Dmitri Sakharov

Is nitric oxide (NO) produced by invertebrate neurones

NADPH-diaphorase (NADPHd) is known to be identical to nitric oxide (NO) synthase in the mammalian nervous system, and is therefore used as a marker of NO-producing neurones. Using the histochemical reaction for NADPHd, we searched for such neurones in a selection of invertebrates. Special emphasis was given to molluscs. No selective neuronal staining was found in representatives of coelenterates, turbellarians, nematodes and urochordates. In all annelids, arthropods and molluscs examined, with the exception of a chiton, specific neurones were selectively stained. The reaction was particularly strong in pulmonate molluscs where scattered positive neurones were found in various ganglia and clustered symmetrically in the paired buccal ganglia. Biochemical assay of NO synthase in osphradia of the gastropod mollusc Lymnaea stagnalis revealed a formation of citrullin that was inhibited by the specific NO synthase N omega-nitro-L-arginine (NO2Arg). Both histochemical and biochemical methods indicate that NO can be used as a signal molecule by specific neurones in advanced invertebrates.

Complex avoidance behaviour and its neurochemical regulation in the land snail Cepaea nemoralis

1. In hot plate experiments, the pulmonate land snail Cepaea nemoralis displays a biphasic passive/active avoidance behaviour composed of retraction and subsequent searching mediated by antagonistic muscular systems. The switch, between the behaviours, is under neuronal control. 2. Leu- and met-enkephalin, as well as FMRFamide-antiserum, attenuated the retraction response and potentiated the searching behaviour. Opposite effects were achieved by injection of antisera to the enkephalins. 3. Both retraction and searching behaviours were potentiated by 5-HT. Methysergide antagonized the effects of the enkephalins on the searching behaviour. 4. We conclude that endogenous opioids act antagonistic to FMRFamide in the neuronally controlled switch between passive and active avoidance behaviour.

Serotonin- and FMRFamide-immunoreactive nerve elements in the chiton, Lepidopleurus asellus (Mollusca, Polyplacophora)

The distribution of serotonin-like and FMRFamide-like immunoreactive (5HT-ir and FMRFa-ir, respectively) neurons in the nervous system of the chiton Lepidopleurus asellus (Mollusca, Polyplacophora) was studied using an immunocytochemical technique. The neurons were distributed in characteristic patterns in the central nervous system, the 5HT-ir neurons predominating in the ventral (pedal) cords and FMRFa-ir neurons in the lateral cords. In the body wall including the foot, a tight network of 5HT-ir and FMRFa-ir nerve fibers is found, the former being mostly attributed to the musculature whereas the latter seems to be associated with the blood sinuses. Intraepithelial neurons of both types are abundant in the fore-and hindgut. The presence and general distribution in the central and peripheral nervous system of the 5HT-ir and FMRFa-ir elements seems thus to be similar in simple and advanced molluscs. The relationship between these neurons and their targets in the body also appears to be well conserved in molluscs.

Patterns of enkephalin immunolabeling in the pulmonate snailCepaea nemoralis and related molluscs

In the land pulmonate snail Cepaea nemoralis, immunocytochemical localization of methionine- and leucine-enkephalin-like substances was demonstrated in specific populations of brain neurons. Methionine-enkephalin reactivity is also present in the peripheral nervous system as (i) abundant axonal projections of central neurons to the sole of the foot, and (ii) sparse local neurons of the head and mantle wall. Similar peripheral methionine-enkephalinergic elements were found immunocytochemically in all pulmonates (Helix aspersa, Lymnaea stagnalis) and prosobranchs (Littorina littorea, Acmaea testudinalis) examined and seem therefore conserved among gastropods. Their distribution was different from those of nervous elements labeled by antisera against serotonin and FMRFamide. The chiton Lepidopleurus asellus considered to be a more primitive mollusc, had both types of enkephalin-immunoreactive elements in the central nervous system whereas peripheral methionine-enkephalin-immunoreactive elements were lacking. It is suggested on the morphological evidence that enkephalinergic (especially methionine-enkephalinergic) neurons are part of a sensory system mediating analgetic effects in molluscs.

TYROSINE HYDROXYLASE-NEGATIVE, DOPAMINERGIC NEURONS ARE TARGETS FOR TRANSMITTER-DEPLETING ACTION OF HALOPERIDOL IN THE SNAIL BRAIN

Summary1.The effects of long term administration of micromolar concentrations of the D2 antagonist haloperidol upon monoaminergic neurons in the snailLymnaea stagnalis was investigated.2.Treatment by bath application with 0.5–2.0 micromolar haloperidol, caused a significant, continuous depletion of dopamine levels in the nervous system as revealed by high performance liquid chromatography.3.A transient depletion of serotonin was also observed, but DOPA and norepinephrine levels were unaffected. Similar depletion of dopamine was observed after the land snail,Achatina fulica, was injected with haloperidol on each of 4 consecutive days.4.The depletion of dopamine as revealed with glyoxylate-induced fluorescence inLymnaea appears to be restricted to a subpopulation of catecholaminergic neurons which are immuno-negative for tyrosine hydroxylase, the synthetic enzyme responsible for the conversion of tyrosine to DOPA.

The activity of isolated neurons and the modulatory state of an isolated nervous system represent a recent behavioural state

ABSTRACT Behavioural/motivational state is known to influence nearly all aspects of physiology and behaviour. The cellular basis of behavioural state control is only partially understood. Our investigation, performed on the pond snail Lymnaea stagnalis whose nervous system is useful for work on completely isolated neurons, provided several results related to this problem. First, we demonstrated that the behavioural state can produce long-term changes in individual neurons that persist even after neuron isolation from the nervous system. Specifically, we found that pedal serotonergic neurons that control locomotion show higher activity and lower membrane potential after being isolated from the nervous systems of hungry animals. Second, we showed that the modulatory state (the chemical neuroactive microenvironment of the central ganglia) changes in accordance with the nutritional state of an animal and produces predicted changes in single isolated locomotor neurons. Third, we report that observed hunger-induced effects can be explained by the increased synthesis of serotonin in pedal serotonergic neurons, which has an impact on the electrical activity of isolated serotonergic neurons and the intensity of extrasynaptic serotonin release from the pedal ganglia. Summary: In the pond snail Lymnaea stagnalis, the behavioural state can produce long-term changes in individual neurons that persist even after neuron isolation from the nervous system as well as alterations in the neurochemical microenvironment of the central ganglia.

Serotonin depletion after prolonged chlorpromazine treatment in a simpler model system

1. Prolonged exposure of the pond snail Lymnaea stagnalis to micromolar concentrations of chlorpromazine (CPZ) results in marked changes in the serotonin (5-HT) content of the central nervous system. 2. High-performance liquid chromatography with electrochemical detection indicates that levels of 5-HT, but not those of dihydroxyphenyl-alanine (DOPA), dopamine or norepinephrine, were significantly decreased (e.g., to less than 40% of normal after 30 days of exposure to 1 microM CPZ in the bathing water). 3. Glyoxylate-induced fluorescence was depressed to undetectable levels in central, serotonergic neurons. 4. Performance of 5-HT-dependent motor behaviors was impaired. 5. The present results, in accord with earlier studies on the effects of chronic exposure to haloperidol, suggest that previously overlooked mechanisms of monoamine downregulation may contribute to long-term effects of antipsychotic drugs.

The activity of isolated snail neurons controlling locomotion is affected by glucose

The involvement of serotonin in mediating hunger-related changes in behavioral state has been described in many invertebrates. However, the mechanisms by which hunger signals to serotonergic cells remain unknown. We tested the hypothesis that serotonergic neurons can directly sense the concentration of glucose, a metabolic indicator of nutritional state. In the snail Lymnaea stagnalis, we demonstrate that completely isolated pedal serotonergic neurons that control locomotion changed their biophysical characteristics in response to glucose application by lowering membrane potential and decreasing the firing rate. Additionally, the excitatory response of the isolated serotonergic neurons to the neuroactive microenvironment of the pedal ganglia was significantly lowered by glucose application. Because hunger has been reported to increase the activity of select neurons and their responses to the pedal ganglia microenvironment, these responses to glucose are in accordance with the hypothesis that direct glucose signaling is involved in the mediation of the hunger-related behavioral state.

PLEURAL-BUCCAL INTERNEURONS IN THE PTEROPOD MOLLUSC CLIONE LIMACINA

Abstract Paired, Phe-Met-Arg-Phe-NH2-ergic pleural-to-buccal projecting neurons of the pleural ganglia were suggested to be responsible for feeding arrest associated with defensive withdrawal in freshwater and terrestrial pulmonate molluscs. In the present study, the pleural-to-buccal projecting cells were, for the first time, identified in a representative opisthobranch, the carnivorous marine pteropod Clione limacina. Two symmetric neurons of its pleural ganglia were found to be similar to the pulmonate pleural-to-buccal projecting neurons in the number of neurons, positions of their cell bodies in the central nervous system, a unique, indirect route of their axon, electrotonic coupling of the left and right cells, and expression of Phe-Met-Arg-Phe-NH2-like immunoreactivity and inhibitory action on neurons participating in the motor program for feeding. In their turn, pleural-to-buccal projecting neurons receive excitatory inputs from the protractor interneurons involved in the feeding rhythm generation. Also, it was demonstrated that the pleural-to-buccal projecting cells activity positively correlates with spontaneous and induced acceleration of the locomotor rhythm. Accordingly, stimulation of the cerebral command neuron for locomotion, cell CPA1, excited pleural-to-buccal projecting neurons. We conclude that the neuronal network underlying feeding behavior in both pulmonate and opisthobranch molluscs is similarly linked to defensive behavior by pleural Phe-Met-Arg-Phe-NH2-ergic neurons, thus indicating evolutionary conservation of these pleural-buccal projections.

Chronic haloperidol : neural correlates of motor disorders in an invertebrate model

To analyse the mechanisms underlying chronic effects of antipsychotic drugs, a simpler invertebrate preparation is proposed. Bathing of leeches for three days in a 1.0 microM aqueous haloperidol (HAL) solution resulted in (i) discoordination of locomotion, including the inability of the posterior sucker to successfully attach to the substratum, and (ii) transmitter depletion in all putative dopamine (DA) neurones of the sucker and in some, but not all, DA neurones of middle body segments. Both behavioural restoration and cellular DA recovery were observed in disabled animals after bathing in 1.25 mM L-DOPA solution. We conclude that chronic HAL-produced motor disorders are causally related to DA deficiency.