Duration of changes in electrical characteristics of command neurons after defensive conditioning in snails

Electrical characteristics of snail command neurons were studied during and after defensive conditioning. Tapping on the shell was used as a conditioned stimulus. A light air blow into the lung cavity orifice (pneumostome) was used as an unconditioned stimulus. The conditioned defensive reflex is known to be retained for 40 days. We have shown earlier the decrease in membrane and threshold potentials of command neurons after defensive conditioning (Gainutdinov et al., 1996). In these experiments it has been found that the decreased level of membrane and threshold potentials are maintained during 40 days after defensive reflex conditioning.     

Effect of selective protein kinase C inhibitor on synaptic plasticity of command neurons of defensive behaviour during sensitization in snails

Polymyxin B (proteinkinase C inhibitor) effects on nociceptive sensitization of semiintact preparation were investigated in LP11 and RP11 snail neurons. It was found that application of sensitizing stimuli to control snail head initiated neural membrane depolarization, increase its excitability as well as depression of neural responses evoked by sensory stimulation during short-term stage. Polymyxin B application suppressed neural responses evoked by sensitizing (nociceptive) stimuli. At the same time changes in neural membrane excitability as well as neural responses evoked by tactile stimulation of snail foot or chemical stimulation of snail head were similar with ones in control snails. Polymyxin Bdid does not change the depression of neural responses evoked by tactile stimulation of snail head during short-term stages of sensitization but significantly suppressed facilitation of neural responses evoked by tactile stimulation of snail head during long-term stage of sensitization. It was suggested that proteinkinase C is involved in regulation of nociceptive mechanisms as well as in plasticity selective induction mechanisms in command neuron synaptic inputs activated by tactile stimulation of snail head.     

Effect of changes in extracellular calcium concentration on electrical characteristics of command neurons after defensive reflex conditioning in snail

Studies of electrical characteristics of command neurons of the defensive reflex have shown that membrane potential does not reliable differ for both naive and learned snails with changing of the calcium concentrations in external solution. With increasing of the concentrations of calcium ions it is observed the increases of threshold potential from 14 + 0.7 mV in 2.5 mM Ca2+ to 21.8 + 0.9 mV in 20 mM Ca2+ for naive snails. Threshold potential decreases from 16.8 + 0.6 mV (saline solution for snail--10 mM Ca2+) to 13.3 + 0.6 mV in 20 mM Ca2+ with increasing of calcium concentrations for learned snails, and under its reduction it is also decreased, but less remarkably, than for naive, to 11.8 + 0.8 mV in 2.5 mM Ca2+. On the same scheme it is changed a critical level of depolarization: for naive snails it decreases with increasing of the calcium concentrations, and for learned snails it increases either with increasing of the calcium concentrations, and so with its reduction.     

Effect of desglycine-arginine vasopressin on the excitability of the command neurons of the defensive reflex in the edible snail

Perfusion of the snail (Helix lucorum L.) CNS with DG-AVP (concentration 10(-6) M) in the course of low frequency intracellular stimulation (2-4-minute interval) of the defensive reflex command neurons led to an increase in the excitability. It was expressed both in the reduction of the spike generation latency, in the increased number of spikes in response to fixed stimuli, and in the activation of pacemaker potentials. If DG-AVP was added to the medium during endoneuronal habituation, there was no increase in the excitability. It is supposed that modification of the neuronal excitability may be caused by the DG-AVP effect on the pacemaker mechanism.     

Mobility of acetylcholine receptors in command Helix lucorum neurons in a cellular analog of habituation

We investigated the role of the mobility of acetylcholine receptors in the depression of an acetylcholine-induced inward current (ACh-current) of Helix lucorum (a land snail) command neurons of defensive behavior in a cellular analog of habituation. The inhibitors of endocytosis and exocytosis, actin microfilaments and cytoskeleton microtubules, serine/threonine protein kinases (PKA, PKG, calcium calmodulin-dependent PK II, p38 mitogen-activated PK), tyrosine kinases (including Src-family kinases), serine/threonine phosphatases (PP1, PP2A, PP2B, PPM1D), and tyrosine protein phosphatases altered the depression of the ACh-current. A comparison of experimentally calculated curves of the ACh-current of these neurons and those obtained by mathematical modeling revealed the following: (a) ACh-current depression is caused by the reduction in the number of membranous ACh-receptors, which results from the shift in the balance of multidirectional transport processes of receptors toward the predominance of ACh-receptor internalization over their recycling; (b) depression of ACh-current depends on the activity of serine/threonine and tyrosine protein kinases and protein phosphatases, whose one of the main targets is the neuron transport system—actin microfilaments and microtubules of cytoskeleton, as well as motor proteins.     

Elementary and compound postsynaptic potentials in the defensive command neurons of Helix lucorum

The present communication concerns with the analysis of elementary and the compound excitatory postsynaptic potentials (eEPSPs and cEPSPs) recorded by intracellular microelectrode from an identified defensive command neuron of the snail Helix lucorum. The eEPSPs were evoked by single presynaptic action potentials (APs) elicited by cationic current injection into one of the identified sensory neurons synapsing on the respective command neuron. The cEPSPs were elicited by local brief tactile stimuli on the skin or internal organs. It was shown that the cEPSPs amplitudes depend mainly on the number of activated sensory neurons. Compound EPSPs depend also on frequency and the number of APs in the bursts occurring in a single neuron. Presynaptic APs having frequency 2-10 Hz evoke high frequency depression of that eEPSPs after an interval is followed by post-tetanic potentiation of single eEPSPs. Preceding stimulation of a pneumostom area facilitates the cEPSPs elicited by repeated stimulation of viscera. The eEPSPs from the same visceral area demonstrate no heterosynaptic facilitation in experiments with double parallel intracellular recording from responsive sensory and command neurons. The different types of the eEPSPs plasticity are discussed according to their contribution cEPSPs plastic changes.     

Sensitization inHelix snail: Morphofunctional correlates in command neurons of withdrawal behavior

Electrophysiological effects have been studied in command neurons of withdrawal behavior inHelix snail. In parallel, correlated changes in the content of bound calcium (Ca-b), as well as changes in DNA condensation, were investigated using a chlortetracycline fluorescent probe and the fluorescent dye bisbenzimide, respectively. Short-term electrophysiological changes (depolarization of the membrane and elevation of its excitability) in sensitized snails have been found to be accompanied by an increase in the Ca-b level in the cell nucleus and by partial DNA decondensation. Long-term effects were characterized by more pronounced synaptic components of the responses — slow EPSPs evoked by sensory stimuli, as well as by further DNA decondensation and considerable elevation of the Ca-b content in the nucleus and cytoplasm. The results obtained inin vitro conditions have shown that Ca-binding nonhistone proteins of chromatin are components of the cell nucleus whose content may be measured by chlortetracycline fluorescence.Translated from Neirofiziologiya, Vol. 25, No. 2, pp. 150–157, March–April, 1993.     

Interrelationship between grape snail alimentary and defense behavior command neurons]

Food stimulus (carrot juice), releasing feeding behaviour in intact snails, evoked spike discharges in giant meta-erebral cells (considered to be command neurones of feeding behaviour) and subthreshold EPSPs in giant parietal cells (command neurones of avoidance behaviour) of a half-intact preparation. Tactile stimulation, eliciting avoidance reactions in intact snails, evoked hyperpolarization in command neurones of feeding behaviour and a spike discharge in command neurones of avoidance behaviour. Spikes induced into either of command neurones produced no changes in activity of other command cells. Inhibition of command neurones of feeding behaviour is assumed to be the basis of behavioural choice when food and tactile stimulus are presented simultaneously.     

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.     

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.     

Rf 0.58 protein in Helix command neurons during learning

At early stages of aversive conditioning in Helix, a most considerable increase in the acid brain-specific protein Rf 0.58 occurred in LPa3 and PPa3 neurones. Later the content of this protein decreased in the PPa3 but went on increasing in LPa3. In sham learning, the protein content did not increase so obviously. Hence the protein Rf 0.58 metabolism in individual neurones of the snail CNS correlates with the draw step of receptor and effector fields in avoidance conditioning.     

Immunocytochemical study of the hcs2 gene products distribution in the command neurons of grape snail

In the present study the cellular and subcellular distribution of putative protein products of hcs2 gene in the giant command neurons of parietal ganglia of the terrestrial snail Helix lucorum L. were investigated using light- and electron-microscopic immunocytochemistry. The product of hcs2 gene is a hybrid precursor protein belongs to the EF-hand family of the Ca(2+)-binding proteins, whose processed products are neuropeptides. By use of polyclonal antibodys against a synthetic CNP3, CNP4 and C-terminus peptide immunoreactivity was observed in the cytoplasmic secretory granules. The colloidal gold density in the granules for CNP3-4 neuropeptides was twice one for the Ca(2+)-binding protein. These immunocytochemical results point to a specific connection between putative protein products of hcs2 gene and the cell secretory apparatus, that correspond to our early expressed hypothesis that products of hcs2 gene act as neuromodulators or neurotransmitters.     

Conditioned reflex: detector and command neuron

Conditioned reflex is characterized by plasticity resulting in a bilateral selective input-output linking. In simple nervous systems, input stimuli are represented by selective detectors connected with command neurons through plastic synapses strengthened during associative learning and weakened during extinction. The process of associative learning is due to temporal coincidence of excitation in both detector and command neurons. Short-term memory within a plastic synapses is mediated by phosphorilation of postsynaptic receptor molecules not requiring protein synthesis. Long-term synaptic memory parallels expression of immediate early genes that mediates structural gene expression and protein synthesis. A simple detector-command neuron association becomes more complex in the course of evolution. Input mechanism is supplemented with predetector interneurons preceding detectors. Detector selectively tuned to specific input stimulus is converging on a command neuron constitute selectivity mechanism for conditioned reflexes to complex stimuli. The complication also concerns the output mechanisms. Command neurons become more specialized, and an additional link of premotor interneurons is incorporated between command neurons and motor neurons. Via synapses, the command neurons can produce excitation in a particular set of premotor neurons controlling a specific set of motor neurons responsible for behavioral act configuration. Specialization of command neurons in combination with premotor neuron structures increases the variability of outputs. Conditioned reflexes with more complex inputs and more flexible outputs determine the diversity of acquired behaviors.