Selective specificity of calcium-binding proteins calbindin and calretinin expression in the magnocellular neurosecretory hypothalamic nuclei of tortoises and turtles.

We have studied the distribution of calcium-binding proteins in the magnocellular neurosecretory nuclei of nonapeptidergic neurosecretory nuclei of the preoptic-hypothalamic complex in a tortoise (Testudo horsfieldi) and a pond turtle (Emys orbicularis) using immunohistochemistry. We have found that different types of cells in the paraventricular and supraoptic nuclei predominantly express calbindin and, to a lesser extent, calretinin, but not parvalbumin. The selective calbindin/calretinin control of the neurohormone secretion in these hypothalamic nuclei is an evolutionary conservative feature typical of reptiles and mammals.

[Effect of GABA and glycine neuromediator interaction in the central nervous system].

Today it is well accepted that GABA can be co-localized and co-released with glycine in the same synapse. This article provides an overview of GABA and glycine co-localization and the effects of simultaneous activation of GABAA and glycine receptors. The review deals with mechanisms of direct and indirect receptor interaction, as well as with the effect of non-selective activation of glycine receptors by GABA.

[Effect of antagonists of 5-HT receptors on modulation with serotonin of synaptical activity of projectional neurons of dorsolateral nucleus of rat amygdala].

The work deals with study of role of different serotonin (5-HT) receptor types in the process of synaptic activity modulation of rat dorsolateral amygdale projection neurons with serotonin. The selective antagonist of 5-HT1,2 receptors methylsergide maleate was shown to suppress the 5-HT inhibitory action on amplitude of the postsynaptic currents evoked by glutamate and GABA, whereas the antagonist of 5-HT3,4 receptors SDZ202-557 produced no effect on the above-mentioned 5-HT action. The obtained action indicates that the 5-HT modulatory effect on the projectional neuron synaptic inputs is mediated by 5-HT receptors of the 1 and 2 types.

[Role of long-term potentiation in mechanism of the conditioned learning].

The review analyzes the fundamental problem of study of the neuronal mechanisms underlying processes of learning and memory. As a neuronal models of these phenomena there was considered one of the cellular phenomena that has characteristics similar with those in the process of "remembering"--such as the long-term potentiation (LTP). LTP is easily reproduced in certain synapses of the central nervous system, specifically in synapses of hippocampus and amygdala. As to the behavioral model of learning, there was used the conditioned learning, in frames of which production of the context-dependent/independent conditioned reaction was considered. Analysis of literature data has allowed showing that various stages of LTP produced on synapses of hippocampus or amygdala can be comparable with certain phase of the process of learning. Based on the exposed material the authors conclude that plastic changes of synapses of hippocampus and amygdala can represent the morphological substrate of some kinds of learning and memory.

[Gaba- and glycine-immunoreactive synapses in the spinal cord of the frog Rana temporaria].

Postembedding immunogold method was used to examine the distribution of gamma-aminobutyric acid- and glycine-immunoreactives synapses on the motoneurons and primary afferent axons in frog spinal cord. Analysis of all labeled boutons on dendrites and somata of motoneurons showed that 7% were labeled for GABA, 23% only for glycine and approximately 70% were immunoreactive for both GABA and glycine. These results confirm the predominant role of glycine in postsynaptic inhibition of motoneuronal activity. Three populations of synaptic boutons were found on primary afferent axons: GABA-immunoreactive (25%), glycine-immunoreactive (5%) and the majority of the immunoreactive synapses exhibited colocalization of two inhibitory transmitters. Greater proportion of axo-axonal synases was organized in synaptic triads. The possible roles of glycine in the axo-axonal synapses on the primary afferent fibers are discussed.

[Principles of organization and evolution of systems of regulation of functions].

Evolution of living organisms is closely connected with evolution of structure of the system of regulations and its mechanisms. The functional ground of regulations is chemical signalization. As early as in unicellular organisms there is a set of signal mechanisms providing their life activity and orientation in space and time. Subsequent evolution of ways of chemical signalization followed the way of development of ways of delivery of chemical signal and development of mechanisms of its regulation. The mechanisms of chemical regulation of the signal interaction is discussed by the example of the specialized system of transduction of signal from neuron to neuron, of effect of hormone on the epithelial cell and modulation of this effect. These mechanisms are considered as the most important ways of the fine and precise adaptation of chemical signalization underlying functioning of physiological systems and organs of the living organism.

[Th role of inhibitory interneurons in mechanisms of regulation of sensory synapses formed by thalamic and cortical inputs in pyramidal cells of the dorsolateral amygdala nucleus].

The work deals with study of role of inhibitory interneurons in the process of regulation of sensory currents converging on soma of pyramidal cells of the dorsolateral amygdala nucleus as well as of role of these interneurons in mechanism of regulation of plasticity of amygdalar synapses. It has been shown that the part of the spontaneous inhibitory postsynaptic currents recorded on the dorsolateral amygdale pyramidal cells is relatively high and amounts to about a half of the total amount of the recorded events. Analysis of the evoked postsynaptic responses has shown the interneurons to regulate activity and duration of these responses due to the postsynaptic membrane hyperpolarization as a result of activation of GABA(A)-receptors. Also studied was role of interneurons in providing mechanisms of the long-term potentiation of the synaptic responses evoked by stimulation of cortical and thalamic inputs. Block of effect of interneurons with help of picrotoxin has been shown to lead to an increase of evoked potentiation of synaptic responses.

Interaction of the postsynaptic effects of glycine and GABA on spinal cord neurons in the frog Rana temporaria.

Studies were performed on mechanically isolated spinal cord multipolar cells (presumptive motoneurons) from the frog Rana temporaria using patch-clamp methods in the whole-cell configuration. These experiments showed that the amplitudes of transmembrane currents arising in response to simultaneous application of GABA and glycine were smaller than the sums of the amplitudes of the responses of the same neurons to GABA and glycine applied individually. Investigation of the mechanisms of this occlusion showed that superfusion of neurons with glycine solution (0.2 mM) resulted in complete blockade of responses to application of GABA (5 mM) and vice versa. This phenomenon may have resulted from cross-blockade associated with the existence of a single receptor complex sensitive to both GABA and glycine and from the interaction of GABA and glycine receptors.

[The role of group II and III metabotropic glutamate receptors in modulation of miniature synaptic activity in frog spinal cord motoneurons].

The results of present work demonstrate significant modulating effects mediated by group II and III mGluRs on miniature postsynaptic potentials (mPSP) of the frog spinal motoneurons. The mode of group II and III mGluRs ligands influences, i. e. the changes in the mPSPs average frequency without significant changes in their average amplitude, suggests the presynaptic mechanism of modulation by the change in transmitter release. Selective antagonists of group II and III mGluRs (EGLU and MAP4) increased the average frequency of mPSPs by 52.8 +/- 30.2% (in 4 of 6 motoneurons) and by 54.7 +/- 23.7% (in all 7 motoneurons), respectively. Application of the group III mGluRs agonist LAP4 decreased the mPSPs frequency by 21.8 +/- 5.2% in 3 of 5 motoneurons. The efficiency of the antagonist usage and comparative low efficiency of the agonist suggest that presynaptic mGluRs at motoneuronal synapses under normal condition possess some level of tonic activity. The lack of group II mGluR antagonist effect on some motoneurons appears to be explained by specific localization of the group II mGluRs in preterminal area which is distant from the transmitter release site. The hetero-receptor modulation of pharmacologically isolated inhibitory miniature activity and its glycine- and GABAergic fractions by group III mGluRs was investigated. MAP4 application has been shown to increase the glycine-mediated mlPSPs frequency more than GABA-mediated mlPSPs frequency: in average by 97.6 +/- 20.7% (n = 7) and 54.6 +/- 20.8% (n = 5), respectively. This difference may be due to the segregation of the postsynaptic glycine- and GABA-receptors. The preliminary examination of the convergence of the presynaptic mGluRs and metabotropic GABA(B) receptors influences on GABA-mediated IPSPs was undertaken. It has been shown that presynaptic GABA(B) receptors are tonically active under normal condition. Under condition of GABA(B) receptor blockage by phaclofen, the application of group III mGluR agonist L-AP4 elicited typical effect which was completely taken off by subsequent application of the group III mGluRs antagonist MAP4. This result is in accordance with the assumption that the effects mediated by GABA(B) receptors and mGluRs are independent.

[Dorsal root afferent fiber termination in the spinal cord of the turtle Testudo horsfieldi and 3-dimensional reconstruction of the sensory-motoneuron connection].

HRP tracing methods and computer reconstruction were used to study the structural organization of sensory-motoneuron connections in the turtle. HRP was applied through suction electrodes to thin dorsal and ventral root filaments of superfused isolated lumbar spinal cord of the turtle Testudo horsefieldi. Single motoneurons were labeled iontophoretically with the use of intracellular glass microelectrodes. Labeled elements were examined by light microscopy. The Eutectic Neuron Tracing System and its associated program were used for 3-D reconstructions and morphometry. The distribution of dorsal root afferent fibers and their terminations were presented in a new scheme in which, beside the well known zones, new ones were shown in the Lissauer zone, motor nuclei, ventrolateral funiculus and in the contralateral medial gray matter (IV--V laminae). Unlike in the frog, the motoneuron dendritic field in the turtle was restricted to an ellipsoid space having a short axis in the rostro-caudal direction (300-500 microm). The dorsal root afferent fibers connected to motoneurons produced very short branches (50-70 microm) in a restricted rostro-caudal direction. One dorsal root fiber collateral had about 80 synapselike enlargements (approximately 10-fold fewer than in the frog). The putative sensory-motoneuron contacts were found on the I--VII order dendritic segments of the dorsal and ventromedial dendritic trees. It was shown that in the turtle only one first order collateral of the dorsal root fiber participated in the sensory-motoneuron connection with a small number (about 4) of putative contacts, which was also less than in the frog by a factor of 10. The simplicity of the synapse structure in the turtle is likely to be compensated through the higher efficiency of the signal transmission which is comparable to that in mammals.

[Immunoreactivity of the synapses on the primary afferent axons and sensory neurons of the spinal cord Lampetra fluviatilis].

The existence of GABA-like immunoreactivity in the synapses on the primary afferent axons and GABA- and glutamate immunoreactive synapses on the dorsal cell somatic membrane was shown using double postembedding immunogold cytochemistry. These morphological findings suggest that control of the sensory information in the lamprey spinal cord is realized by means of presynaptic inhibition through the synapses on the primary afferent axons as well as directly through the synapses on the somata of the sensory neurons.

[Transmembranous currents of isolated spinal cord neurons of ammocete--larva of the lamprey Lampetra fluviatilis].

The work is carried out with aid of the patch-clamp method in the "whole cell" modification. There were studied potential-activated and chemosensitive currents of isolated spinal neurons of the lamprey larva--ammocete. The described properties of the currents indicate their similarities with those in adult lampreys.

Three types of inhibitory miniature potentials in frog spinal cord motoneurons: possible GABA and glycine cotransmission.

Miniature inhibitory postsynaptic potentials (mIPSP) of motoneurons in isolated frog spinal cord were recorded in conditions of blockade of the conduction of nerve spikes and ionotropic glutamate receptors (TTX, 1 microM, CNQX, 25 microM, D-AP5, 50 microM). Three types of mIPSP were identified: those with fast and slow time characteristics and mIPSP with two-component decays. Two-component mIPSP accounted for 8.7% of all selected responses, fast mIPSP for 64.5%, and slow mIPSP for 26.8%. Blockade of GABA(A) receptors with bicuculline (20 microM) led to decreases in the numbers of slow and two-component mIPSP and an increase in the number of mIPSP with fast kinetics. Strychnine (1 microM), a blocker of glycine receptors, led to a reduction in the number of fast receptors and an increase in the number of slow potentials. These data suggest that frog spinal cord motoneurons have three types of inhibitory mIPSP, mediated by GABA, glycine, and simultaneous release of these two transmitters from the same presynaptic terminals.

[Peculiarities of dopamine receptors on the membrane of spinal cord multipolar neurons of the brook lamprey Lampetra planeri].

On isolated multiporal neurons of spinal cord of amniocoete larva of the brook lamprey Lampetra planeri, by the patch-clamp method in configuration "the whole cell", a modulating effect of dopamine on potential-activated Na+ currents was studied. Application of dopamine (10 microM) was shown to produce a complex action on the sodium current amplitude. In some cases a decrease of the amplitude, on average, by 13.5 +/- 2.2% was found, while in others--an increase, on average, by 8.6 +/- 6.1%. The modulation dopamine effect was not accompanied by any changes either of the threshold of the current appearance or of resistance of neuronal cell membranes. Pharmacological analysis with use of dopamine agonist has shown that the agonist of D1-receptors (-)-SKF-38393 (10 microM) decreases the Na+ current amplitude, whereas the agonist of D2-receptors (-)-quinpirole (10 microM) can produce in different cells both an increase, by 30.7 +/- 17.0 %, and a decrease, by 13.2 +/- 3.1%, of the Na+ current amplitude. The obtained data indicate the existence of D1- and D2-receptors on the membrane of multipolar spinal neurons of the amniocoete larva of the brook lamprey. Study of action of antagonists has shown that the antagonist of D1-receptors (+)-SCH-23390 (10 microM) does not affect action of the agonist of D1-receptors (-)-SKF-38393 (10 microM); the antagonist of D2-receptors (-)-sulpiride (10 microM) blocks completely effects both of the agonist of D1-receptors (-)-SKF-38393 (10 microM) and of the agonist of D2-receptors (-)-quinpirole (10 microM). The antagonist of D1-receptors (+)-SCH-23390 (10 microM) produced no effect on action of the agonist of D1-receptors (-)-SKF-38393 (10 microM). The obtained data indicate peculiarities of dopamine receptors of Cyclostomata as compared with those in mammals.

[SECOND MESSENGERS IN PRESYNAPTIC REGULATION OF GLYCINERGIC SYNAPSE ON THE FROG MOTONEURON].

The possible pathways of inhibitory influences mediated by two kinds of metabotropic receptors, group III metabotropic glutamate receptors (mGluRs III) and GABAB receptors (GABABRs) to the miniature glycinergic events were investigated in the isolated spinal cord of the frog Rana ridibunda. The glycinergic events prevailed within the miniature inhibitory activity of motoneurons [3]. Selective agonists of GABABRs (baclofen) and group III mGluR (LAP4) reduce the frequency of miniature events to 48.0 ± 5.56% (n = 8) and 48.5 ± 8.6% (n = 5) respectively. The mean amplitude of miniature potentials was not modified significantly which indicates the presynaptic mode of their action on the synaptic transmission. To reveal the stages of metabotropic receptors regulatory effects on the glycine exocitosis the application of their selective agonists was performed after selective blockade of putative links of glycinergic transmission modulation. It was shown that the mGluRsIII influences are due to the negative feedback with adenylyl cyclase (AC) whereas the following step is formed by protein kinase A (PKA), because their selective blockade eliminate the effect of group III mGluRs activation. The AC is not involved in transdusing the GABABRs signal to the glycine miniature activity because SQ22536, an AC-inhibitor does not eliminate the inhibitory action of baclofen. The action of GABABRs depends on the activity of the phospholipase C (PLC) because its inhibition with U73122 prevents the effect of baclofen. The next possible link of this pathway could be the protein kinase C (PKC); but it is not involved in the realization of GABABRs influences because the PKC blocker (GF 109302X) does not modify the baclofen effect. The common link of group III mGluRs and GABABRs influences on the glycinergic miniature activity realization can be the inositol trisphosphate receptors (IP3Rs) that regulate the Ca2+ outflow from the nerve terminal depots and are connected (according to the literature data) with PKA, cAMP and PLC. In our experiments 2-APB, an IP3Rs antagonist, reduced to 26 ± 8% (n = 7) the frequency of glycinergic miniature activity and prevented the inhibitory effects of group III mGluRs and GABABRs. Our data suggest that collision and cross-talk of these Glu- and GABA-metabotropic pathways which was described earlier [2] may take place at this level.

[INTERACTION OF GLUTAMATE AND GABA RECEPTORS IN THE CENTRAL NERVOUS SYSTEM].

The article provides an overview of postsynaptic interaction of GABA and glutamate receptors in central neurons. Co-localization of GABA and glutamate in synapses, structure and function of their receptors and interaction of both ionotropic and metabotropic glutamate and GABA receptors are being discussed.