Inhibition of high-voltage-activated calcium currents by acute hypoxia in cultured retinal ganglion cells.

Hypoxia is a common factor of numerous ocular diseases that lead to dysfunctions and loss of retinal ganglion cells (RGCs) with subsequent vision loss. High-voltage-activated calcium channels are the main source of calcium entry into neurons. Their activity plays a central role in different signaling processes in health and diseases, such as enzyme activation, gene transcription, synaptic transmission, or the onset of cell death.

Insulin modulates the paired-pulse plasticity at glutamatergic synapses of hippocampal neurons under hypoinsulinemia.

Hypoinsulinemia is a pathological consequence of diabetes mellitus that can cause a number of complications of the central and peripheral nervous system. Dysfunction of signaling cascades of insulin receptors under insulin deficiency can contribute to the development of cognitive disorders associated with impaired synaptic plasticity properties. Earlier we have shown that hypoinsulinemia causes a shift of short-term plasticity in glutamatergic hippocampal synapses from facilitation to depression and apparently involves mechanisms of glutamate release probability reduction.

Protein kinase C mediates hypoxia-induced long-term potentiation of NMDA neurotransmission in the visual retinocollicular pathway.

The identification of processes and mechanisms underlying the early stage of hypoxic injury of the retinocollicular pathway may be beneficial for the future prevention and treatment of navigation, orientation, and visual attention impairments. Previously, we have demonstrated that short-term hypoxia led to long-term potentiation (LTP) of NMDA neurotransmission in the background of long-term depression of GABA retinocollicular transmission. Here, we sought to obtain insight into the mechanisms of hypoxia-induced LTP of NMDA retinocollicular neurotransmission and the role of the protein kinase C (PKC) signaling pathway in it.

Short-term hypoxia induces bidirectional pathological long-term plasticity of neurotransmission in visual retinocollicular pathway.

Using the paired patch-clamp technique, we studied the effects of short-term hypoxia on retinocollicular synaptic transmission in an originally-developed coculture of dissociated retinal cells and superficial superior colliculus (SSC) neurons. Pharmacologically isolated N-methyl-D-aspartate receptor (NMDA)-, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)- and gamma-aminobutyric acid receptor (GABA)-mediated postsynaptic currents (PSCs) were evoked in SSC neurons by generation action potentials in presynaptic retinal ganglion cells. Spontaneous and miniature PSCs were recorded in SSC neurons in the absence of presynaptic stimulation.

ANALYSIS OF QUANTAL PARAMETERS OF GABA RELEASE DURING SHORT-TERM DEPRESSION AND FACILITATION OF SYNAPTIC TRANSMISSION.

Changes in amplitudes of evoked inhibitory postsynaptic currents (eIPSCs) from rat cultured hippocampal neurons were studied using whole-cell patch-clamp technique in postsynaptic neuron and local extracellular electrical paired pulse stimulation of single presynaptic axon. Paired pulse depression (PPD) and paired pulse facilitation (PPF) were observed in studied 43 neurons. According to coefficient of variance (CV) analysis was found that CV of second respond was significantly larger than CV first by 57 % (n 26) during depression and significanty smaller by 27 % (n = 17) during facilitation.

EXCITABILITY PROPERTIES OF TRIGEMINAL GANGLION NEURONS.

The firing properties of small neurons (with diameters of soma less than 25 µm) were investigated using patch-clamp technique in whole-cell configuration in primary culture of trigeminal ganglia (TG) of postnatal rats. TG neurons were divided into three groups according to their firing responses to long-lasting depolarizing pulses: adaptive neurons (AN) characterized by a strongly adaptive responses; tonic neurons (TN) characterized by a multiple tonic firing; neurons with a delay before initiation of AP generation, namely, NDG. AN, TN and NDG also differed in AP electrophysiological and pharmacological characteristics.

[EFFECT OF PEPTIDE SEMAX ON SYNAPTIC ACTIVITY AND SHORT-TERM PLASTICITY OF GLUTAMATERGIC SYNAPSES OF CO-CULTURED DORSAL ROOT GANGLION AND DORSAL HORN NEURONS].

The influence of long-term culturing (12 days in vitro) of dorsal root ganglion (DRG) and dorsal horn (DH) neurons with peptide Semax on the level of synaptic activity at co-cultures, as well as short-term plasticity in sensory synapses were studied. It has been shown that neuronal culturing with peptide at concentrations of 10 and 100 µM led to increasing the frequency of spontaneous glutamatergic postsynaptic currents in DH neurons to 71.7 ± 1.

[EFFECT OF HYPOXIA ON SYNAPTIC TRANSMISSION BETWEEN RETINAL GANGLION CELLS AND SUPERIOR COLLICULUS NEURONS IN COCULTURE].

In this study we conducted a series of experiments to characterize the effect and define the mechanisms of hypoxia on synaptic transmission between retinal ganglion cells and superior colliculus (SC) neurons. Application of hypoxic solution leads to a long lasting potentiation (LTP) NMDA-mediated synaptic transmission. Analysis of the oxygen deficiency effect on the spontaneous and miniature postsynaptic currents (sPSC and mPSC respectively) revealed an increase in the frequency of their occurrence and the appearance of the second peak in the mPSC histogram distribution.

Presynaptic Ca²⁺-permeable AMPA-receptors modulate paired-pulse depression in nociceptive sensory synapses.

The role of Ca(2+)-permeable AMPA-receptors (CP-AMPARs) in the induction of paired-pulse depression (PPD) at glutamatergic nociceptive sensory synapses was examined in co-culture of rat's dorsal root ganglion and dorsal horn neurons. CP-AMPARs make a considerable contribution to excitatory postsynaptic currents recorded in DH neurons following action potential generation in nociceptive DRG neurons. Activation of CP-AMPARs during fast synaptic transmission induces strong PPD (with a 200 ms inter-pulse interval).

Kv7 potassium channel subunits and M currents in cultured hippocampal interneurons.

Potassium channels of the Kv7 family that mediate the non-inactivating M current regulate the excitability of many types of neurons in the central nervous system, including some in the hippocampus. We report here that individual interneurons from newborn rat hippocampi in long-term culture strongly express messenger RNA specific for Kv7.2 and Kv7.

Kv3 channels modulate calcium signals induced by fast firing patterns in the rat retinal ganglion cells.

Expression of non-inactivating Kv3.1/Kv3.2 potassium channels determines fast-spiking phenotype of many types of neurones including retinal ganglion cells (RGCs); furthermore Kv3 channels regulate neurotransmitter release from presynaptic terminals.

Calcium-dependent changes of paired-pulse modulation at single GABAergic synapses.

Whole-cell patch-clamp and local electrical stimulation were used for measuring of monosynaptic GABAergic currents from rat hippocampal neurons in culture. Under control conditions (normal extracellular calcium, 2 mM) paired-pulse depression with 150 ms interpulse interval was observed. The mean current amplitude for both 1st and 2nd IPSCs displayed bell-shaped dependency from the stimulus amplitude.

Activation of ryanodine receptors influences the paired-pulse depression in cultured rat hippocampal neurons.

Role of intraterminal calcium stores in modulation of short-term plasticity of evoked inhibitory postsynaptic currents (IPSCs) was studied in synaptically connected cultured hippocampal neurons using patch-clamp technique in whole-cell configuration. Currents were induced by voltage stimulation which were applied externally to presynaptic fiber. Paired stimuli resulted in paired-pulse depression (n=18) or facilitation (n=7) of the second IPSC at interpulse intervals 150 and 500 ms.

Regulation of GABA release by depolarisation-evoked Ca2+ transients at a single hippocampal terminal.

We correlated dynamic changes in free cytosolic [Ca2+] ([Ca2+]i) within single presynaptic terminals of cultured hippocampal neurones with the postsynaptic GABA-mediated currents. The local changes in [Ca2+]i and evoked inhibitory postsynaptic currents (eIPSCs) were recorded simultaneously using Fura-2 fluorescence and whole-cell patch-clamp respectively. The Ca2+ signals and eIPSCs were evoked by direct extracellular electrical stimulation of a single presynaptic terminal by short depolarising pulses.

Quantal GABA release in hippocampal synapses: role of local Ca2+ dynamics within the single terminals.

Results of recent studies dedicated to the mechanisms of neurotransmission at a single inhibitory synaptic terminal in cultured neurones support the hypothesis that multiple quanta of neurotransmitter are released during excitation of inhibitory and excitatory central synapses. This is an important consideration as previous less direct measurements have suggested that a synapse can release no more than one quantum. Neurotransmitter release during long stimuli may occur at certain times with maximal probability, keeping the mean inter-release interval constant.

Ca(2+) dynamics in the lumen of the endoplasmic reticulum in sensory neurons: direct visualization of Ca(2+)-induced Ca(2+) release triggered by physiological Ca(2+) entry.

In cultured rat dorsal root ganglia neurons, we measured membrane currents, using the patch-clamp whole-cell technique, and the concentrations of free Ca(2+) in the cytosol ([Ca(2+)](i)) and in the lumen of the endoplasmic reticulum (ER) ([Ca(2+)](L)), using high- (Fluo-3) and low- (Mag-Fura-2) affinity Ca(2+)-sensitive fluorescent probes and video imaging. Resting [Ca(2+)](L) concentration varied between 60 and 270 microM. Activation of ryanodine receptors by caffeine triggered a rapid fall in [Ca(2+)](L) levels, which amounted to only 40--50% of the resting [Ca(2+)](L) value.

Temporal regularity of neurotransmitter release at single terminal in cultured hippocampal neurons.

The whole-cell GABA-mediated inhibitory postsynaptic currents were studied using the patch-clamp technique on synaptically connected cultured hippocampal neurons. The stimulus-evoked inhibitory postsynaptic currents were recorded in the tetrodotoxin-containing solution in response to the low-amplitude long (10-20ms) extracellular depolarization of a single presynaptic terminal. During each depolarization the postsynaptic response in a form of several superimposed independent events was recorded.

Single-bouton-mediated synaptic transmission: postsynaptic conductance changes in their relationship with presynaptic calcium signals.

Most central neurons contact their dendritic targets at several sites. However, it is not known whether all synapses formed by a single parent axon make the same contribution to the postsynaptic response. In order to answer this question it is necessary to isolate the synaptic currents generated by individual axon terminals.

Possibility of multiquantal transmission at single inhibitory synapse in cultured rat hippocampal neurons.

Miniature, spontaneous and evoked inhibitory postsynaptic currents were studied using the whole-cell patch-clamp technique on synaptically connected cultured hippocampal neurons, at a holding potential of -75 mV. All experiments were done in tetrodotoxin-containing solution to exclude an action potential generation. Spontaneous miniature inhibitory postsynaptic currents were observed in Ca2+-free solution.

Relationship between presynaptic calcium transients and postsynaptic currents at single gamma-aminobutyric acid (GABA)ergic boutons.

Postsynaptic responses to stereotyped activation of single axons are known to fluctuate, but the origin of synaptic variability in the vertebrate central nervous system is still unclear. To test the hypothesis that fluctuations of inhibitory postsynaptic currents reflect variations in presynaptic Ca2+ concentration, we examined single GABAergic axodendritic contacts in low-density cultures. Collicular neurons from rat embryos were loaded with the Ca2+ indicator Oregon Green 488 BAPTA-1.

Voltage-operated potassium currents in the somatic membrane of rat dorsal root ganglion neurons: ontogenetic aspects.

Whole-cell transmembrane potassium currents were studied in somatic membrane of freshly isolated rat dorsal root ganglion neurons. We defined three types of potassium currents, which were separated on the basis of their different potential dependence of activation and sensitivity to external tetraethylammonium and 4-aminopyridine. The potential dependence of kinetic and steady-state properties of a fast inactivating potassium current, a slow inactivating potassium current and a non-inactivating delayed rectifier current were described by the Hodgkin-Huxley equations.

Fast local superfusion technique.

A system for rapid, local superfusion of cultured neurones and their neurites with various different test drugs is elucidated. An area of down to 30 micron diameter was superfused with the aid of two micropipettes, one for delivering the test solution and the other for its removal. Active removal of solution within the deadspace of the delivery pipette guarantees, on the one hand, fast and flexible pressure control and, on the other, enables the quick exchange (<1 s) of multiple solutions.

Glutamate-produced long-term potentiation by selective challenge of presynaptic neurons in rat hippocampal cultures.

Excitatory postsynaptic long-term potentiation (LTP) was observed after restricted glutamate or metabotropic agonist application to somata and proximal neurites of identified presynaptic neurons. LTP-induction in this way entailed delays of minutes that correlated with lengths of afferent fibres. Potentiation failed to occur in cells pretreated with colchicine to degrade their microtubular transport matrix.

Comparative analysis of ionic currents in the somatic membrane of embryonic and newborn rat sensory neurons.

Inward currents in the somatic membrane of dissociated rat dorsal root ganglion neurons have been studied in two groups of animals (17 days of embryonic development and the first day after birth) by suction pipette (whole-cell configuration) and voltage-clamp techniques. Altogether 157 neurons were examined. Four components in the inward currents have been identified: fast tetrodotoxin-sensitive (INaf) and slow tetrodotoxin insensitive (INas) sodium, low-(ICal) and high-threshold (ICah) calcium currents.

Pharmacological characterization of calcium currents and synaptic transmission between thalamic neurons in vitro.

We recorded from pairs of cultured, synaptically connected thalamic neurons. Evoked excitatory postsynaptic currents (EPSCs) reversed at +17 mV and were blocked reversibly by 1 mM kynurenic acid, a glutamate receptor antagonist. NMDA and non-NMDA receptors mediated excitatory post-synaptic responses, as shown by selective block of EPSC components with 50 microM (+/-)-2-amino-5-phosphonopentanoic acid and 10 microM 6,7-dinitroquinoxaline-2,3-dione, respectively.