lucorum. Antibodies to these proteins were found to completely suppress the behavioral reactions and responses of the defensive behavior command neurons evoked by a presentation of a certain conditioning stimulus: a carrot. The action of the antibodies was specific, as they did not change the snail's reactions to presentation of another conditioning stimulus: an apple. The effect developed 1.5 h after the administration of antibodies to Np-3.5, and remained for 80–110 min, with subsequent complete restoration of the conditioned reflex. The antibodies to Np-3.5 did not affect the nutritive behavior of untrained snails. The antibodies to Np-3.5 were found to label cytoplasm in the command neurons, whereas in the snails trained to reject carrot the label appeared in the nuclei of the cells. It has been suggested that the Np-3.5 proteins selectively participate in the molecular-genetic processes responsible for neurophysiological mechanisms of an information draw from the long-term memory.     

Acoustic response of the B cell in noctuid moths

Spike activity of the B cell, a unit in the tympanic organ of noctuid moths, was monitored during both stationary flight and acoustic stimulation. This neuron was previously considered (Treat and Roeder, 1959) unresponsive to sound and was thought to be a sensor for thoracic deformations accompanying flight movements. The present study did not reveal changes in B cell spike repetition rate related to active wing flapping, but did reveal a decline in the tonic repetition rate with the onset of an acoustic stimulus. Experimental results indicate that this inhibition of B spike activity is, at least in part, indirect, with the acoustic sense cells serving as the primary receptors. B cell inhibition is most marked with rapidly pulsed, relatively intense bursts of ultrasound. Lowintensity continuous tones evoke little or no change in the B spike repetition rate. The relationship between B spike activity and that of the acoustic sense cells (A cells) is complex. There is roughly an inverse correlation between the two spike activities with the onset and with the cessation of an acoustic stimulus, but B spike activity changes during the stimulus with no concomitant change in A cell spike activity. The possible significance of this complex functioning is considered in relation to avoidance behaviour in flying moths.     

The theta modulation of rabbit hippocampal neurons and its correlation with other indices of spontaneous and evoked activity]

Reliability of the existing functional criteria for differentiation of pyramidal ("complex spike neurones") and inhibitory ("theta neurones") cells in the hippocampus of waking rabbit is evaluated on the basis of statistical analysis of neuronal spontaneous and evoked activity. The analysis shows, that the criteria of mean frequency, presence of theta modulation, neuronal behaviour in situations provoking EEG theta rhythm (e.g., excitation or inhibition during presentation of sensory stimuli), effects of medial septum and intrahippocampal stimulation do not permit reliable identification of the hippocampal neuronal types in the waking rabbit. The data on functional classification of the hippocampal neurones are discussed in connection with existing suggestions about their state in situations inducing theta rhythm generation.     

The effect of the small cardioactive peptide (b) on synaptic transmission efficiency and on the excitability of command neurons in the defensive behavior of the edible snail]

Small cardioactive peptide (b) (SCPb) application in concentration 5 X 10(-8) mol/l into the saline surrounding the snail CNS leads to an increase of amplitude of summate excitatory postsynaptic potential in the command neurones (CN) elicited by the intestinal nerve stimulation. Besides, SCPb causes an increase of excitability of the CN. Described effects can change the threshold of the neuronal net underlying avoidance behaviour. The possibility of integrative effects of peptides in low concentration on the behaviour is discussed.     

Electrophysiological study of effects of chronic injection of caffeine on defensive reflex conditioning in grape snail

It was found that chronic injection of caffeine to grape snail increases a velocity of elaboration of conditioned defensive reflex. It was shown that after daily injection of caffeine immediately after procedure of learning the conditioned defensive reflex elaborated faster than daily injection before procedure of learning. It has been shown, that chronic injection of caffeine both in naive as well as learned snails led to depolarizing shift of membrane potential and to decrease of threshold potential of command neurons of the defensive behaviour of grape snails. It was also found that addition of caffeine in bath solution led to decrease of threshold of generation of action potential of command neurons both in intact and learned snails. The resting membrane potential of command neurons was not changed.     

Microelectrode investigations of learning phenomena in snail (Helix pomatia) neurones.

We have examined changes of postsynaptic potentials and of pattern activity of the identified silent and oscillatory snail neurones in Helix pomatia during conditioning. Local changes of EPSP or IPSP have been recorded during association following the first stimulus in the silent cells, whereas spike discharges could be observed in response to the 2nd stimulus. In the oscillatory neurones changes of pattern activity have been recorded following the 2nd stimulus, while the first stimulus proved to be ineffective. The formation of temporary connections of snail neurones seemed to be a specific phenomenon, because it was necessary to pair stimuli of different inputs for the development of these modifications. These plastic changes seemed to depend on the interstimulus as well as on the intertrial intervals. Our experimental data underline the probable role of the stimulus parameters and of the electrical properties of neurones during the formation of learned neuronal responses.