[THE REGULATING EFFECT OF DIPEPTIDES ON CELL PROLIFERATION IN NERVE TISSUE CULTURE IN MAMMALS AND ON ASSOCIATIVE LEARNING IN INSECTS].

The effect of dipeptides AspPro and AspSer and of their composing amino acids (asparagine acid--Asp, proline--Pro, serin--Ser) on the proliferative activity in the explants of cortex and subcortical structures of the rat brain and on the functional activity of CNS of the honeybee was studied. The square index defined as a proportion of the whole explant square to the square of its central zone was determined. The number of bees responded with the conditional reaction (proboscis extension in the direction to aromatized solution) after 1 min (short-term memory) and 180 min (long-term memory) was detected after single learning procedure. Both dipeptides, as well as the asparagine acid, stimulated an increase of the growth zone of the subcortical structure explants in rats and of the number of honeybees with retention of conditional reaction in the short-term/long-term memory independently of the effect of the second member of the dipeptide. The unidirectionality of the effect suggests the existence of common mechanisms of reception and signal transduction established during evolution that require the further study.

[Metabotropic receptor of the group I of the 5th subtype (ImGLuR5) in honeybee associative olfactory learning].

The work deals with study of character of localization in the honeybee head ganglion of metabotropic receptor ImGluR5 and its role in memory formation. With aid of pharmacological method (injections of antisense oligonucleotide and of selective receptor agonist and antagonist) and of behavioral criterion (formation and testing of preservation in memory of conditioned alimentary reflex for olfactory stimulus), there is first shown participation of the studied receptor in formation of the honeybee long-term memory. By using the immunohistochemical method, there is first revealed the predominant expression of the ImGluR5 receptor in the mushroom body Canyon cells responsible for the insect integrative activity. The present study, together with the previous ones, allows concluding about the presence in the honeybee head ganglion of the group I of metabotropic glutamate receptors with two subtypes 1 and 5 (ImGluR1,5) that have similar with mammalian pharmacological properties favoring preservation of the individually acquired experience in the long-term memory.

[On epigenetic regulation of process of formation of long-term memory].

The review summarizes current concepts on role of several covalent posttranslation chromatin modifications in the process of memory formation in vertebrate and invertebrate animals. There is described a chain of intracellular events from activation of receptors and signal pathways to change of the functional state of genome.

Effect of tripeptide Lys-Glu-Asp on physiological activity of neuroimmunoendocrine system cells.

Tripeptide Lys-Glu-Asp stimulates proliferation and inhibits apoptosis in organotypic cultures of neuroimmunoendocrine system cells. Lys-Glu-Asp accelerates cell renewal processes (decrease of apoptosis marker p53 and increase of proliferation marker Ki-67) in the pineal gland; this effect is more pronounced in cultures derived from old animals than in young cultures. The tripeptide induces the expression of low-differentiated lymphocyte marker CD5 and macrophage marker CD68, but in "old" cultures this effect is less pronounced than in "young" ones. Thus, in tissue culture Lys-Glu-Asp primarily affects the nervous and endocrine tissues during aging and produces a less pronounced effect on the nervous tissue. Physiological activity of the tripeptide consists in modulation of associative learning of honey bee in the model of short-term and the long-term memory.

[Metabotropic glutamate receptors in the mechanisms of plasticity of central nervous system of the honeybee Apis mellifera].

Localization of metabotropic glutamate receptors (MGR) in head ganglion of honeybee Apis mellifera, and mechanisms of participation of activated MGR in CNS plasticity are investigated by means of complex approach using immunochemical, electrophysiological and behavioral methods. Influense of MGR activation on GABAergic system and ionotropic glutamate receptors (IGR) of AMPA- and NMDA-subtypes in studied. MGRa are revealed in lateral and medial calices of mushroom bodies. The inhibiting influence of MGR on AMPA- and NMDA receptors is shown using method of conditioned reflex. Previous activation of MGR neutralizes the inhibiting effect of GABA. Modulating role of heterogeneous MGR population in mechanisms of CNS plasticity on the level of glutamate-ergic synapse, and at interaction with GABAergic system is discussed.

[Behavioral and molecular effects of endogenic kynurenines deficit in the honeybee (Apis mellifera L.)].

The importance of tryptophan endogenous metabolites kynurenines in the long-term memory and functioning of the signal cascade GluR - LIMK1 - F-actin, mediating the long-term memory trace storage, was demonstrated. The deficit of kynurenines induced by allopurinol (tryptophanoxygenase inhibitor) suppressed the long-term memory, decreased the LIMK1 expression, and paradoxically increased the F-actin content in the honeybee brain. These data agree with the earlier findings in drosophila mutant vermilion (the mutation of tryptophanoxygenase gene).

Comparative analysis of the locations of the NR1 and NR2 NMDA receptor subunits in honeybee (Apis mellifera) and fruit fly (Drosophila melanogaster, Canton-S wild-type) cerebral ganglia.

The locations of the NR1 and NR2 subunits of the GABA receptor were studied in brain structures in insects--honeybees and fruit flies--using an immunohistochemical method. The specificities of the antibodies to the NR1 and NR2 subunits were confirmed by the antisense knockdown method for the NR1 subunit and western blotting. The data obtained here lead to the conclusion that the distributions of the NR1 and NR2 subunits of the NMDA receptor complex in the cerebral ganglia of the honeybee and fruit fly are similar; areas with the highest concentrations of NR1 and NR2 subunits were identified, and these were found to be different in the different insects. This is associated with the behavioral characteristics of these two insect species.

[Mutations in structural genes of tryptophan metabolic enzymes of the kynurenine pathway modulate some units of the L-glutamate receptor--actin cytoskeleton signaling cascade].

Methods of immunohistochemistry and fluorescent staining was used to study the localization and amounts of protein components of the signal cascade connecting the receptor link (NMDA-subtype glutamate receptor) with actin of the cytoskeleton in the head ganglia of Drosophila strain Canton-S (wild type, control) and strains carrying mutations vermilion, cinnabar, and cardinal, which sequentially inactivate tryptophan-hydrolyzing enzymes during its metabolism into ommochrome. The obtained data are evidence for modulatory effects of genes controlling the kynurenine pathway of tryptophan metabolism on the major components of the signal cascade: the initial link (NMDA receptor, postsynaptic density protein-95, a structural protein involved in receptor localization and internalization), the intermediate link (limkinase-l, the key neuronal enzyme in actin remodeling) and the final link (f-actin, the critical factor in the morphogenesis of synaptic structures and, hence, in the processes of synaptic plasticity, learning and memory). It is suggested that kynurenine acid (an endogenous nonspecific antagonist of L-glutamate receptor) and 3-hydroxykynurenine capable of inducing a nonspecific stimulating effect are biochemical intermediates of the effects of these genes.

Electrophysiological characteristics of a population of mushroom body neurons in Apis Mellifera honeybees in kynurenine deficiency.

Electrophysiological methods were used to study the neurophysiological characteristics of mushroom body neurons in snow (laranja) mutant and wild-type bees. The snow (laranja) mutation, which produces a sharp reduction in the activity of the enzyme tryptophan oxygenase, thus creating a deficiency of all kynurenines in the body, modifies the bioelectrical properties of mushroom body neurons. The parameters most dependent on the snow (laranja) mutation and, thus, the level of endogenous kynurenines, were the duration of action potential afterdepolarization recorded from neurons in the calyx and the amplitude of the postsynaptic potential of neurons in the calyx evoked by focal stimulation of the antennal lobes. There was also a tendency to an increase in the spontaneous spike frequency. These data lead to the conclusion that the effect of the mutation is recessive in nature and appears only in homozygotes (bearing two mutant alleles).

[Comparative analisys of localization NR1 and NR2 subunits of NMDA-receptor in the brain structures in honeybee (Apis mellifera L.) and Drosophila (Drosophila melanogaster, wild type canton-S)].

Localization NR1 and NR2 subunits of NMDA-receptor was studied in brain structures of the honeybee and Drosophila by immunohistochemistry. The Western-blotting and NR1 subunit antisense-knockdown confirmed specificity of antibodies to NR1 and NR2 subunits. The data obtained demonstrated similar distribution of NR1 and NR2 subunits of NMDA-receptor in the insect brain (cranial ganglion). The brain regions with the highest expression NR1 and NR2 were different in the honeybee and Drosophila. This can be associated with behavioral repertoire peculiarities in these insects.

Characteristics of neuron activity in the honey bee (Apis mellifera L.) in conditions of kynurenine deficiency.

Neuron activity was studied electrophysiologically in the antennal lobes, mushroom bodies, and cervical connective during ontogenesis (days 1, 3, 7, and 25) in snow(laranija) mutant bees, which suffer kynurenine deficiency, and in wild-type bees. Neuron activity recorded from the cervical connective was found to show the greatest dependence on kynurenine content. Mutation in the structural gene for tryptophan oxygenase, the first and key enzyme in the kynurenine pathway for tryptophan metabolism (KPTM) and which leads to kynurenine deficiency, decreased the frequency of neuron spike activity recorded from the cervical connective. This would appear to be associated with a possible deficiency of glutamic acid; it was independent of the deficiency of kynurenic acid and kynurenine, a point subject to further studies. Clear manifestation of the effect of the mutation requires the presence of two mutant alleles in the bee genome (homozygosity). The effect of the mutation, to inhibit neuron activity in the cerebral ganglion, corresponds to the effect seen previously at the behavioral level. Less clear effects were obtained from recordings of neuron activity from the antennal lobes and mushroom bodies. This may be associated with the complexity of the spectrum of biochemical lesions in different parts of the brain resulting from the mutation.

[Electrophysiological characteristics of a population of mushroom body neurons in Apis mellifera L. honey bee under conditions of kynurenines deficit].

Neurophysiological characteristics of mushroom body neurons were examined by electrophysiological methods in mutants snow(laranja) and wild-type honey bees. Mutation snow(laranja) causes a drastic decrease in the activity of enzyme tryptophane oxygenase that results in deficiency of all kynurenines. It also modifies bioelectrical properties of neurons in the mushroom bodies. The duration of afterdepolarization in spikes recorded from calyx neurons and the amplitude of postsynaptic potentials in these neurons evoked by focal stimulation of antennal lobes were shown to be most dependent on the mutation and, consequently, on the content of endogenous kynurenines. A tendency to an increase in the frequency of spontaneous spikes was also observed. The effect of the mutation on neurophysiological characteristics under study was recessive, i.e. it was observed only in homozygous individuals.

[Neuronal activity in a honey bee (Apis mellifera L.) with kynurenine deficiency]. / Osobennosti neironnoi aktivnosti u medonosnoi pchely (Apis mellifera L.) v usloviiakh defitsita kinureninov.

Neuronal activity of the antennal lobes, mushroom bodies, and cervical connective in wild-type honey bees and snowlaranija mutants was recorded at different stages of the ontogeny (on the 1st, 3rd, 7th, and 25th days). The mutation snowlaranija affects the structural gene of tryptophane oxygenase, the first key exzyme in the kynurenine pathway of tryptophane metabolism, and leads to a deficit of kynurenines. Changes in neuronal activity in nutant bees were most pronounced in the cervical connective. A significant decrease in the pulse rate was revealed only in homozygous but not in heterozygous individuals. This finding is in accordance with previously reported inhibitory effect of the mutation at the behavioral level. Less pronounced effects were obtained when the neuronal activity was recorded in the antennal lobes or mushroom bodies. This may be related to a complex character of biochemical changes in different parts of mutants brain.

Central non-NMDA receptors of a honey bee with hereditary kynurenine deficiency.

Pharmacological characteristics of non-NMDA receptors involved in associative learning were studied in wild honey bees (normal) and carriers of snow laranja mutation (kynurenine deficiency) by pharmacological analysis and behavioral criteria. The effects of systemic injections of non-NMDA receptor agonists (AMPA, kainic, quisqualic, and domoic acids), AMPA receptor antagonist (NS257-HCl), and AMPA receptor modulator (cyclothiaside) on retention of conditioned reflexes in short-term memory (1 min after the end of learning) were studied. The pharmacological characteristics of non-NMDA receptors were changed in snow laranja mutants with kynurenine deficiency.