[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.

[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.

[NMDA-Receptors in the central nervous system of the honey bee with kynurenine deficiency]. / NMDA-Retseptory tsentral'noi nervnoi sistemy medonosnoi pchely v usloviiakh defitsita kinureninov.

Effects of some drugs on the short-term memory were tested. In kynurenine deficit, mutant snow laranja manifested the same pharmacological profile and 10 to 100-fold enhancement of sensitivity of the NMDA receptors' different sites. The data obtained suggest that the gene controlling the key enzyme activity of the kynurenine pathway of tryptophan metabolism is involved in regulation of the CNS NMDA receptors' functional condition. The kynurenines may be classified as endogenous modulators of the NMDA receptor sensitivity.

Genetic approaches to the study of memory in insects.

This article provides a short summary of studies carried out on mutant Drosophila with defects in learning ability, including our own experimental data on the role of the tryptophan oxygenase gene (this is a key enzyme, and is the first enzyme in the tryptophan-ommochrome metabolic pathway) in the inherited determination of learning ability and memory in the honey bee. A set of allelic mutations was used which inhibit the activity of this enzyme to different extents, resulting in the complete lack of kynurenines or particular levels of kynurenine deficiency in the mutant organisms. The effects of mutations at the snow locus (snow, s, snowlaranja, sla) on the dynamics of memory trace formation after single training sessions were studied in the honey bee and were related to the activity of the enzyme responsible for hydrolysis of cyclic nucleotides (phosphodiesterase). Relationships were found between the level of disruption in the dynamics of memory trace formation and changes in kynurenine content and phosphodiesterase activity.

[Genetic approaches to the study of memory in insects]. / Geneticheskie podkhody k izucheniiu pamiati u nasekomykh.

The paper presents some experimental data on the structural gene of tryptophanoxygenase and its role in hereditary determination of learning ability and memory in honey bees. Changes in the memory trace formation were shown to depend on the degree of breaks in the kynurenine and phosphodiesterase activity.