Calcium-Dependent Desensitization of NMDA Receptors.

Glutamate receptors play the key role in excitatory synaptic transmission in the central nervous system (CNS). N-methyl-D-aspartate-activated glutamate receptors (NMDARs) are ion channels permeable to sodium, potassium, and calcium ions that localize to the pre- and postsynaptic membranes, as well as extrasynaptic neuronal membrane. Calcium entry into dendritic spines is essential for long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission. Both LTP and LTD represent morphological and functional changes occurring in the process of memory formation. NMDAR dysfunction is associated with epilepsy, schizophrenia, migraine, dementia, and neurodegenerative diseases. Prolonged activation of extrasynaptic NMDARs causes calcium overload and apoptosis of neurons. Here, we review recent findings on the molecular mechanisms of calcium-dependent NMDAR desensitization that ensures fast modulation of NMDAR conductance in the CNS and limits calcium entry into the cells under pathological conditions. We present the data on molecular determinants related to calcium-dependent NMDAR desensitization and functional interaction of NMDARs with other ion channels and transporters. We also describe association of NMDARs with lipid membrane microdomains.

Downregulation of Purkinje Cell Activity by Modulators of Small Conductance Calcium-Activated Potassium Channels In Rat Cerebellum.

Small-conductance calcium-activated potassium channels (SK channels) are widely expressed in CNS tissues. Their functions, however, have not been well studied. Participation of SK channels in Purkinje cell (PC) pacemaker activity has been studied predominantly in vitro. Here we studied for the first time the effects of SK channel activation by NS309 or CyPPA on the PC simple spike frequency in vivo in adult (3 - 6 months) and aged (22 - 28 months) rats using extracellular microelectrode recordings. Both pharmacological agents caused a statistically significant decrease in the PC simple spike frequency. The maximum value of the decrease in the simple spike frequency did not depend on age, whereas a statistically significant inhibition of the spike frequency was achieved faster in aged animals than in adult ones. In experiments on cultured neurons PCs were identified by the expression of calbindin as the PC-specific marker. Registration of transmembrane currents in cerebellar neurons revealed the direct action of NS309 and CyPPA on the SK channels of PC consisted in the enhancement of outward potassium currents and action potential after-hyperpolarization. Thus, SK channel activators can compensate for age-related changes of the autorhythmic functions of the cerebellum.

[HOMOCYSTEINE-INDUCED MEMBRANE CURRENTS, CALCIUM RESPONSES AND CHANGES OF MITOCHONDRIAL POTENTIAL IN RAT CORTICAL NEURONS].

Homocysteine, a sulfur-containing amino acid, exhibits neurotoxic effects and is involved in the pathogenesis of several major neurodegenerative disorders. In contrast to well studied excitoxicity of glutamate, the mechanism of homocysteine neurotoxicity is not clearly understood. By using whole-cell patch-clamp, calcium imaging (fluo-3) and measurements of mitochondrial membrane potential (rhodamine 123) we studied transmembrane currents, calcium signals and changes in mitochondrial membrane potential induced by homocysteine versus responses induced by NMDA and glutamate in cultured rat cortical neurons. L-homocysteine (50 µM) induced inward currents that could be completely blocked by the selective antagonist of NMDA receptors - AP-5. In contrast to NMDA-induced currents, homocysteine-induced currents had a smaller steady-state amplitude. Comparison of calcium responses to homocysteine, NMDA or glutamate demonstrated that in all cortical neurons homocysteine elicited short, oscillatory-type calcium responses, whereas NMDA or glutamate induced sustained increase of intracellular calcium. Analysis of mitochondrial changes demonstrated that in contrast to NMDA homocysteine did not cause a drop of mitochondrial membrane potential at the early stages of action. However, after its long-term action, as in the case of NMDA and glutamate, the changes in mitochondrial membrane potential were comparable with the full drop of respiratory chain induced by protonophore FCCP. Our data suggest that in cultured rat cortical neuron homocysteine at the first stages of action induces neurotoxic effects through activation of NMDA-type ionotropic glutamate receptors with strong calcium influx through the channels of these receptors. The long-term action of homocysteine may lead to mitochondrial disfuction and appears as a drop of mitochondrial membrane potential.

[The features of postsynaptic currents in primary culture of rat cortical neurons].

The generation features of postsynaptic currents were studied in primary culture of cortical neurons at 7-20 days in vitro (DIV). The use of specific blockers of postsynaptic ion channels after 10 DIV revealed all types of electrical activity found in adult cortex including miniature inhibitory (mIPSCs), excitatory (mEPSCs) and spontaneous giant excitatory currents and spikes. The frequency of mEPSCs increased exponentially from 7 to 20 DIV doubling every 2.2 days in parallel with changes in action potentials generation. The mEPSCs generated by NMDA and AMPA or by only AMPA receptor activation were found. The inhibition of NMDA receptors by magnesium ions or AP5 were shown to modulate the frequency and amplitude of mEPSCs, which differ primary culture from brain slices possibly because of the lack of glial control of synaptic transmission.

Effects of intranasal administration of epitalon on neuron activity in the rat neocortex.

This report discusses the properties of the synthetic tetrapeptide epitalon (Ala-Glu-Asp-Gly), synthesized on the basis of an epiphyseal peptide extract. Intranasal administration of epitalon was selected as a noninvasive means of applying the agent to the CNS by bypassing the blood-brain barrier. The aim of the present work was to assess the characteristics of the action of epitalon on the frequency of spontaneous neuron activity in the cerebral cortex of white rats. Studies were performed using male Wistar rats anesthetized with urethane (1 g/kg). Extracellular activity of cortical neurons was recorded with a glass microelectrode of resistance 1-2 MOmega. Recording of spontaneous neuron discharges for 10-15 min was followed by intranasal administration of epitalon solution and recording of neuron activity to 30 min after doses of 30 ng per animal. Significant activation of neuron activity was seen several minutes after dosage, with an increase (by factors of 2-2.5) in discharge frequency. In some experiments, the effect of epitalon was multiphasic. The first peak of increased neuron discharge frequency at 5-7 min was followed by peaks at 11-12 and 17-18 min. The increase in discharge frequency occurred because of an increase in the discharge frequency of neurons which were already active and the recruitment of previously silent neurons. At least the first peak of increased neuron activity following exposure to epitalon was found to be associated with the direct action of the peptide on cortical cells.

Reflection of the pattern of cortical activation in the phase structure of the human EEG.

A total of 22 healthy subjects in the EEG laboratory and 62 patients in the clinical functional diagnostic unit were studied. Spontaneous EEG recordings were made using the 10-20 scheme relative to combined ear electrodes in the state of rest with the eyes closed and open and during various functional loads. Traces were analyzed by computer animation of the EEG phase structure. The main concept of the method for extracting phase structure was based on not using a single reference lead. Time shifts were measured only between neighboring electrodes, with the result that the oscillations being compared were highly coherent. Time discordance was assessed in terms of the shift in the peak of the cross-correlation function. The results showed that from the point of view of phase structure, the differences between the high-and low-frequency EEG rhythms were purely quantitative. Qualitatively, the properties of the rhythms were identical and were reduced to slow (in the seconds range) oscillations of phase shifts. Low-frequency activity was characterized by large (in absolute terms, msec) phase shifts from electrode to electrode as compared with high-frequency activity. The phase shifts of potentials formed a structure which was overall very similar in different subjects and was reproduced in different leads. The initial appearance of EEG waves was statistically linked with the main sensory projections--the visual (occipital areas), auditory (temporal areas), and somatic (parietal areas), with addition of the frontal areas. Rearrangement of phase leadership in favor of the occipital pole at the expense of both temporal areas was observed on opening the eyes. This appears to depend on the level of sensory influx to this cortical area from the thalamus. It is suggested that the direction of the phase gradient reflects a gradient of cortical current density parallel to the surface. This can be used to locate compact sources lying close to the surface.

[Reflection of cortical activation pattern in the human EEG phase structure].

Investigation was fulfilled on healthy subjects (22) and on outpatients (62). The EEG by the standard scheme as recorded at resting with open and closed eyes and under different functional loads. These records were processed in accordance with the EEC phase structure with the aid of computer animation technology. The main idea of the phase structure technology consists in rejection of one supporting lead. Time shifts were measured only between two neighbouring electrodes, so that the oscillations under comparison were always highly coherent. Time errors were evaluated according to crosscorrelation function maximum shift. The differences between high- and low-frequency EEG rhythms were shown to be only quantitative from the phase structure viewpoint. Qualitatively, the rhythm properties were equal and came to slow (second) phase structure oscillations. Low frequency activity compared to high frequency one was characterized by greater phase shifts from electrode to electrode. Phase shifts of potentials are forming the structure which, as a whole, is very similar in different people and is reproduced in different states. Initial EEG waves appearance is statistical linked with main sensory projections: visual (occiput), auditory (temples) and somatic (parietal region) with addition of frontal region. Redistribution of phase leadership in favor of occipital pole and to both temporal regions when eyes are open is described. It is apparently dependent on the sensory surge level from thalamus to a given cortex region. Phase gradient direction seems to reflect the cortex current density gradient which is parallel to surface. It can be used for localization of compact sources lying near to cortex.

[Intranasal epitalon infusion modulates neuronal activity in the rat neocortex].

Properties of tetrapeptide epitalon (Ala-Glu-Asp-Gly) constructed on the basis of pineal peptide extract, have been studied. The intranasal infusions: a noninvasive way to deliver this peptide to CNS hypassing the blood-brain barrier, was used. The aim of the study is to estimate epitalon action on rat motor cortex spontaneous activity. Wistar male rats were anesthetized with urethane (1 g/kg). Extracellular unit recording was made using glass microelectrodes (1-2 MOhm). After recording of spontaneous activity (10-15 min), epitalon intranasal infusion (2 ng) was followed by 30-minute recording. Within a few minutes after the infusion, significant activation of neural activity was observed (2-2.5-fold higher frequency of neuronal spikes). Complex response consisting of several phases was identified in some recordings. The spikes frequency growth during 5 to 7 min (first phase) after the infusion was followed by the second (11-12 min) and the third (17-18 min) phases. An increase of neuronal spontaneous activity was conditioned by the higher frequency of already active units and by the involvement of previously silent cells. At least the first phase of epitalon action can be explained by direct action of the peptide on the cells of the motor cortex.