Cdk5 activation induces hippocampal CA1 cell death by directly phosphorylating NMDA receptors

CA1 pyramidal neurons degenerate after transient forebrain ischemia, whereas neurons in other regions of the hippocampus remain intact. Here we show that in rat hippocampal CA1 neurons, forebrain ischemia induces the phosphorylation of the N-methyl-D-aspartate (NMDA) receptor 2A subunit at Ser1232 (phospho-Ser1232). Ser1232 phosphorylation is catalyzed by cyclin-dependent kinase 5 (Cdk5). Inhibiting endogenous Cdk5, or perturbing interactions between Cdk5 and NR2A subunits, abolished NR2A phosphorylation at Ser1232 and protected CA1 pyramidal neurons from ischemic insult. Thus, we conclude that modulation of NMDA receptors by Cdk5 is the primary intracellular event underlying the ischemic injury of CA1 pyramidal neurons.     

Changes of pyridoxal kinase expression and activity in the gerbil hippocampus following transient forebrain ischemia

In the previous study, we observed chronological alterations of glutamic acid decarboxylase (GAD), which is the enzyme converting glutamate into GABA. GAD isoforms (GAD65 and GAD67) differ substantially in their interactions with cofactor pyridoxal 5'-phosphate, which is catalyzed by pyridoxal kinase (PLK). In the present study, we examined the chronological changes of PLK expression and activity in the hippocampus after 5 min transient forebrain ischemia in gerbils. PLK immunoreactivity in the sham-operated group was detected weakly in the hippocampus. Ischemia-related change of PLK immunoreactivity in the hippocampus was significant in the hippocampal cornu ammonis (CA1)region, not in the hippocampal CA2/3 region and dentate gyrus. PLK immunoreactivity was observed in non-pyramidal GABAergic neurons at 30 min to 3 h after ischemic insult. At 12 h after ischemic insult, PLK immunoreactivity was shown in many CA1 pyramidal cells as well as some non-pyramidal cells. At this time point, PLK immunoreactivity and protein content was highest after ischemia. Thereafter, PLK immunoreactivity and protein content is decreased time-dependently by 4 days after ischemic insult. Four days after ischemia, some astrocytes expressed PLK in the CA1 region. The specific PLK activity was not altered following ischemic insult up to 2 days after ischemic insult. Thereafter, the specific PLK activity decreased time-dependently. However, total activity of PLK was significantly increased 12-24 h after ischemic insult, and thereafter total activity of PLK decreased. Therefore, we suggest that the over-expression of PLK in the CA1 pyramidal cells at 12 h after ischemia may induce increase of GAD in the CA1 pyramidal cells, which plays an important role in delayed neuronal death via the increase of GABA or enhancement of GABA shunt pathway.     

Alteration in the immunoreactivity of the calcineurin subunits after ischemic hippocampal damage.

Dephosphorylation processes of target proteins are critical to the reversible regulation of intracellular signal transduction systems. Further, brain damage such as ischemic insult induces marked changes in protein kinase activity. To study these changes more thoroughly, specific monoclonal antibodies of the A and B subunits of calcineurin (protein phosphatase 2B) were raised, and regional alterations in the immunoreactivity of calcineurin in the rat hippocampus were investigated after a transient forebrain ischemic insult causing selective and delayed hippocampal CA1 pyramidal cell damage. In normal rats it was found that both the calcineurin A and the B subunits showed high immunoreactivity in the dendritic fields of the hippocampal formation. The immunoreactivity of subunit A in the strata oriens, the radiatum of the CA1 subfield and in the stratum lucidum of the CA3 subfield was most intense, whereas the immunoreactivity in the other CA3 subfields and in the dentate gyrus was relatively low. In contrast, the dendritic fields of the hippocampal formation were equally immunoreactive to calcineurin subunit B, although the stratum lucidum of the CA3, where the mossy fibers from the dentate granule cells terminate, showed a very high immunoreactivity of the B subunit. After transient forebrain ischemia in the CA1 subfield, where selective pyramidal cell death occurred two days after this ischemia, a marked loss of immunoreactivity in both subunits was observed, along with morphological pyramidal cell damage. A recovery of the immunoreactivity of A and B subunits in the strata oriens and radiatum was later noted 30 days after ischemia. In the stratum lucidum of the CA3, the immunoreactivity of both the A and B subunits was transiently depressed from 6 to 24 h, followed by a marked immunoreactivity enhancement from four to 30 days after ischemia. Further, in the histologically intact dentate gyrus, both the immunoreactivity of the A and B subunits in the molecular layer were transiently enhanced from four to 14 days after ischemia, particularly in the supragranular layer. The results clearly indicate that the protein dephosphorylation systems were markedly altered in the whole hippocampal formation during the recirculation period following ischemia. Further, the transient depression in the calcineurin immunoreactivity seen in the mossy fiber terminals may reflect modulated synaptic activity of the dentate granule cells, which may play a pivotal role in the delayed and selective death of the CA1 pyramidal cells. Thus, calcineurin appears to be an excellent marker enzyme for the detection of neuronal activity and synaptic plasticity after brain damage, such as an ischemic insult.     

Increased intracellular Ca2+ concentration in the hippocampal CA1 area during global ischemia and reperfusion in the rat: a possible cause of delayed neuronal death

The crucial role of free cytosolic Ca2+ in ischemic neuronal damage has been studied in recent years. In the present report, changes in the intracellular Ca2+ concentration in the hippocampal CA1 area during transient global ischemia and reperfusion were measured using in vivo Ca2+ fluorometry with fura-2 in the four-vessel occlusion and reperfusion model in halothane-anesthetized rats. Marked changes were seen during 10-min global ischemia, with the intracellular Ca2+ concentration increasing gradually following application of the ischemic insult and rapidly about 2 min after the beginning of ischemia, and continuing to increase until reperfusion. On reperfusion, the intracellular Ca2+ concentration began to decrease and returned to the pre-ischemic level within 15 min. Induction of severe global ischemia was confirmed by the complete suppression of synaptic activity and the decrease in hippocampal temperature in the CA1 area. After seven days, CA1 pyramidal cell loss was observed histopathologically in the same rats which had undergone measurement of the intracellular Ca2+ concentration changes. In the present study, a temporal profile of the free cytosolic Ca2+ dynamics during ischemic and early post-ischemic period was determined in vivo. The results demonstrate that the intracellular Ca2+ concentration in the hippocampal CA1 area is transiently and markedly increased during a brief ischemia-inducing delayed neuronal death, implying that Ca2+ overload during cerebral ischemia is a possible cause of the delayed cell death of CA1 pyramidal neurons.     

Selective translocation of diacylglycerol kinase zeta in hippocampal neurons under transient forebrain ischemia

The molecular mechanisms responsible for differential neuronal vulnerability to ischemic injury are incompletely understood. Previous studies have reported that the expression and activity of protein kinase C (PKC), some subtypes of which are activated by Ca(2+) and diacylglycerol (DG), are altered after ischemic insults. Therefore, DG kinase (DGK), which is responsible for controlling PKC activity through DG metabolism, may also be involved in this process. DGKzeta, which is abundantly expressed in the brain, contains a nuclear localization signal (NLS), suggesting its involvement in some nuclear processes in neuronal cells. To elucidate the functional implications of DGKzeta in ischemia, we examined detailed localization of DGKzeta in rat brain after ischemic insults. We used an ischemic model of global cerebral ischemia for 20 min by bilateral common carotid artery occlusion combined with hypotension and followed time-points of reperfusion. DGKzeta expression was evaluated by immunohistochemistry using affinity-purified anti-DGKzeta antibody. In sham-operated rats, a strong DGKzeta-immunoreactivity was observed in the nucleus of neurons in various parts of the brain. In the global ischemic model DGKzeta-immunoreactivity was reduced in intensity in the hippocampal formation and detected in the cytoplasm of CA1 pyramidal neurons throughout reperfusion time courses. Change in the subcellular localization was restricted to the pyramidal cells in CA1 and later in CA3, but not observed in other areas of hippocampus. No change was observed in the cerebral and cerebellar cortices. The present study suggests that DGKzeta might be involved in the process of selective vulnerability of hippocampal pyramidal neurons in postischemic brain.     

Ca2+ signaling in gerbil CA3 hippocampal neurons following transient in vivo ischemia

Using the gerbil model of post-ischemic neuron death in the hippocampal CA1 region, it was recently shown that there is a strong down-regulation of voltage-gated Ca2+ influx in neurons examined at 2 days after the ischemic insult (Connor, J.A., Razani-Boroujerdi, S., Greenwood, A.C., Cormier, R.J., Petrozzino, J.J. and Lin, R.C., Reduced voltage-dependent Ca2+ signaling in CA1 neurons after brief ischemia in gerbils, J. Neurophysiol., 81 (1999) 299-306). The aim of the present study was to determine whether a similar change occurs in pyramidal neurons of the CA3 region that are relatively resistant to transient ischemia. In vitro intracellular recordings and fluorometric Ca2+ measurements were made from CA3 neurons in coronal slices prepared from controls and 1 or 2 days following in vivo ischemia. In slices from control and post-ischemic animals, the electrophysiological properties of CA3 neurons were consistent with significant voltage-gated Ca2+ influx, leading to spike frequency adaptation. Quantitative results indicated no significant difference in Ca2+ transients evoked by action potential trains. This Ca2+ signaling was compared with responses in CA1 neurons from the same preparations, which showed substantially diminished Ca2+ influx at 2 days post-ischemia. These findings suggest that diminished Ca2+-signaling is not a general feature of pyramidal neurons following ischemia, but is characteristic of neurons destined to die.     

The tumor suppressor p53 and its response gene p21WAF1/Cip1 are not markers of neuronal death following transient global cerebral ischemia

The tumor suppressor protein p53 is implicated in cell cycle arrest and DNA repair as well as in apoptosis. In the CNS, p53 has been associated with neuronal cell death following various insults, including cerebral ischemia. We investigated the expression of p53 messenger RNA and protein, and the messenger RNA expression of the p53-responsive gene p21(WAF1/CiP1, in specific hippocampal regions following 15 min of normothermic and neuroprotective hypothermic (33 degrees C) global forebrain ischemia in the rat. Both p53 and p21WAF1/Cip1 messenger RNAs were transiently induced in ischemia resistant regions following normo- and hypothermic ischemia. In the ischemia sensitive CA1 region, p53 and p21WAF1/Cip1 messenger RNAs were up-regulated throughout reperfusion following the normothermic insult. The p53 protein levels increased following the insult, most markedly in ischemia-resistant CA3 neurons after normothermic ischemia, and in the CA1 neurons following hypothermic ischemia. Concomitantly, the protein was translocated to nuclei. These findings indicate that p53 and p21WAF1/Cip1 are not markers of neuronal death following global cerebral ischemia. Their rapid and transient induction correlates with cell survival, and suggests a possible role in DNA repair.     

Convergence of stress granules and protein aggregates in hippocampal cornu ammonis 1 at later reperfusion following global brain ischemia

The delayed and selective vulnerability of post-ischemic hippocampal cornu ammonis (CA) 1 pyramidal neurons correlates with a lack of recovery of normal protein synthesis. Recent evidence implicates sequestration of translational machinery into protein aggregates and stress granules as factors underlying persistent translation arrest in CA1 neurons. However, the relationship between protein aggregates and stress granules during brain reperfusion is unknown. Here we investigated the colocalization of protein aggregates and stress granules using immunofluorescence microscopy and pair-wise double labeling for ubiquitin/T cell internal antigen (TIA-1), ubiquitin/small ribosomal subunit protein 6 (S6), and TIA-1/S6. We evaluated the rat dorsal hippocampus at 1, 2 or 3 days of reperfusion following a 10 min global brain ischemic insult. At 1 day of reperfusion, ubiquitin-containing aggregates (ubi-protein clusters) occurred in neurons but did not colocalize with stress granules. At 2 days' reperfusion, only in CA1, cytoplasmic protein aggregates colocalized with stress granules, and ubiquitin-containing inclusions accumulated in the nuclei of CA1 pyramidal neurons. Functionally, a convergence of stress granules and protein aggregates would be expected to sustain translation arrest and inhibit clearance of ubiquitinated proteins, both factors expected to contribute to CA1 pyramidal neuron vulnerability.     

Protein disulfide isomerase immunoreactivity and protein level changes in neurons and astrocytes in the gerbil hippocampal CA1 region following transient ischemia

We investigated the temporal and spatial alterations of protein disulfide isomerase (PDI) immunoreactivity and protein level in the hippocampus proper after 5 min transient forebrain ischemia in gerbils. PDI immunoreactivity was significantly altered in the hippocampal CA1 region. PDI immunoreactivity in the sham-operated animals was found in non-pyramidal cells. At 30 min after ischemia, PDI immunoreactivity was shown in the pyramidal cells of the stratum pyramidale (SP): the PDI immunoreactivity in the pyramidal cells was increased up to 12 h after ischemia. Thereafter PDI immunoreactivity was decreased, and the PDI immunoreactivity was shown in non-pyramidal cells 2 days after ischemia. Four to 5 days after ischemia, almost pyramidal cells in the CA1 region were lost because the delayed neuronal death occurred. At this time period, PDI immunoreactivity was expressed in some astrocytes as well as some neurons. The results of the Western blot analysis were consistent with the immunohistochemical data. These findings suggest that increase of PDI in pyramidal cells may play a critical role in resistance to ischemic damage at early time after ischemic insult, and that expression of this protein in astrocytes at late time after ischemic insult is partly implicated in the acquisition of tolerance against ischemic stress.     

Permanent reduction of seizure threshold in post-ischemic CA3 pyramidal neurons

The effects of ischemia were examined on CA3 pyramidal neurons recorded in hippocampal slices 2-4 mo after a global forebrain insult. With intracellular recordings, CA3 post-ischemic neurons had a more depolarized resting membrane potential but no change of the input resistance, spike threshold and amplitude, fast and slow afterhyperpolarization (AHP) or ADP, and firing properties in response to depolarizing pulses. With both field and whole-cell recordings, synaptic responses were similar in control and post-ischemic neurons. Although there were no spontaneous network-driven discharges, the post-ischemic synaptic network had a smaller threshold to generate evoked and spontaneous synchronized burst discharges. Thus lower concentrations of convulsive agents (kainate, high K(+)) triggered all-or-none network-driven synaptic events in post-ischemic neurons more readily than in control ones. Also, paired-pulse protocol generates, in post-ischemics but not controls, synchronized field burst discharges when interpulse intervals ranged from 60 to 100 ms. In conclusion, 2-4 mo after the insult, the post-ischemic CA3 pyramidal cells are permanently depolarized and have a reduced threshold to generate synchronized bursts. This may explain some neuropathological and behavioral consequences of ischemia as epileptic syndromes observed several months to several years after the ischemic insult.     

Sevoflurane immediate preconditioning alters hypoxic membrane potential changes in rat hippocampal slices and improves recovery of CA1 pyramidal cells after hypoxia and global cerebral ischemia

Pretreatment with anesthetics before but not during hypoxia or ischemia can improve neuronal recovery after the insult. Sevoflurane, a volatile anesthetic agent, improved neuronal recovery subsequent to 10 min of global cerebral ischemia when it was present for 1 h before the ischemia. The mean number of intact hippocampal cornus ammonis 1 (CA1) pyramidal neurons in rats subjected to cerebral ischemia without any pretreatment was 17+/-5 (neurons/mm+/-S.D.) 6 weeks after the ischemia; na?ve, non-ischemic rats had 177+/-5 neurons/mm. Rats pretreated with either 2% or 4% sevoflurane had 112+/-57 or 150+/-15 CA1 pyramidal neurons/mm respectively (P<0.01) 6 weeks after global cerebral ischemia. In order to examine the mechanisms of protection we used hypoxia to generate energy deprivation. Intracellular recordings were made from CA1 pyramidal neurons in rat hippocampal slices; the recovery of resting and action potentials after hypoxia was used as an indicator of neuronal survival. Pretreatment with 4% sevoflurane for 15 min improved neuronal recovery 1 h after the hypoxia; 90% of the sevoflurane-pretreated neurons recovered while none (0%) of the untreated neurons recovered. Pretreatment with sevoflurane enhanced the hypoxic hyperpolarization(-6.4+/-0.6 vs. -3.3+/-0.3 mV) and reduced the final level of the hypoxic depolarization (-39+/-6 vs. -0.3+/-2 mV) during hypoxia. Chelerythrine (5 muM), a protein kinase C/protein kinase M inhibitor, blocked both the improved recovery (10%) and the electrophysiological changes with 4% sevoflurane preconditioning. Two percent sevoflurane for 15 min before hypoxia did not improve recovery (0% recovery both groups) and did not enhance the hypoxic hyperpolarization or reduce the final depolarization during hypoxia. However if 2% sevoflurane was present for 1 h before the hypoxia then there was significantly improved recovery, enhanced hypoxic hyperpolarization, and reduced final depolarization. Thus we conclude that sevoflurane preconditioning improves recovery in both in vivo and in vitro models of energy deprivation and that preconditioning enhances the hypoxic hyperpolarization and reduces the hypoxic depolarization. Anesthetic preconditioning may protect neurons from ischemia by altering the electrophysiological changes a neuron undergoes during energy deprivation.     

Increase of fragmented DNA transport in apical dendrites of gerbil CA1 pyramidal neurons following transient forebrain ischemia by mild hypothermia

Mild hypothermia (38 degrees C) accelerated transport of fragmented DNA in apical dendrites of the gerbil CA1 pyramidal neurons and increased dendrite-terminal fragmented DNA pooling in the apoptotic process following transient forebrain ischemia. The specific DNA fragmentation after the ischemic insult in gerbil hippocampus was examined by in situ nick-end-labeling method, and fluorescence DNA detection technique by DAPI was also performed. There is a precise temperature dependence for the migration of fragmented DNA from nuclei into apical dendrites of CA1 pyramidal cells during apoptosis following transient forebrain ischemia. Increase of fragmented DNA pooling is highly temperature sensitive, occurring at 38 degrees C, while at 39 degrees C there is a marked decrease in DNA pooling.     

Impairment of protein ubiquitination may cause delayed neuronal death

The hippocampus is a brain structure specifically vulnerable to short periods of transient cerebral ischemia, and which displays delayed neuronal necrosis. Protein ubiquitination is a posttranslational modification of proteins and an important factor in heat shock response and a regulator of ATP-dependent protein degradation. Using affinity purified antibodies against ubiquitin and ubiquitin-protein conjugates we have found that the ubiquitin immunoreactivity (UIR), normally present in all neurons of the hippocampus, disappears in the early recirculation period following cerebral ischemia from all hippocampal cells except the interneurons. Later UIR reappears in the different hippocampal regions over a 72 h period in the following order: granule cells-CA3 pyramidal cells-CA2 pyramidal cells. This is the inverse order of sensitivity of these cells to ischemia. The UIR never recovers in the CA1 pyramidal neurons where a 95% neuronal necrosis is seen following three days of recovery. We propose that the loss of UIR in the pyramidal neurons in the CA1 region signifies a persistent impairment of protein ubiquitination, and thus a change in the turnover of structural and regulatory proteins, which could be an essential part of the mechanism of slow neuronal death following cerebral ischemia.     

Neuroprotective mechanisms of lidocaine against in vitro ischemic insult of the rat hippocampal CA1 pyramidal neurons

To compare neuroprotective effects of lidocaine and procaine against ischemic insult, intracellular recordings were made from rat hippocampal CA1 pyramidal neurons in slice preparations. Superfusion of the slices with oxygen- and glucose-deprived medium (in vitro ischemia) produced a rapid depolarization 6 min from the onset. When oxygen and glucose were reintroduced, the membrane depolarized further until it reached 0 mV, and thereafter the membrane showed no functional recovery. Pretreatment with lidocaine (10 microM), but not procaine (50 microM), restored the membrane potential after the reintroduction of oxygen and glucose. Lidocaine, compared to procaine, significantly inhibited the reduction in both tissue ATP content and flavoprotein fluorescence during and after in vitro ischemia. Under electron microscopy, only lidocaine well preserved the structure of mitochondria in the CA1 pyramidal cell body. Extracellular recordings revealed that procaine reduced the field postsynaptic potential whereas lidocaine augmented it. Both drugs reduced the presynaptic volley dose-dependently. Neither lidocaine nor procaine significantly affected a rapid rise of the intracellular Ca2+ level produced by in vitro ischemia in the CA1 region. All the results suggest that the neuroprotective lidocaine action is due to the protection of the mitochondria to maintain the tissue ATP content during and after in vitro ischemia.     

2-脱氧葡萄糖诱导的内质网应激预处理对大鼠脑缺血再灌注损伤的保护作用

目的研究2-脱氧葡萄糖(2-DG)诱导内质网应激(ERS)预处理对大鼠脑缺血再灌注的保护作用。方法 64只雄性SD大鼠随机均分为假手术组(SH组)、缺血/再灌注组(I/R组)、2-DG诱导的ERS预处理组(IP组)、IP+I/R组。采用TUNEL法检测CA1区凋亡细胞,免疫组化法、Western-Blot法检测p-JNK蛋白在海马CA1区的表达变化。结果与对照组比较,I/R组海马CA1区锥体神经元排列紊乱及变性坏死,形态正常椎体细胞百分数减少,凋亡细胞数目明显增加,脑组织p-JNK表达水平增加;与I/R组相比,IP+I/R组形态正常锥体细胞明显增加,凋亡细胞数目明显减少,脑组织表达较I/R组明显减少。结论 2-DG诱导的ERS对脑缺血再灌注损伤有保护作用,可能与其抑制细胞凋亡和p-JNK表达相关。     

Prolonged translation arrest in reperfused hippocampal cornu Ammonis 1 is mediated by stress granules

Global brain ischemia and reperfusion cause phosphorylation of the alpha subunit of eukaryotic initiation factor 2alpha, a reversible event associated with neuronal translation inhibition. However, the selective vulnerability of cornu Ammonis (CA) 1 pyramidal neurons correlates with irreversible translation inhibition. Phosphorylation of eukaryotic initiation factor 2alpha also leads to the formation of stress granules, cytoplasmic foci containing, in part, components of the 48S pre-initiation complex and the RNA binding protein T cell internal antigen-1 (TIA-1). Stress granules are sites of translationally inactive protein synthesis machinery. Here we evaluated stress granules in rat hippocampal formation neurons after 10 min global brain ischemia and 10 min, 90 min or 4 h of reperfusion by double-labeling immunofluorescence for two stress granule components: small ribosomal subunit protein 6 and TIA-1. Stress granules in CA3, hilus and dentate gyrus, but not CA1, increased at 10 min reperfusion and returned to control levels by 90 min reperfusion. Dynamic changes in the nuclear distribution of TIA-1 occurred in resistant neurons. At 4 h reperfusion, small ribosomal subunit protein 6 was solely localized within stress granules only in CA1 pyramidal neurons. Both TIA-1 and small ribosomal subunit protein 6 levels decreased approximately 50% in hippocampus homogenates. Electron microscopy showed stress granules to be composed of electron dense bodies 100-200 nm in diameter, that were not membrane bound, but were associated with endoplasmic reticulum. Alterations in stress granule behavior in CA1 pyramidal neurons provide a definitive mechanism for the continued inhibition of protein synthesis in reperfused CA1 pyramidal neurons following dephosphorylation of eukaryotic initiation factor 2alpha.     

Apamin-sensitive conductance mediates the K(+) current response during chemical ischemia in CA3 pyramidal cells

Pyramidal cells typically respond to ischemia with initial transient hyperpolarization, which may represent a neuroprotective response. To identify the conductance underlying this hyperpolarization in CA3 pyramidal neurons of rat hippocampal organotypic slice cultures, recordings were obtained using the single-electrode voltage-clamp technique. Brief chemical ischemia (2 mM 2-deoxyglucose and 3 mM NaN(3), for 4 min) induced a response mediated by an increase in K(+) conductance. This current was blocked by intracellular application of the Ca(2+) chelator, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA), reduced with low external [Ca(2+)], and inhibited by a selective L-type Ca(2+) channel inhibitor, isradipine, consistent with the activation of a Ca(2+)-dependent K(+) conductance. Experiments with charybdotoxin (10 nM) and tetraethylammonium (TEA; 1 mM), or with the protein kinase C activator, phorbol 12,13-diacetate (PDAc; 3 microM), ruled out an involvement of a large conductance-type or an apamin-insensitive small conductance, respectively. In the presence of apamin (1 microM), however, the outward current was significantly reduced. These results demonstrate that in rat hippocampal CA3 pyramidal neurons an apamin-sensitive Ca(2+)-dependent K(+) conductance is activated in response to brief ischemia generating a pronounced outward current.     

Protective actions of various local anesthetics against the membrane dysfunction produced by in vitro ischemia in rat hippocampal CA1 neurons

Local anesthetics block not only the Na(+) but also the K(+) and Ca(2+) channels in the mammalian neurons. It is well known that lidocaine has neuroprotective actions against the ischemic insult of neurons in the central nervous system. In order to elucidate how other local anesthetics as well as lidocaine show the neuroprotective effects against in vitro ischemic insult, intracellular recordings were made from CA1 pyramidal neurons in rat hippocampal slices. Superfusion with the medium deprived of oxygen and glucose (in vitro ischemia) produced a rapid depolarization after 5 min of exposure. When the normal medium was immediately reintroduced after the rapid depolarization, the membrane depolarized further (persistent depolarization), the neurons showed no functional recovery. Pretreatment with tetracaine, bupivacaine, procaine, lidocaine, mepivacaine, or dibucaine (10 or 300 microM) prolonged the latency of the rapid depolarization, and most of the drugs partially restored the membrane potential toward the pre-exposure level after the reintroduction. Judging from the neuroprotective actions such as the prolongation and the potential recovery by these drugs, lidocaine, bupivacaine, and dibucaine are candidates for the therapeutic use against the ischemic insult. Suppression of the regenerative Na(+) conductance is somehow involved in the neuroprotective actions of local anesthetics.     

Regional and cellular distribution of neural visinin-like protein immunoreactivities (VILIP-1 and VILIP-3) in human brain

Neural visinin-like proteins (VILIPs) are members of the neuronal subfamily of intracellular EF-hand calcium sensor proteins termed the NCS family, which are thought to play important roles in cellular signal transduction. While numerous studies suggest a wide but uneven distribution of these proteins in rat and chicken brain, their location in, and possible significance for, the human brain, remains to be established. We used specific polyclonal antisera to map the human brain for VILIP-1 and VILIP-3 immunoreactivities. VILIP-1 was detected in cortical pyramidal cells and interneurons, septal, subthalamic and hippocampal neurons (subfields CA1 and CA4 pyramidal cells and especially hilar interneurons) as well as in cerebellar Golgi, basket, granule, stellate and dentate nucleus neurons. Purkinje cells were free of immunoreaction. VILIP-3 was more restricted in its distribution. It was identified in cerebellar Purkinje cells and a subpopulation of granule neurons. Further, neurons belonging to different nuclei of the brain stem and multiple subcortical nerve cells stained for visinin-like protein 3. A weak immunoreaction appeared in cortical and hippocampal neurons. Intracellularly the immunoreactivity appeared in the perikarya, dendrites and some axons. Sometimes, immunostaining was found in the neuropil. Glia did not express visinin-like proteins. Our findings support, from a neuroanatomical viewpoint, the idea that these calcium sensor proteins may be of relevance for neuronal signalling in the human CNS.     

Expression and function of A1 adenosine receptors in the rat hippocampus following transient forebrain ischemia

We investigated how transient cerebral ischemia affects the gene expression, immunoreactive protein levels, and the function of the A1 subtype of adenosine receptor in the rat hippocampus at different times following reperfusion. A1 receptor mRNA levels were altered significantly in different hippocampal subfields as early as 6 h following insult. However, these changes in mRNA levels were not paralleled at the protein level, as western blotting with A1 receptor-specific antibodies revealed that hippocampal A1 adenosine receptor prevalence did not differ from sham control at either 6 or 24 h following insult. The lack of change in A1 receptor prevalence was consistent with functional examinations, as only marginal changes were observed in the ability of A1 receptors to attenuate excitatory post-synaptic potentials in the CA1 subfield at 24 h following reperfusion. These data illustrate that although the mRNA expression levels of the A1 adenosine receptor are altered by transient cerebral ischemia, the immunoreactive prevalence and function of this receptor are maintained in the post-ischemic hippocampus at times preceding the death of the vulnerable neurons.