Effects of FMRFamide on the activity of command neurons in defensive behavior of fed and fasting snails, Helix pomatia

FMRFamide (Phe-Met-Arg-Phe-NH2) micropneumophoresis changed bimodally the activity of LPa2, LPa3, PPa2 and PPa3 neurones in fasting and fed Helix pomatia. In fasting creatures peptide application elicited hyperpolarization and decreased the neuronal membrane excitability and responses to tactile stimulation. In fed snails peptide application caused depolarization, decreased membrane resistance and increased the neuronal membrane excitability and responses to tactile stimulation. Neurophysiological mechanisms underlying FMRFamide effects on feeding and defense behaviour are discussed.     

The monosynaptic connection: the modulating effect of opioid peptides on the plasticity of presynaptic neurons and identified synapses]

Study of opioid peptides (leucine-enkephalin and methionine-enkephalin) action on plastic properties of the system of monosynaptically connected neurones LPa7--LPa3, PPa3 and LPa8--LPa3, PPa3 was conducted in the snail brain. It has been shown that all three links in the system studied (presynaptic neurone, postsynaptic neurone and synapse) manifest one and the same type of plasticity--habituation to rhythmic stimulation. Enkephalins have a modulating action on plastic properties of the presynaptic neurone and synapse: they retard the habituation of the presynaptic neurone to intracellular stimulation and retard the development of habituation at synaptic level. However, changes in the character of postsynaptic response in the presence of enkephalins are not a direct consequence of their influence on plastic properties of the presynaptic neurone. Besides, enkephalines reduce the effectiveness of synaptic transmission in the given system: they reduce EPSP duration in the postsynaptic neurone.     

Postsynaptic potentials in the myocardium of snails in the genus Helix

Cardioregulating neurones in the right parietal and visceral ganglia of the snail evoke postsynaptic potentials of various duration, amplitude and polarity in the auricular and ventricular myocardium. Inhibitory neurones with a marked background activity (1-2 imp/s) evoke IPSPs with a duration of 150-200 msec and a latent period of 160-220 msec in the auricle, these potentials being blocked by tubocurarine. EPSPs of approximately the same duration may be recorded in the ventricle during stimulation of the commanding neurones of the pneumostome LPa3 and PPa/3, as well as unidentified neurones. Action potentials in some other identified cardiostimulating neurones (PPa7, V1, V6) induce slow and sustained depolarization in the myocardium. Functional specificity of elements within fast and slow regulatory systems is suggested: discrete IPSPs and EPSPs account mainly for coordination of the systolic contractions of the auricle and ventricle, whereas long-lasting PSPs affect the frequency and intensity of the whole heart.     

Calmodulin blockaders weaken the short-term plasticity of the neuronal cholinoreceptors in the edible snail

By means of recording transmembrane ion currents of identified snail neurones PPa3 and LPa3 a reversible weakening was shown of the speed and depth of extinction of neuronal cholinoreceptor membrane reactions to repeated iontophoretic applications of acetylcholine to the soma by a number of calmodulin blockaders: R24571 (20-50 mmol/l), trifluoperazine (50-200 mmol/l), chlorpromazine (20-60 mmol/l) and prenylamine lactate (30-400 mmol/l). The obtained results testify to a positive control by calmodulin of short-term cholinoreceptors plasticity of the studied neurons.     

Desensitization of choline receptors of somatic membrane command neurons in the snail

Desensitization of somatic membrane receptors to rhythmic application of acetylcholine was studied in command neurons PPa2, PPa3, LPa2, and LPa3 of the snail. The results indicate that desensitization of choline receptors is a relatively simple, short-term phenomenon usually not accompanied by more complex, long-term intracellular reorganizations. With short intervals between applications, however, densensitization may lead to a significant decrease in the reaction of the postsynaptic neuron and display certain properties characteristic also for depression of the synaptic reaction. On this basis we assume that in habituation, postsynaptic receptors participate in the processes of short-term decrease in synaptic efficiency.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 4, pp. 530–538, July–August, 1985.     

Distribution of the LPa3 and PPa3 neuronal processes in the pedal ganglia of the edible snail

By means of intracellular injection of the dye Lucifer yellow processes were revealed of the snail command neurones of escape behaviour LPa3 and RPa3 in pedal nerves ipsilateral to these neurones. A process of the neurone LPa3 was also found in the contralateral Nervus cutaneus pedalis secundus. Blockade of central chemical synapses by cadmium chloride did not lead to disappearance of motor reactions evoked by electrical stimulation of LPa3 and RPa3 neurones both on ipsi- and contralateral sides of the animal foot; this allowed to suggest a presence in the contralateral muscular pedal nerves of these neurones processes unrevealed by the used dyeing method.     

The monosynaptic connection: identified synapses in the CNS of the edible snail

By electrophysiological and microanatomical methods of analysis of snail CNS small neurones it was shown that a number of neurones form a monosynaptic connection (MSC) with the gigantic polyfunctional neurone LPa3. By using cobalt and nickel staining, the structure of MSC cells LPa7--LPa3 was studied. Six identified synapses in two LPa3 processes zones were found. Physiological analysis showed that the revealed MSC was plastic. The described MSC with identified synapses is convenient for studying synaptic transmission mechanisms.     

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.     

EPYLRFamide-mediated reduction of acetylcholine-induced inward currents in Helix lucorum-identified neurones: role of NAADP-dependent and IP3-dependent Ca2+ release from internal stores,calmodulin and Ca2+/calmodulin-dependent protein kinase II

The effect of seven compounds intracellularly applied by spontaneous diffusion were investigated on the EPYLRFamide-induced reduction of acetylcholine-induced inward current (ACh-current) recorded from identified neurones from Helix lucorum. Inward currents were recorded from neurones LPa2, LPa3, RPa3 and RPa2 in isolated ganglia preparations using two-electrode voltage clamp technique. ACh was applied ionophoretically. Heparin, an antagonist of IP(3) receptors (IP(3)Rs), and IP(3), the agonist of IP(3)Rs, decreased the effect of EPYLRFamide. Thio-NADP, a blocker of NAADP-induced Ca(2+) release, beta-NAADP, Ca(2+) releaser, R24571, W-7 (both calmodulin antagonists), and KN-62, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, did not change the modulatory effect of EPYLRFamide. These data suggest that EPYLRFamide decreases ACh-current through elevation of the basal intracellular level of the putative endogenous agonist of IP(3)Rs which activates release of Ca(2+) from intracellular stores. It is concluded that intracellular free Ca(2+) acts on ACh receptor/ionic channel without activation of calmodulin and Ca(2+)/calmodulin-dependent protein kinase II.     

A comparative analysis of the effect of an analog of vasopressin on functionally different neurons in the grape snail

Application of desglycine-argininvasopressin (DG-AVP) differently influenced different types of cells of snail isolated central nervous system. In neurosecretory cells an increase of spontaneous impulse activity took place and, as a rule, bursts of impulses appeared, most often of synaptic origin, excluding PPa1 neurones and one of the neurosecretory cells of the left parietal ganglion. The increase of the bursts activity in these cells was based on the increase of the amplitude of membrane potential waves. Under the influence of neurosecretory cells system activation, EPSPs frequency and amplitude in secondary-sensory neurones increased, which led to a greater probability of the action potentials appearance. At prolonged action the spontaneous EPSPs in these cells began to group in bursts. Excitability and membrane resistance of these cells remained unchanged. DG-AVP had no influence on primary-sensory neurones and motoneurones.     

Identified motor neuron closing the neurostome as a component of the defensive reaction of Helix pomatia

In the suboesophagal complex of Helix pomatia, a neurone (LPa33) was identified as a member of the system of neurones which trigger the defensive behaviour, one of the most responsive components of which is the reaction of the pneumostome closing. Spike activity of the LPa33 unit triggers the pneumostome closing in the form of a coordinated reaction of certain pneumostome muscles, which compose the neurone motor field. It is suggested that neurone LPa33 innervates separate muscles of its motor field directly with its axones.     

Arachidonic acid and its acyclic derivatives regulate the short-term plasticity of the cholinoreceptors of the neurons in the edible snail]

On identified Helix neurones RPa3 and LPa3 using the method of double-electrode clamp technique on the membrane the influence was shown of eicosanoids on the dynamics of inward current extinction caused by the repeated ionophoretic applications of acetylcholine to soma. Extracellular influence of arachidonic acid (50-100 microM) increased the extinction. Phospholipase A2 inhibitor quinacrine hydrochloride (100-600 microM) decreasing the content of arachidonic acid in the cell acted differently. Inhibitor of lipoxygenase oxidation of arachidonic acid (nordihydraquiaretic acid) (3-10 microM) weakened the extinction. Blockader of cyclooxygenase oxidation of arachidonic acid--indomethacin (10-50 microM) did not influence the extinction. All the studied composition decreased the amplitude of input current caused by acetylcholine. The obtained results allowed to suppose that arachidonic acid and its acyclic metabolites formed as a result of lipoxygenase oxidation regulated short-term plasticity of snail neurones cholinoreceptors. Cyclic eicosanoids formed at cyclooxygenase oxidation of arachidonic acid had no regulating influence on cholinoreceptors plasticity.     

Calcium regulation of the short-term plasticity of the cholinoreceptors in neurons RPa3 and LPa3 of the edible snail]

Pharmacological influences, changing intracellular content of Ca2+, reversibly change the speed and depth of extinction of the input current of the Helix RPa3 and LPa3 neurones, elicited by a repeated iontophoretic application of acetylcholine to the soma. Suppression by extracellular medium, devoid of Ca2+ and by verapamyl (100-150 mumol/l) of Ca2+ input to the cell, induced by cholinoreceptors activation, reversibly weakens the extinction. Raise of intracellular Ca2+ level by blockade with ruthenium red (5-10 mumol/l) of specific Ca2+ transport by mitochondria and by mobilization with caffeine (1-4 mmol/l) of Ca2+, deposited by endoplasmic reticulum, accelerates and intensifies the extinction. The obtained results testify that the short-term cholinoreceptors plasticity of the above neurones is positively controlled by Ca2+ entering the cell by chemically controlled ion channels and mobilized from intracellular Ca-depot.     

Characteristics of photosensitization of Pheophorbide a in liposomal media

Pheophorbide a (PPa), a decomposition product of chlorophyll a, is a photosensitizer. The photosensitization mechanisms (Type I and Type 11) of PPa in simple buffer solutions and in buffer solutions containing double-layered DPPC liposomes have been studied using techniques of ESR, spin-trapping, spin-counteraction and laser flash photolysis. The results showed that adding DPPC liposomes to the buffer solution caused an increase of efficiency of generating¹O₂ and PPa-by photoactivating PPa. The increase could be ascribed to the disaggregation of hydrophobic PPa caused by the addition of liposomes and the protective effect of liposomal media on the triplet state of PPa. It is concluded that the photosensitization of PPa in liposomal systems is different from that in simple aqueous solutions, and shows higher efficacy. The results will be useful to elucidating the mechanisms of photodynamic therapy of cancer.     

The role of cGMP in extinguishing the reactions of identified neurons in the edible snail to acetylcholine

Possible role of cGMP is studied in control of extinction of snail neurones RPa4, RPa3 and LPa3 reactions to acetylcholine (ACh), applied rhythmically to neurone soma by means of microiontophoresis. It is shown that guanylate cyclase activators which raise the cGMP level in the cell--Na nitroprusside and Na azide (5,10(-4)-10(-3) mol/l)--intensify at extracellular application the extinction of inward transmembrane current and membrane depolarization in response to ACh. Suggestion is made about participation of cGMP-dependent phosphorylation of membrane proteins in control of the development rate, depth and duration of neurone cholinoreceptors short-term plasticity.     

Characteristics of photosensitization of Pheophorbide a in liposomal media

Pheophorbide a (PPa), a decomposition product of chlorophyll a, is a photosensitizer. The photosensitization mechanisms (Type Ⅰ and Type Ⅱ) of PPa in simple buffer solutions and in buffer solutions containing double-layered DPPC liposomes have been studied using techniques of ESR, spin-trapping, spin-counteraction and laser flash photolysis. The results showed that adding DPPC liposomes to the buffer solution caused an increase of efficiency of generating ~1O_2 and PPa~- by photoactivating PPa. The increase could be ascribed to the disaggregation of hydrophobic PPa caused by the addition of liposomes and the protective effect of liposomal media on the triplet state of PPa. It is concluded that the photosensitization of PPa in liposomal systems is different from that in simple aqueous solutions, and shows higher efficacy. The results will be useful to elucidating the mechanisms of photodynamic therapy of cancer.     

Biochemical investigation to the reliability of the histochemical demonstration of microsomal arylsulphatase activity in cryostat sections

Summary Polyacrylamide gel-electrophoresis was performed with an extract from cultivated skin fibroblasts. Arylsulphatase activity is measured and visualised using the biochemical substrate dehydroepiandrosterone sulphate and the histochemical substrate 6-bromo-2-naphthyl sulphate respectively. The histochemical substrate was hydrolysed at Rf=0.49 and 0.58 while the biochemical substrate was hydrolysed only at 0.49. We conclude that two different microsomal arylsulphatases exist: a sulphatase able to hydrolyse steroid sulphatases (Rf=0.49) and one unable to hydrolyse steroid sulphatases (Rf=0.58). In consequence it is recommended to carry out an electrophoresis experiment after the histochemical investigation, in order to discriminate between these two types of sulphatase.     

Protein kinase C is not involved in regulating the short-term cholinoreceptor plasticity of the PPa3 and LPa3 neurons of the edible snail

A voltage clamp technique on identified Helix lucorum's RPa3 and LPa3 neurons has been used to negate the effect of protein kinase C on extinction of response to repeated iontophoretic applications of acetylcholine to soma. Extracellular influence of phorbol ether, protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate, 0.1-10 mumol/l), or polymyxin B, its blocker (100-500 mumol/l), do not affect the extinction of acetylcholine-induced neuronal response. The data show that protein kinase C is not involved into molecular mechanisms responsible for the regulation of short-term plasticity of RPa3 and LPa3 neuronal cholinoreceptors in Helix lucorum.     

The sensory innervation of the foregut of Panulirus argus (Decapoda Crustacea)† ‡

Ths structure of the stomatogastric neuromuscular system in Panulirus argus, Callinectes sapidus and Homarus americanus has recently been described (Maynard and Dando, 1973). We attempt here to describe the sensory innervation of the foregut in Panulirus argus and, by combining this information with previous published data and less systematic observations on Callinectes and Homarus, to provide in addition a summary of the stomatogastric sensory systems in these types of Decapoda Crustacea (Figure 1, Table I).

Some anatomical problems remain unresolved and there is variation in the structure of the sense organs in different species, but we are able to recognize six major receptor groups in all of the species examined. These are (i) mechanoreceptors which monitor movements of the lower oesophagus and mouth (Receptor reference Nos. 1, 2, 3); (ii) probable chemoreceptors in the higher oesophagus and ventral cardiac sac (Rf. Nos. 6, 9, 11); (iii) cells located in or near the stomatogastric ganglion which monitor movements of the gastric mill (Rf. No. 8); (iv) neurones of the posterior stomach nerve (Rf. No. 15) which monitor movements of the gastric mill; (v) neurones innervating muscles near the cardio‐pyloric valve (Rf. No. 16); (vi) neurones innervating the hepatopancreas duct and the initial part of the intestine (Rf. Nos. 18, 19).

In such a restricted system it should be possible to determine the precise role that the various sensory systems play in the control of the simple movements of the foregut. This research must necessarily involve the investigation, with intracellular techniques, of the central events in the commissural ganglia as these ganglia appear to be the major co‐ordination centres of the stomatogastric nervous system.