N. A. Lozovaya

Extrasynaptic NR2B and NR2D subunits of NMDA receptors shape ‘superslow’ afterburst EPSC in rat hippocampus

In conditions of facilitated synaptic release, CA3/CA1 synapses generate anomalously slow NMDA receptor‐mediated EPSCs (EPSCNMDA). Such a time course has been attributed to the cooperation of synapses through glutamate spillover. Imitating a natural pattern of activity, we have applied short bursts (2–7 stimuli) of high‐frequency stimulation and observed a spike‐to‐spike slow‐down of the EPSCNMDA kinetics, which accompanied synaptic facilitation. It was found that the early component of the EPSCNMDA and the burst‐induced late component of the EPSCNMDA have distinct pharmacological properties. The competitive NMDA antagonist R‐(−)‐3‐(2‐carboxypiperazine‐4‐yl)‐propyl‐1‐phosphonic acid (D‐CPP), which has higher affinity to NR2A than to NR2B subunits and lowest affinity at NR2D subunits, significantly slowed down the decay rate of the afterburst EPSC while leaving the kinetics of the control current unaffected. In contrast, ifenprodil, a highly selective NR2B antagonist, and [±]‐cis‐1‐[phenanthren‐2yl‐carbonyl]piperazine‐2,3‐dicarboxylic acid (PPDA), a competitive antagonist that is moderately selective for NR2D subunits, more strongly inhibited the late component of the afterburst EPSCNMDA. The receptors formed by NR2B and (especially) NR2D subunits are known to have higher agonist sensitivity and much slower deactivation kinetics than NR2A‐containing receptors. Furthermore, NR2B is preferentially and NR2D is exclusively located on extrasynaptic membranes. As the density of active synapses increases, the confluence of released glutamate makes EPSC decay much longer by activating more extrasynaptic NR2B‐ and NR2D‐subunit‐containing receptors. Long‐term potentiation (LTP) induced by successive rounds of burst stimulation is accompanied by a long‐term increase in the contribution of extrasynaptic receptors in the afterburst EPSCNMDA.

Protective cap over CA1 synapses: extrasynaptic glutamate does not reach the postsynaptic density

Numerous data indicate that nonsynaptic release of glutamate occurs both in normal and pathophysiological conditions. When reaching receptors in the postsynaptic density (PSD), glutamate (Glu) could affect the synaptic transmission. We have tested this possibility in the hippocampal CA1 synapses of rats, either by applying exogenous Glu to the CA1 neurons or by disruption of Glu transporter activity. L-Glu (400 microM) was directly applied to the hippocampal slices acutely isolated from the rats. It produced a strong inhibition of both ortho- and antidromically elicited action potentials fired by CA1 neurons while the excitatory postsynaptic current (EPSC) measured in these neurons remained totally unaffected. The optical isomer D-Glu which is not transported by the systems of Glu uptake inhibited not only orthodromic and antidromic spikes, but also EPSC. Non-specific glutamate transporter inhibitor DL-threo-beta-hydroxyaspartic acid (THA, 400 microM) mimicked the effects of exogenous Glu and produced strong inhibition of both orthodromic and antidromic spikes, without any influence on the amplitude of EPSCs. Dihydrokainate (DHK, 300 microM), selective inhibitor of GLT-1 subtype of glutamate transporter, exerted a significant inhibitory action on the orthodromically evoked spikes and also on the EPSC. Our results indicate that extrasynaptic and PSD membranes of CA1 neurons form separate compartments differing in the mechanisms and efficiency of external Glu processing: the protection of PSD markedly prevails.

Persistently enhanced ratio of NMDA and non-NMDA components of rat hippocampal EPSC after block of A1 adenosine receptors at increased [Ca2+]o[Mg2+]o

NMDA and non-NMDA receptor-mediated components of excitatory post-synaptic current (EPSC) were studied by in situ whole-cell voltage-clamp recordings in the CA1 field of rat hippocampus. We found that the amplitudes ratio of the NMDA to the non-NMDA components can be strongly increased by blocking A1 adenosine receptors. The necessary conditions for this effect are both, increased Ca2+ and lowered Mg2+ in the external medium. The so achieved increase in the NMDA/non-NMDA ratio of EPSC components is irreversible and no longer depends on the activity of A1 adenosine receptors.

NMDA receptor-mediated synapses between CA1 neurones : activation by ischaemia

Using an in situ patch clamp in hippocampal CA1 mini-slices, we measured excitatory postsynaptic currents (EPSC) by varying the strength of the stimulus applied to the axons of CA3 neurones. The kinetics of the EPSC was initially independent of the stimulus strength. Post-ischaemic potentiation of the EPSC was observed 60-80 min after brief periods (10 min) of anoxia/aglycaemia. The decay of the EPSC slowed significantly in most of the examined neurones. In 11 of 17 cells the EPSC kinetics became dependent on stimulus strength: a slower decay corresponded to a stronger stimulus. This effect was not abolished by N-methyl-D-aspartate (NMDA) or a non-NMDA receptor blocker (D-2-amino-5-phosphonovaleric acid or 6-cyano-7-nitroquinoxaline-2,3-dione respectively) indicating the polysynaptic nature of the modified EPSC: transient ischaemia led to the long-term recruitment of previously inactive, possibly latent NMDA synapses between CA1 neurones.

Post-synaptic N-methyl-d-aspartate signalling in hippocampal neurons of rat: spillover increases the impact of each spike in a short burst discharge

High-frequency burst discharges in hippocampus typically consist of less than ten spikes fired at frequencies too high to be followed by a post-synaptic neuron. How significant are these numbers for synaptic signalling? We have measured the N-methyl-d-aspartate (NMDA) component of the excitatory post-synaptic current (EPSC(NMDA)) in hippocampal CA1 neurons of rat after burst discharge of variable duration. The synaptic facilitation is accompanied by a slow-down of the EPSC(NMDA) which develops on a spike-to-spike basis. Consequently the charge transferred by the after-burst EPSC(NMDA) is increased with each spike. The phenomenon is most probably due to the spillover-mediated recruitment of extrasynaptic NMDA receptors. In terms of post-synaptic signalling it dramatically increases the impact of each spike in a short burst discharge.

New channel blocker BIIA388CL blocks delayed rectifier, but not A-type potassium current in central neurons

A new substance (R,S)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-yl)-2-cyclohexyl-N-(3,3-diphenylpropyl)-acetamide hydrochloride (BIIA388Cl), which demonstrates neuroprotective properties in animal models, was examined for its action on K(+) currents in acutely isolated rat hippocampal neurons using the patch-clamp/concentration clamp techniques in the whole-cell configuration. The delayed rectifier K(+)-current (I(DR)) was strongly inhibited by externally applied BIIA388Cl, while the transient A-current (I(A)) remained virtually unaffected. Block of I(DR) by the pre-applied BIIA388Cl was revealed as a rapid decay of the current indicating direct interaction of the drug with the open state of the channel. The removal of the block upon repolarization was also rapid (tau=22 ms). The dose-response relationship for the blocking action of BIIA388Cl revealed an IC(50) value of 300 nM for the peak I(DR), whereas the IC(50) value for I(DR) measured 300 ms after the onset of depolarization was 120 nM. The blocking action of BIIA388Cl on I(A) was at least 200 times less potent. These data allow us to conclude that BIIA388Cl is an effective and selective blocker of I(DR). This current is the main pathway for the loss of intracellular potassium by depolarized neurons. Selective obstruction of this pathway could be useful for neuroprotection.

Antioxidant-caused changes in the permeability of proton-gated ion channels for sodium and calcium

Using a patch-clamp technique in the whole-cell configuration, we studied the effect of an exogenous antioxidant, dithiothreitol (DTT), on transmembrane currents in isolated cells obtained from the rat spinal ganglia. We demonstrated that this antioxidant (DTT) is capable of modulating the proton-gated current. In most neurons, proton-gated currents increased in the presence of the antioxidant. Since proton-gated receptor-channel complexes of sensory neurons are involved in different processes of signalling and transmission of sensory information in the peripheral nervous system, we hypothesize that the influences mediated by alterations of the concentrations of antioxidants participate in the formation of the state of algesia under normal physiological conditions and of that of hyperalgesia in pathological states. In addition, oxidative stress, which causes a shift in the balance of concentrations of antioxidants, accompanies numerous abnormal pathophysiological states, in particular diabetes, ischemia, and inflammation. Since proton-gated channels are permeable for calcium ions, an antioxidant-induced increase in calcium signalling can be significantly important for a number of biochemical processes occurring in tissues.

Effects of Long-Term Hypoxia/Hypoglycemia on Synaptic Transmission between the CA3 and CA1 Zones in Rat Hippocampal Slices

On rat hippocampal slices using a standard patch-clamp technique in the whole-cell configuration, we studied the effects of long-term (40 to 60 min) hypoxia/hypoglycemia (HH) on excitatory postsynaptic currents (EPSC) evoked by stimulation of Schaffer collaterals in the cells of the CA1 zone. In addition to the earlier described effect of an immediate drop in the EPSC amplitude, a significant transient increase in its amplitude 30-50 min after the beginning of HH was observed. A pharmacologically isolated NMDA component of excitatory synaptic events underwent similar changes: 30-50 min after the blockade of NMDA receptor-mediated current, a fast recovery of its amplitude to the control (or even higher) values occurred. A blocker of NMDA/glutamate (Glu) receptors, D-aminophosphonovaleric acid (D-APV), and a competitive nonspecific antagonist of metabotropic Glu receptors, (RS)-α-methyl-4-carboxyphenylglycine – (RS)-MCPG – did not influence the HH-induced initial suppression of synaptic transmission but completely eliminated its delayed recovery. Our findings allow us to suppose that NMDA receptors, as well as metabotropic Glu receptors, play important roles in the cascade of biochemical reactions resulting in death of hippocampal pyramidal cells in the course of and after long-term ischemia in vivo.

Modulating Action of Hyperforin on the P-Type Calcium Channels in the Membranes of Rat Cerebellar Purkinje Neurons

A modulating action of hyperforin (an active compound of the extract from Hypericum perforatum) on a high-threshold component of the calcium current, sensitive to application of 100 nM ω-Aga-IVA toxin and identified as P current, was studied on freshly isolated Purkinje neurons with the use of a patch-clamp technique in the whole-cell configuration. It was shown that extracellular application of 0.8 μM hyperforin caused a shift of the current-voltage (I-V) relationship of P current by -(8 ± 2) mV, slowdown of the activation kinetics, and a decrease in the amplitude of this current. The shift of the I-V relationship and slowdown of activation kinetics developed for less than 10 sec, while the P-current amplitude decreased for a much longer time (several minutes) and depended on the intracellular concentration of Ca2+ ions. ω-Aga-IVA toxin at the concentration of 100 nM completely blocked the recorded inward current in the presence of 0.8 μM hyperforin. In experiments with intracellular perfusion of Purkinje neurons, we found that interaction of hyperforin with its binding site occurs at the external side of the cell membrane. The study of the mechanisms involved in the hyperforin-induced P-current modulation revealed that 1 mM GTPγS (activating GTs proteins, as well as activating or blocking GMs proteins) or 1-2 mM GDPβS (blocking GTs and GMs proteins) in the intracellular solution did not affect the hyperforin-induced modulation of P current. Hyperforin-induced Ca2+-independent shift of the I-V relationship and slowdown of the activation kinetics of P current were abolished in the presence of 0.5 μM calmidazolium in the extracellular medium.

Effect of cannabinoids on glycine-activated currents in pyramidal neurons of the rat hippocampus

Using electrophysiological techniques (a patch-clamp technique in the whole-cell configuration and intracellular perfusion of neurons), we studied the effect of cannabinoids on the characteristics of glycine-activated currents in freshly isolated pyramidal neurons of the rat hippocampus. We found that endocannabinoids (anandamide and 2-arachidonoyl glycerol), as well as a synthetic cannabinoid, WIN 55,212-2, when applied in physiological concentrations, decreased the amplitude of glycine-activated currents. The agents under study accelerated the kinetics of activation and desensitization of glycine-induced Cl− currents. The characteristics of the currents recovered after washout from cannabinoids. Changes in the kinetics of desensitization of glycine-activated currents depended noticeably on the holding potential; at positive potentials the sensitivity to cannabinoids was higher. These effects of cannabinoids were also observed in the presence of antagonists of CB1/CB3 receptors and an inhibitor of G proteins, GDPβS. These data indicate that under our experimental conditions cannabinoids exerted direct effects on glycine receptors.