The action of the neurotoxins 5,6-dihydroxytryptamine and p-chlorophenylalanine on the electrical activity parameters of the command neurons during long-term sensitization and learning in the snail]

The influence of 5,6-dihydroxytryptamine (5,6-DHT), which selectively destroyed serotonin terminals, and p-chlorphenylalanine, which inhibited serotonin synthesis, was studied on the long-term sensitization (LTS) in a snail. The membrane mechanisms were analyzed by measuring electrical characteristics of command neurons of defensive behavior LPa3, RPa3, LPa2, and RPa2. Snails injected with saline served as an active control. It was shown that the injected drugs inhibited the LTS in certain concentrations. A significant increase was observed in the membrane potential and the threshold of the action potential generation in the command neurons after 5,6-DHT injection in the doses of 20 and 30 mg/kg (in comparison with the active control). Sensitization of snails injected with saline solution led to the LTS and decrease in the membrane and threshold potentials of the command neurons. After the LTS, changes in membrane and threshold potentials in snails injected with 5,6-DHT were negligible in comparison with those injected with 5,6-DHT but without the LTS. Neither the LTS nor subsequent learning resulted in a further decrease in membrane and threshold potentials. Thus, the neurotoxin injection led to an increase in excitability of command neurons and their depolarization, and the LDS did not elicit further excitability increase. Since the shifts of the threshold and membrane potentials were the same, it was concluded that the increase in membrane excitability was induced by the depolarizing shift of the membrane potential.     

Comparative investigation of influence of injections of chlorpromazine and 5,6-dihydroxytryptamine on locomotion, defensive reactions of snall helix lucorum and excitability of command neurons in long-term sensitization

Comparative analysis of the action of chlorpromazine (CPZ) and neurotoxin 5,6-dihydroxytryptamine (5,6-DHT) on defensive reactions and locomotion of grape snail and elaboration of long-term sensitization (LTS), was carried out. Long-term (chronic) injection of chlorpromazine led to significant increasing of a pneumostome closing time and to changing of motor behaviour towards decrease of the velocity of the locomotion. Daily injections of 5,6-DHT in small doses within a week were accompanied by the gradual decrease of the velocity of snails locomotion, which was kept for a week. Similar effect was observed in injection of neurotoxin (30 mgs/kg). Injections of CPZ prevents elaboration of LTS, as well as injections of 5,6-DHT. After the action of CPZ, LTS, LTS followed by CPZ, and also during elaboration of LTS after injection of CPZ, the velocity of locomotion directly depended on the length of leg. During elaboration of LTS after injection of 5,6-DHT, such dependency is not retained. Electrophysiological study revealed that chronic injections of CPZ led to depolarizing shift of membrane potential and decrease of the threshold of action potential generation in command neurons as after injection of neurotoxin 5,6-DHT. Therefore, the action of neuroleptic drug CPZ on the defensive behaviour, locomotion of grape snail and electrical characteristics of identifying neurons is comparable with the action of toxic analogue of serotonin.     

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.     

False transmitter, 5,6-dihydroxytryptamine as a tool in selecting serotonergic Helix neurons for studies with isolated,dialysed cells

1. Dialysed serotonergic neurons were identified, isolated from the ganglia of 5,6-dihydroxytryptamine (5,6-DHT) treated snail, Helix pomatia L. Twenty-four to 40 days after injection of 5,6-DHT into the animal, serotonergic neurons show a specific brown pigmentation, which stays there for several weeks. After protease digestion (0.5–1.0 mg/ml for 10–12 min) the labelled neurons can be easily separated. This method ensures the reliable identification of serotonergic neurons for intracellular dialysis.2. We showed that isolated serotonergic neurons maintain their membrane characteristics, and ion-currents can be registered under voltage clamp, just as from neurons of untreated animals. The threshold concentration of serotonin (10 ⁻⁷ M) and the survival time of pigment labelled dissociated cells were the same as for the control cells.3. Following 5-HT application, the voltage activated Ca-currents were either increased or decreased, depending on the neuron used.4. The different responses are probably caused by different receptors on the cell membrane or by the presence of different types of Ca-channels.5. The deactivation time constant of the Ca-current, calculated from the tail current, was also altered in the pigment labelled neuron following serotonin treatment.     

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.     

The duration of the storage of the electrical characteristics of command neurons in the acquisition of long-term sensitization in the snail

The retention of the long-term sensitization (LTS) of defensive reflex and dynamics of change in electric characteristics (membrane potential (Vm) and action potential generation threshold (Vt)) of command neurons of defensive reflex was studied in a snail during behavioral tests. The membrane mechanisms were analyzed by measuring electrical characteristics of the LPa3, RPa3, LPa2, and RPa2 command neurons on the 1st, 4th, 7th, 10th, and 14th days after the LTS formation and 1 month later. The membrane potential and threshold potential in sensitized snails (-54.1 +/- 2.0 and 24.5 +/- 1.4 microV, respectively) were significantly (p < 0.001) decreased in comparison with the control animals (-60.9 +/- 0.8 and 19.9 +/- 0.6 microV respectively). These changes retained within 14 days after the LTS formation. The results suggest the long-term retention of the increased excitability of command neurons. A month after the LTS formation, the duration of the defensive reflex returned to the initial level and the electric characteristics of command neurons did not significantly differ from the control (-61.1 +/- 2.0 and 19.3 +/- 1.4 microV, respectively).     

The short term effects of 5,7-dihydroxytryptamine on peripheral serotonin stores in Rhodnius prolixus and their long-term recovery

The serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) appears to affect invertebrate systems differently from vertebrate ones. The basis for toxicity in vertebrates appears to involve the intraneuronal actions of monoamine oxidase (MAO) upon the toxin. In insects, MAO is not present in appreciable amounts. In this study, we demonstrate that in vitro 5.7-DHT competitively inhibits the uptake of [³H]serotonin by serotonergic neurohaemal areas. The apparent K_M increases from 4.9 × 10⁻⁷ to 1.7 × 10⁻⁶ M. This neurotoxin also causes a significant release of previously accumulated [³H]serotonin in nominally Ca²⁺-free saline. While 5,7-DHT does not affect the uptake of [³H]tryptophan, it reduces the subsequent synthesis of [³H]serotonin. In vivo, the tissues appear to have recovered 2 weeks after toxin treatment, as determined by immunohistochemistry. At 24 h, 1 week and 2 weeks after injection, the tissues are able to take up and release [³H]serotonin normally. 1 and 2 weeks after injection, insects ingest a normal-sized blood meal, a behaviour acutely disrupted by 5,7-DHT treatment. The results of this and other invertebrate studies suggest that 5,7-DHT does not destroy serotonergic neurons, as it does in vertebrates. 5,7-DHT may be a more useful tool to study the functions of serotonin in invertebrates as one may transiently affect serotonin stores.     

Neurochemical mechanisms of food aversion conditioning consolidation in snail helix lucorum

Effects of cycloheximide, protein synthesis inhibitor as well as serotonin receptor antagonist and NMDA receptor antagonist, on food aversion conditioning consolidation were studied in snail Helix lucorum. Food aversion conditioning was absent in snails after application of cycloheximide. Repeated training produced no food aversion conditioning for the same type of food in these snails without cycloheximide application. Food aversion conditioning was absent in snails after metiotepin, nonselective serotonin receptors antagonist, or after MK-801, NMDA glutamate receptors antagonist, applications. At the same time, repeated training produced facilitated food aversion conditioning for the same type of food in these snails. Our experiments were the first which showed that effect on different molecular mechanisms evoked reversible or irreversible disruption of long-term memory consolidation during the same learning. It was suggested that suppression of retrieval produced reversible effect whereas disruption of memory storage initiated irreversible effect on long-term memory consolidation.     

Activity of neurons in the pedunculopontine nucleus during defensive instrumental conditioning

The activity of 109 neurons in the compact and diffuse parts of the pedunculopontine tegmental nucleus (PPTg) was recorded in freely moving rabbits during the acquisition and actualization of the defensive instrumental conditioned reflex. It was found that 47% of the recorded neurons responded to a conditioned stimulus (CS). This finding suggests the involvement of the PPTg in the instrumental conditioning. A significant prevalence of the excitatory conditioned responses to the CS suggests the predominantly activating influence of the PPTg on its projection structures during conditioning. The neuronal responses to the CS were classified into several basic patterns reflecting stimulus effects, the structure of the behavioral act, and the nature of the reinforcement. They indicated the involvement of the PPTg in attention, motor learning, and reinforcement. A significant decrease in the reactivity of the CS of the PPTg neurons as a result of learning specialization was shown. The revealed differences in the associative and reactive properties (with respect to CS) between the neurons of the compact and diffuse PPTg parts testify to the functional heterogeneity of this structure and suggest the leading role of the cholinergic compact part of the PPTg in the instrumental defensive conditioning. Thus, the obtained evidence suggests the involvement of the PPTg in the mechanisms of attention and acquisition of the active defensive motor conditioning.     

Selective disturbance of associative memory reactivation by serotonin or NMDA glutamate receptor antagonists in snail

Effects of serotonin or glutamate receptors antagonists on reactivation of food aversion conditioning were studied in snail Helix lucorum. Metiotepin (nonselective serotonin receptor antagonist, 0.1 mg/snail) or MK-801 (NMDA glutamate receptor antagonist, 0.005 mg/snail) were injected 24 hours after 3 days of food aversion conditioning, then reminding stimulus (banana, "conditioned" food) was presented and food aversion conditioning was tested. Long-term impairing (more then 2 weeks) of food aversion conditioning was found 3 hours after concurrent reminding and inhibitors injection. Injection of receptor antagonists without reminding stimulus did not influence on food aversion conditioning retrieval. Besides, in snails with amnesia after metiotepin/reminder, facilitation of repeated elaboration aversion conditioning on banana is revealed. The repeated training of snails with amnesia caused by MK-801/reminder did not result in food aversion conditioning. It is was suggested that 5-HT5,6,7 serotonin receptors are involved in mechanisms of memory "trace" extraction of food aversion conditioning, whereas NMDA glutamate receptor - in processes of its storage in snail.     

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.     

Polarographic Measurements of Spontaneous and Mitochondria-Promoted Oxidation of 5,6-and 5,7-Dihydroxytryptamine

Abstract: Spontaneous oxygen consumption by 5,6- and 5,7-DHT (dihydroxytryptamine), related indoleethylamines, and 6-hydroxydopamine and oxygen consumption by these compounds in the presence of rat liver mitochondria were measured by the polarographic oxygen electrode technique. 5,6- and 5,7-DHT react with oxygen at very different rates (2.7 nmol O₂/min and 33.4 nmol O₂/min, respectively) when incubated in buffer, pH 7.2, at a concentration of 1 mm and with different kínetic characteristics. While the oxidation of 5,7-DHT obeys a reaction of second-order type, the oxidation of 5,6-DHT is more complex and characterized by autocatalytic promotion. Coloured quinoidal oxidation products appeared during the degradation of both indoleamines. Glutathione, ascorbate, dithiothreitol, cysteine, albumin, and superoxide dismutase partially prevented 5,6- and 5,7-DHT from oxidative destruction. Catalase saved oxygen only in the case of 5,6-DHT by recycling of O₂ released from near-stoichiometrically formed H₂O₂ during oxidation of 5,6-DHT: 5,7-DHT did not generate H₂O₂ in measurable amounts. Oxygen consumption rates of 5,6- and 5,7-DHT were enhanced after addition of rat liver mitochondria to the incubation medium; this resulted in an accelerated formation of quinoidal products. This stimulatory effect on the oxidation rates of both 5,6- and 5,7-DHT was blocked by cyanide, but not rotenone, and was abolished by boiling of the mitochondria fraction. The observed increase in oxygen consumption in the presence of mitochondria was found not to be influenced by monoamine oxidase-dependent deamination of 5,6- and 5,7-DHT. It is postulated that 5,6- and 5,7-DHT are capable of participating in the electron transfer of the mitochondrial respiration chain beyond complex III. Results obtained in determinations of ADP:0 ratios in respiratory control experiments exclude a possible interference of 5,6-DHT, 5,7-DHT, and 6-OH-DA with phosphorylating sites. During the activated state of respiration, no signs of electron transfer inhibition by 5,6- and 5,7-DHT were detectable. A comparison and evaluation of the autoxidation rates of various hydroxylated indoleethylamines, of their affinity to the 5-HT transport sites, and their neurotoxic potency in vivo reveals that interaction of these compounds with oxygen at restricted reaction velocity is a prerequisite for efficient toxicity in monoaminergic neurons following active accumulation in these neurons via the high-affinity uptake systems.     

Functional recuperation of the serotoninergic innervation in the rat locus coeruleus

1. 5,6-dihydroxytryptamine (5,6-DHT) or a lesion of the raphe centralis superior (RCS) cause significant decreases in the serotonin (5-HT) content and significant increases in the tyrosine hydroxylase activity in the locus coeruleus (LC) of the rat. This suggests that noradrenaline (NA) synthesis is controlled by serotonin-containing neurons in the raphe system via their terminals in the LC. 2. Radioautography after intraventricular infusion of tritiated serotonin (3H-5-HT) and biochemical determinations of endogenous 5-HT content showed an almost complete disappearance of serotoninergic axonal varicosities and content in the LC region 10-15 days after intraventricular administration of 75 micrograms of 5,6-DHT. Two to 4 months after neurotoxin administration, 5-HT fibers had regrown in the LC but, contrary to the normal innervation pattern, the majority of them invaded the medial most portion of the nucleus and the adjacent subependymal region. The LC region regained almost all of its endogenous 5-HT content in the same time period. 3. Functional recuperation of these 5-HT fibers was demonstrated by the fact that the RCS had, after regeneration, the same functional control on NA synthesis as in the normal animal.     

Immediate and Long-Term Effects of 5,7-Dihydroxytryptamine on Rat Striatal Serotonergic Neurons Measured Using In Vivo Voltammetry

The immediate and long-term effects of the selective serotonergic neurotoxin 5,7-dihydroxytryp-tamine (5,7-DHT) on rat striatal serotonergic neurons were examined after its intracerebroventricular administration using in vivo voltammetry. Extracellular concentration of 5-hydroxyindoles increased immediately following intracerebroventricular 5,7-DHT injection (200 g in 24 l, 18 min), peaked at 1.5-2 h, and returned to normal by 4 h. 5,7-DHT diffused to the contralateral striatum in detectable amounts 9 to 12 min after the start of injection and returned to basal levels by 1.5 h. Three to 6 days after 5,7-DHT lesions, 5-hydroxytryptophan administration produced an increase in striatal 5-hydroxyindoles that was greater than that produced in pre-lesioned rats. This effect was maximal at 14 to 17 days post-lesion, and remained even after 50 days. The short-term effect of 5,7-DHT may be attributable to increased serotonin release, inhibition of uptake, or monoamine oxidase inhibition. The long-term effect of 5,7-DHT lesions may attributable to increased synthesis of serotonin or decreased reuptake in remaining serotonergic neurons.     

Involvement of mesencephalic reticular formation neurons in cat conditioned reflexes

Traditional defensive and operant food reflexes were used to investigate neuronal responses of the mesencephalic reticular formation. It was found that these neurons may be divided into different groups according to function, depending on how they respond to positive conditioning stimuli. Of the two main groups of neurons with sustained tonic reactions one is activated in response to positive acoustic conditioning stimulation; it no longer reacts to the same stimulus after extinction of the reflex, while the other only becomes involved in response to positive stimulation accompanying the initiation of movement. Neurons belonging to the second group begin to respond directly to acoustic stimulation after extinction of the conditioned reflex. Neurons of the mesencephalic reticular formation can thus exercise additional tonic ascending effects both in the production and inner inhibition of the conditioned reflex. The group of neurons with a phasic reaction, i.e., a double response (a direct response to sound and another produced by movement) displayed a drop in spontaneous activity during the shaping of inhibition of differentiation and of extinction in particular. It was found that the initial changes in the spike response of reticular formation neurons during conditioning and pseudo-conditioning are similar. There are thus grounds for stating that neurons of the mesencephalic reticular formation participate in the shaping, production, and inner inhibition of traditional and operant conditioned reflexes in a differentiated capacity rather than as a population reacting identically.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 161–171, March–April, 1986.     

The effect of 5,6-dihydroxytryptamine on post-decapitation convulsions

Previous data (1) have shown that L-DOPA increases the duration of the clonic phase of post-decapitation convulsions (PDC) in mice. It was suggested that this effect is produced by depleting 5-hydroxytryptamine (5-HT) in the inhibitory bulbospinal pathways and thus enhancing reflex activity in the spinal cord. If this were true then L-DOPA administration should not influence clonic PDC in animals whose 5-HT pathways were destroyed. We therefore tested the effects of L-DOPA on mice 3 weeks after pretreatment with the 5-HT neurotoxin, 5,6-dihydroxytryptamine (5, 6-DHT) (50 μg/kg, intracerebroventricularly). All mice were given the peripheral decarboxylase inhibitor, Ro 4-4602. 5,6-DHT halved the brain 5-HT levels and significantly increased the duration of clonic PDC. The administration of L-DOPA (320 mg/kg i.p.) to 5,6 DHT treated mice did not produce any further significant increases in duration. The administration of 5-hydroxytryptophan (5-HTP) (100 mg/kg, i.v.) to 5,6-DHT treated mice, however, increased 5-HT to above control levels and reduced convulsions to control levels. Administration of both 5-HTP and L-DOPA to 5,6-DHT treated mice resulted in 5-HT levels and convulsion times which were also not significantly different from the controls. These data give additional indication that intact 5-HT nerve terminals are necessary for L-DOPA to prolong the duration of clonic PDC.     

Differential effects of serotonin depletion on sensitization and dishabituation in the leech, Hirudo medicinalis.

The goal of these experiments was to test the hypothesis that serotonin (5-HT) is involved in facilitation of the shortening reflex in the leech Hirudo medicinalis. For this reason, we have used the toxin 5-hydroxytryptamine (5,7-DHT) to deplete serotonin from the nervous systems of intact leeches and have assessed the effect on early facilitation, dishabituation, and sensitization of the touch-elicited shortening reflex using behavioral procedures previously developed in our lab (Boulis and Sahley, 1988). We find that 5,7-DHT lesions completely attenuate early facilitation and sensitization but only reduce dishabituation of the touch-elicited shortening reflex. Histological analyses of the ganglia from these leeches using glyoxilic acid staining procedures revealed an absence of staining in the Retzius cell of experimental leeches. All other serotonin-containing neurons showed glyoxilic acid staining comparable to that observed in the control leeches.     

Differential effects of serotonin depletion on sensitization and dishabituation in the leech,Hirudo medicinalis

The goal of these experiments was to test the hypothesis that serotonin (5-HT) is involved in facilitation of the shortening reflex in the leech Hirudo medicinalis. For this reason, we have used the toxin 5-hydroxytryptamine (5,7-DHT) to deplete serotonin from the nervous systems of intact leeches and have assessed the effect on early facilitation, dishabituation, and sensitization of the touch-elicited shortening reflex using behavioral procedures previously developed in our lab (Boulis and Sahley, 1988). We find that 5,7-DHT lesions completely attenuate early facilitation and sensitization but only reduce dishabituation of the touch-elicited shortening reflex. Histological analyses of the ganglia from these leeches using glyoxilic acid staining procedures revealed an absence of staining in the Retzius cell of experimental leeches. All other serotonin-containing neurons showed glyoxilic acid staining comparable to that observed in the control leeches.     

Serotonin involvement in the inhibition of luteinizing hormone (LH) release during immobilization in castrated male rats

The involvement of serotonin in mediating the inhibitory effect of immobilization stress on LH secretion in castrated male rats was examined by employing p-chlorophenylalanine (PCPA, 320 mg/kg, ip), an inhibitor of serotonin synthesis, and 5,6-dihydroxytryptamine (5,6-DHT, 50 micrograms, icv), a drug toxic to the indoleaminergic system. Immobilization stress suppressed pulsatile LH release and decreased mean plasma LH levels. Pretreatment with PCPA or 5,6-DHT apparently eliminated the inhibitory effect of immobilization stress on LH release. These results suggest the possible involvement of a serotoninergic mechanism in mediating the suppression of LH release induced by immobilization stress in castrated male rats.