O. V. Godukhin

Neuronal hyperexcitability induced by repeated brief episodes of hypoxia in rat hippocampal slices: involvement of ionotropic glutamate receptors and L-type Ca2+ channels

Repeated exposures of rat hippocampal slices to short episodes of hypoxia induce a sustained decrease in the threshold of the development of stimulus-evoked epileptiform discharges in CA1 pyramidal neurons. We have previously demonstrated that the K(+)(o)-induced hyperexcitability required functional L-type voltage-dependent Ca(2+) channels and NMDA-receptors, but was independent of AMPA/kainate-receptor activation. As hypoxia/ischaemia can lead to increased K(+)(o), the epileptiform activity observed after exposure to these challenges could also result from high K(+)(o). The purpose of this study was: (i) to determine whether ionotropic glutamate receptors and L-type Ca(2+) channels are involved in the development of epileptiform activity induced by repeated exposures of hippocampal slices to hypoxia; and (ii) to compare the properties of hypoxia- and high K(+)(o)-induced hyperexcitability. Population spike of presynaptic fibres with field excitatory postsynaptic potential from the stratum radiatum, and population spike of CA1 pyramidal neurons from the stratum pyramidale, were recorded simultaneously in the CA1 area of rat hippocampal slices in response to electrical stimulation of the Schaffer collateral/commissural fibres. Repeated, brief hypoxic episodes induced a sustained decrease in the threshold for development of evoked epileptiform discharges that was associated with long-term potentiation of the CA3-CA1 synapses, but without EPSP-spike potentiation (i.e. in contrast to high K(+)(o)-induced hyperexcitability). The selective antagonist of NMDA receptors, D-APV (25 microM), and the selective blocker of L-type Ca(2+) channels, nifedipine (10 microM) depressed the development of hypoxia-induced hyperexcitability. However, in contrast to high K(+)(o)-induced hyperexcitability, hypoxia-induced hyperexcitability was also blocked by the AMPA/kainite-receptor antagonist, CNQX (5 microM). The present findings confirm that repeated, brief episodes of hypoxia, like exposure to high extracellular K(+), can induce a pro-epileptic state in the CA1 neuronal network, but that the mechanisms leading to hyperexcitability are different for the two stimuli.

Involvement of cAMP- and Ca2+/calmodulin-dependent neuronal protein phosphorylation in mechanisms underlying genetic predisposition to audiogenic seizures in rats

It was shown that increased excitability in neurons underlying epilepsies would be maintained by abnormalities in protein phosphorylation systems. This study was initiated to compare the functioning of Ca(2+)/calmodulin- and cAMP-dependent systems of protein phosphorylation in homogenates of neocortex and hippocampus in three animal groups: genetically prone to audiogenic seizures (GPAS) rats, GPAS rats exposed to daily repeated audiogenic seizures (AGPAS rats) and nonepileptic Wistar ones. We found significant differences in phosphorylation of 270, 58, 54 and 42 kDa proteins in neocortex and hippocampus of GPAS rats in comparison with Wistar ones. Daily repeated seizures induced further modifications of phosphorylation of these proteins in only hippocampus of AGPAS rats as compared with GPAS ones. Ca(2+)-independent, functional CAMKII activity was considerably increased in hippocampus but decreased in neocortex of GPAS rats in comparison with Wistar ones. The activity of PKA was increased both in neocortex and hippocampus of GPAS rats. Daily repeated audiogenic seizures induced the decrease of Ca(2+)-independent CAMKII activity in hippocampus and the increase of PKA activity in neocortex of AGPAS rats in comparison with GPAS ones. The present results indicate that modification of 270, 58, 54, and 42 kDa proteins phosphorylation as well as altered CAMKII and PKA activities might be involved in mechanisms of genetic predisposition to audiogenic seizures.

Neuroprotective effects of interleukin-10 and tumor necrosis factor-α against hypoxia-induced hyperexcitability in hippocampal slice neurons

In our previous experiments we have demonstrated that repeated exposures of rat hippocampal slices to brief episodes of hypoxia induce a sustained decrease in the threshold of stimulus-evoked epileptiform discharges in CA1 pyramidal neurons. The aim of this study was to investigate the comparative effects of interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) on the hyperexcitability of CA1 pyramidal neurons induced by brief episodes of hypoxia in the rat hippocampal slices. The method of field potentials measurement in CA1 region of hippocampal slices have been described in our previous work [O. Godukhin, A. Savin, S. Kalemenev, S. Levin, Neuronal hyperexcitability induced by repeated brief episodes of hypoxia in rat hippocampal slices: involvement of ionotropic glutamate receptors and L-type Ca2+ channels, Neuropharmacology 42 (2002) 459-466]. The principal results of our work are summarized as follow. Pro-inflammatory cytokine TNF-alpha (0.8, 4 and 20 ng/ml) and anti-inflammatory cytokine IL-10 (1 and 10 ng/ml) significantly reduced the hyperexcitability in CA1 pyramidal neurons induced by brief episodes of hypoxia in the rat hippocampal slices. The neuroprotective effects of IL-10 and TNF-alpha against the hypoxia-induced hyperexcitability were mediated by anti-hypoxic actions of these cytokines through, possibly, mechanism of preconditioning.

Interleukin-10 modulates [Ca2+]i response induced by repeated NMDA receptor activation with brief hypoxia through inhibition of InsP3-sensitive internal stores in hippocampal neurons

The goal of this study was to evaluate an effect of interleukin-10 (IL-10) on the Ca(2+) response induced by repeated NMDA receptor activation with brief hypoxia in cultured hippocampal neurons. We focused on the importance of internal Ca(2+) stores in the modulation of this Ca(2+) response by IL-10. To test this, we compared roles of InsP(3)- and ryanodine-sensitive internal stores in the effects of IL-10. Measurements of intracellular cytosolic calcium concentration ([Ca(2+)](i)) in cultured hippocampal neurons were made by imaging Fura-2AM loaded hippocampal cells. Repeated episodes of NMDA receptor activation with brief hypoxia induced the spontaneous (s) [Ca(2+)](i) increases about 3 min after each hypoxic episode. The amplitude of the s[Ca(2+)](i) increases was progressively enhanced from the first hypoxic episode to the third one. IL-10 (1 ng/ml) abolished these s[Ca(2+)](i) increases. Exposure of cultured hippocampal neurons with thapsigargin (1 μM) or an inhibitor of phospholipase C (U73122, 1 μM) for 10 min also abolished the s[Ca(2+)](i) increases. On the other hand, antagonist of ryanodine receptors (ryanodine, 1 μM) did not affect this Ca(2+) response. These studies appear to provide the first evidence that Ca(2+) release from internal stores is affected by anti-inflammatory cytokine IL-10 in brain neurons. It is suggested that these data increase our understanding of the neuroprotective mechanisms of IL-10 in the early phase of hypoxia.

Repeated brief episodes of hypoxia modulate the calcium responses of ionotropic glutamate receptors in hippocampal neurons

The aim of this study was to evaluate the intracellular cytosolic calcium concentration ([Ca(2+)](i)) changes induced by activation of ionotropic glutamate receptors in cultured hippocampal neurons after repeated brief episodes of hypoxia. To investigate what kinds of ionotropic glutamate receptors are involved we used specific agonists for AMPA- and NMDA-type glutamate receptors. Measurements of [Ca(2+)](i) in cultured hippocampal neurons were made by imaging Fura-2AM loaded hippocampal cells. In the rat hippocampal slice method, field potential measurements in CA1 pyramidal neurons were used. The main result of our study is that brief hypoxic episodes progressively depress the [Ca(2+)](i) increases induced by agonists of AMPA and NMDA glutamate receptors in cultured hippocampal neurons. An effectiveness of this depression is increased from the first hypoxic episode to the third one. Hypoxic preconditioning effect is observed during 10-20 min after termination of hypoxic episode and depends on [Ca(2+)](i) response amplitudes to agonists before hypoxia. In contrast to AMPA receptor activation, NMDA receptor activation before hypoxia induce the spontaneous [Ca(2+)](i) increase about 3 min after each hypoxic episode. These spontaneous [Ca(2+)](i) increases may be an indicator of the development of posthypoxic hyperexcitability in hippocampal neurons. Our results suggest that brief hypoxia-induced depression of the glutamate receptor-mediated [Ca(2+)](i) responses contributes to the development of rapid hypoxic preconditioning in hippocampal CA1 neurons.

Anti-inflammatory cytokines, TGF-β1 and IL-10, exert anti-hypoxic action and abolish posthypoxic hyperexcitability in hippocampal slice neurons: Comparative aspects

The aim of this study was to investigate the comparative effects of transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) on the repeated brief hypoxia-induced alterations in the activity of hippocampal slice CA1 pyramidal neurons. The method of field potentials measurement in CA1 region of hippocampal slices was used. The principal results of our work are summarized as follow. 1. TGF-β1 reduces the depressive effect of brief hypoxia on the population spike amplitude more effectively than IL-10. 2. During TGF-β1 exposure (in contrast to IL-10), three 3-min hypoxic episodes do not induce the rapid hypoxic preconditioning. 3. TGF-β1 and IL-10 equally abolish posthypoxic hyperexcitability induced by repeated brief episodes of hypoxia in CA1 pyramidal neurons. These findings indicated that TGF-β1 and IL-10 are able to evoke anti-hypoxic effect and abolish the development of posthypoxic hyperexcitability induced by repeated brief hypoxic episodes in hippocampal CA1 pyramidal neurons. Our results also demonstrated that TGF-β1 reduced the effectiveness of hypoxia to depress neuronal activity more effectively than IL-10. We suggest that the present findings allow to explain the certain neuroprotective mechanisms of IL-10 and TGF-beta1 in the early phase of hypoxia and indicate that a therapeutic anti-inflammatory approach using these substances can provide neuroprotection in the brain hypoxic conditions.

Kindling-like state in rat hippocampal CA1 slices induced by the repeated short-term extracellular K+ increases: the role of L-type Ca2+-channels

The repeated 30 s applications (3-6 episodes) of K+ (up to 20 mM) induced the appearance of single electric pulse-triggered population spike bursts in the rat CA1 hippocampal slices. This kindling-like state kept for a long time after the last K(+)-application and was limited by the time of slice survival. Disconnection of CA3 region did not significantly eliminate the effect of repeated short-term [K+]o increases on the establishment and maintenance of the kindling-like state in the CA1 region. This state correlated with the increases in the efficiency of the excitatory postsynaptic potential (EPSP)-spike transfer (the excitability of CA1 pyramidal neurons) but not in the glutamatergic synaptic efficiency. The selective blocker of L-type Ca2+ channels nimodipine (10 microM) abolished the development of the kindling-like state as well as the increases in the efficiency of the EPSP-spike transfer in rat hippocampal CA1 slices. Taken together, these data indicate that the described model of kindling in vitro can be useful for a study of the role of different sites of Ca2+ entry into hippocampal neurons for the cellular mechanisms of epileptogenesis.

Comparative roles of ATP-sensitive K+ channels and Ca2+-activated BK+ channels in posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro.

The aim of this study was to investigate the comparative effects of glibenclamide (GC), a selective blocker of K(+)(ATP) channels, and iberiotoxin (IbTX), a selective blocker of BK(+)(Ca) channels, on the repeated brief hypoxia-induced posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro. The method of field potentials measurement in CA1 region of the rat hippocampal slices was used. In contrast to GC (10 microM), IbTX (10nM) significantly abolished both posthypoxic hyperexcitability and rapid hypoxic preconditioning induced by brief hypoxic episodes. These effects of IbTX did not depend on its ability to reduce the hypoxia-induced decrease of population spike (PS) amplitude during hypoxic episodes since GC (10 microM), comparatively with IbTX (10nM), significantly reduced the depressive effect of hypoxia on the PS amplitude during hypoxic episodes but did not abolish both posthypoxic hyperexcitability and rapid hypoxic preconditioning in CA1 pyramidal neurons. Our results indicated that BK(+)(Ca) channels, in comparison with K(+)(ATP) channels, play a more important role in such repeated brief hypoxia-induced forms of neuroplasticity in hippocampal CA1 pyramidal neurons as posthypoxic hyperexcitability and rapid hypoxic preconditioning.

Anti-inflammatory cytokine interleukin-10 increases resistance to brain ischemia through modulation of ischemia-induced intracellular Ca2+ response

It is suggested that anti-inflammatory cytokine interleukin-10 (IL-10) mediates the delayed protective effects through activation of Jak-Stat3, PI3K-Akt and NF-κB signaling pathways. However, our previous experiments have demonstrated that IL-10 is capable to exert the rapid neuroprotective action through modulation of hypoxia-induced intracellular Ca(2+) ([Ca(2+)]i) response. The first purpose of the present study was to evaluate the neuroprotective effects of IL-10 using three models of the ischemic insults in rats: permanent middle cerebral artery occlusion, ischemia in acute hippocampal slices in vitro and ischemia in cultured hippocampal cells in vitro. The second purpose of the study was to elucidate a role of [Ca(2+)]i changes in the mechanisms underlying IL-10 elicited protection of neurons and astrocytes from ischemia-induced death in cultures of primary hippocampal cells. The data presented here shown that anti-inflammatory cytokine IL-10 is capable to induce a resistance of the brain cells to ischemia-evoked damages in in vivo and in vitro models of the ischemic insults in rats. This protective effect in cultured hippocampal cells is developed rapidly after application of IL-10 and strongly associated with the IL-10 elicited elimination of [Ca(2+)]i response to ischemia. Thus, our results provide the evidence that anti-inflammatory cytokine IL-10, in addition to an activation of the canonical signaling pathways, is capable to exert the rapid neuroprotective effects through transcription-independent modulation of ischemia-induced intracellular Ca(2+) responses in the brain cells.

Epileptiform activity and EPSP-spike potentiation induced in rat hippocampal CA1 slices by repeated high-K+ : involvement of ionotropic glutamate receptors and Ca2+/calmodulin-dependent protein kinase II

We have previously demonstrated that repeated brief increases in extracellular K(+) (K(+)(o)) induce a hyperexcitability in CA1 pyramidal cells that persists for a long time after the final application of K(+) [Neurosci. Lett. 223 (1997) 177; Epilepsy Research (2000) 75]. This epileptiform activity, which was associated with a lasting excitatory postsynaptic potential (EPSP)-spike potentiation, presented some of the characteristic features of traditional in vivo kindling. We have also found that Ca(2+) influx through L-type voltage-sensitive Ca(2+) channels is essential for the development of both in vitro kindling and EPSP-spike potentiation. The aims of this study were to investigate the involvement of ionotropic glutamate receptors, especially those of the NMDA subtype, and the requirement for Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in these phenomena. Field EPSPs with presynaptic fibre volleys from the stratum radiatum, and population spikes from the stratum pyramidale, were recorded in the CA1 area of rat hippocampal slices in response to electrical stimulation of the Schaffer collateral/commissural fibres. Repeated (three episodes) brief (30 s) increases in extracellular K(+) induced a sustained decrease in the threshold for development of evoked epileptiform discharges (i.e. an in vitro kindling-like state) and a lasting potentiation of the EPSP-spike transfer in CA1 pyramidal neurons (EPSP-spike potentiation). The selective antagonist of NMDA receptors, APV (50 microM), blocked the EPSP-spike potentiation, depressed the induction phase of the in vitro kindling-like state, and blocked the maintenance phase of this state. In contrast to APV, the blockade of AMPA/kainate receptors by CNQX (10 microM) had no effect. Like APV, KN62 (3 microM), a selective membrane permeable inhibitor of CaMKII, blocked the EPSP-spike potentiation and the maintenance phase of the in vitro kindling-like state. Our previous and present results therefore demonstrate that Ca(2+) influxes through L-type voltage-dependent-and NMDA receptor-dependent-Ca(2+) channels contribute differentially to the development of an in vitro kindling-like state, and both induce EPSP-spike potentiation in CA1 hippocampal pyramidal cells in response to repeated brief increases in K(+)(o). It is suggested that these effects of intracellular Ca(2+) on the maintenance phase of the in vitro kindling-like state and EPSP-spike potentiation are mediated by CaMKII-dependent mechanisms.