Histochemical study of Ca2+-ATPase activity in ischemic CA1 pyramidal neurons in the gerbil hippocampus

Although cytosolic Ca²⁺ accumulation plays a pivotal role in delayed neuronal death, there have been no investigations on the role of the cellular Ca²⁺ export system in this novel phenomenon. To clarify the function of the Ca²⁺-pump in delayed neuronal death, the plasma membrane Ca²⁺-ATPase activity of CA1 pyramidal neurons was investigated ultracytochemically in normal and ischemic gerbil hippocampus. To correlate enzyme activity with delayed neuronal death, histochemical detection was performed at various recirculation times after 5 min of ischemia produced by occlusion of the bilateral carotid arteries. At 10 min after ischemia, CA1 pyramidal neurons showed weak Ca²⁺-ATPase activity. Although enzyme activity had almost fully recovered 2 h after ischemia, it was reduced again 6 h after ischemia. Thereafter, Ca²⁺-ATPase activity on the plasma membrance of CA1 pyramidal neurons decreased progressively, losing its localization on day 3. On day 4 following ischemia, reaction products were diffusely scattered throughout the whole cell body. Our results indicate that, after once having recovered from ischemic damage, severe disturbance of the membrane Ca²⁺ export system proceeds from the early stage of delayed neuronal death and disturbs the re-export of accumulated cytosolic Ca²⁺, which might contribute to delayed neuronal death. Occult disruption of Ca²⁺ homeostasis seems to occur from an extremely early stage of delayed neuronal death in CA1 pyramidal cells.     

A serotonin S2 antagonist, naftidrofuryl, exhibited a protective effect on ischemic neuronal damage in the gerbil

The effect of a serotonin S₂ antagonist, naftidrofuryl, on ischemic neuronal damage was examined in the gerbil. Naftidrofuryl was injected i.p. 5 min prior to a single 5-min forebrain ischemia or immediately after each of three 2-min forebrain ischemic insults at 60-min intervals. In both groups the number of intact hippocampal CA1 neurons were significantly higher than in the saline-treated group. These results indicate that serotonin S₂ antagonists have a protective effect against ischemic neuronal damage.     

NMDA-evoked consumption and recovery of mitochondrially targeted aequorin suggests increased Ca2+ uptake by a subset of mitochondria in hippocampal neurons

Activation of NMDA receptors produces large increases in cytosolic Ca(2+) that are taken up into mitochondria. We used recombinant aequorin targeted to mitochondria to report changes in matrix Ca(2+) in rat hippocampal neurons in culture. Upon binding Ca(2+), aequorin emits a photon in a one-shot reaction that consumes the indicator. Here we show that stimulation with NMDA produced a mitochondrial Ca(2+) response that rapidly inactivated. However, following a 30-min recovery period the response was restored, suggesting the presence of a pool of indicator that was not exposed to high Ca(2+) during the initial stimulus. We speculate that aequorin distant from the Ca(2+) source was protected from microdomains of high Ca(2+) near the plasmalemma and that this aequorin moved, either by movement of individual mitochondria or via the mitochondrial tubular network, to replenish consumed indicator during the recovery time. A large Ca(2+) increase in a subset of mitochondria could produce local changes in energy metabolism, regional Ca(2+) buffering, and foci that initiate neurotoxic processes.     

Removal of the entorhinal cortex protects hippocampal CA-1 neurons from ischemic damage

Summary The excitatory (glutamatergic) innervation seems to determine a nerve cells vulnerability to complete, transient ischemia. Interruption of the excitatory afferents to the hippocampus by removal of the entorhinal cortex prior to ischemia allows examination of this hypothesis. Groups of adult male Wistar rats were subjected to 20 min of ischemia (fourvessel occlusion) 4 days following a sham procedure, unilateral or bilateral entorhinotomy. CA-1 pyramidal cell survival following ischemia was assessed by light microscopic examination (cell counts) 4 days after ischemia. Compared to control animals unilateral entorhinotomy protected 50% of the CA-1 pyramidal neurons ipsilateral to the lesion, whereas bilateral entorhinotomy resulted in 84% protection. The pathophysiology of ischemic brain damage is discussed, and it is suggested that the protection of CA-1 pyramidal neurons after entorhinotomy is due to interruption of the input to the dentate granule cells, which forms a link in the trisynaptic pathway from the entorhinal cortex to the CA-1.Supported by the NOVO foundation and by The Danish Medical Research Council grant no. 12-5285 and no. 12-5704     

Histamine modulates reactivity of hippocampal CA3 neurons to afferent stimulation in vitro

Intracellular activity was recorded from hippocampal CA3 pyramidal cells maintained in vitro. Histamine (HA) produced a slow depolarization associated with minimal conductance changes. In addition, there was an increase in action potential discharge rates and the emergence of bursting firing patterns. EPSP size increased by about 50% and spontaneous dendritic spikes were observed. These effects were markedly reduced by retrodotoxin. Extracellular recording of population spikes revealed a marked difference between CA1 and CA3 regions; in the former HA produced an increase in population spike size whereas in the latter this increase was larger and was associated with the appearance of secondary and tertiary population spikes. It is suggested that HA produces its effects by enhancing release of neurotransmitters from excitatory synapses on the recorded neurons.     

Re-evaluation of ischemia-induced neuronal damage in hippocampal regions in the normothermic gerbil

Summary It has not been discussed whether transient forebrain ischemia of 5-min duration, which is a model frequently used to evaluate pharmacological protection against ischemic injury, is an optimal model in the CA1 field of this animal whose brain temperature is maintained at normothermic levels. The temperature of the brain during an ischemic insult strongly affects the extent of the resulting neuronal injury. If the brain temperature is not regulated, it usually falls in the gerbil by 2°–4°C during 5-min ischemia. However, the brain temperature during ischemic insult was not regulated in many previous studies. In the present study, the effects of transient (1 to 5 min) forebrain ischemia on the development of neuronal degeneration in hippocampal regions of the gerbil whose brain temperature was maintained at 37°C were examined. In the CA1 field of the hippocampus, transient ischemia of 3- and 4-min duration caused almost the same maximal damage (88%–91% neuronal loss) as observed in the gerbils subjected to 5-min ischemia. Transient ischemia of 2-and 2.5-min duration provoked substantial neuronal damage in 25% and 55% of experimental gerbils, respectively. These results indicate that 5-min bilateral forebrain ischemia is more than is necessary to examine ischemiainduced neuronal degeneration in hippocampal CA1 field of the gerbil whose brain temperature is maintained at normothermic levels. In the normothermic gerbil brain, an ischemic period of 3-min already induces extensive neuronal death in the CA1 and, thus, constitutes a sensitive model to evaluate faint protective effects of drugs against ischemic injury in the normothermic gerbil.Supported by Grant-in-Aid for Encouragement of Young Scientist (03857019) from the Ministry of Education, Science and Culture of Japan and the Sasakawa Health Science Foundation to A.M., and Grants-in-Aid for General Scientific Research (01400004 and 03557007) from the Ministry of Education, Science and Culture of Japan, Japan Foundation for Aging and Health and Mitsui Life Social Welfare Foundation to K.K.     

Nanomolar concentrations of lead inhibit glutamatergic and GABAergic transmission in hippocampal neurons

To investigate whether lead (Pb2+) affects the tetrodotoxin (TTX)-sensitive release of neurotransmitters, the whole-cell mode of the patch-clamp technique was applied to cultured hippocampal neurons. Pb2+ (>/=10 nM) reversibly blocked the TTX-sensitive release of glutamate and gamma-aminobutyric acid (GABA), as evidenced by the reduction of the amplitude and frequency of glutamate- and GABA-mediated postsynaptic currents (PSCs) evoked by spontaneous neuronal firing. This effect of Pb2+, which occurred 2-3 s after exposure of the neurons to Pb2+-containing external solution, was not related to changes in Na+-channel activity, and was quantified by measurements of changes in the amplitude of PSCs evoked when a 50-micros, 5-V stimulus was applied via a bipolar electrode to a neuron synaptically connected to the neuron under study. With an IC50 of approximately 68 nM, Pb2+ blocked the evoked release of glutamate and GABA. This effect was most likely mediated by Pb2+'s actions on extracellular targets, because there was a very short delay (<3 s) for its onset, and it could be completely reversed by the chelator ethylene diaminetetraacetic acid (EDTA). Given that Pb2+-induced blockade of evoked transmitter release could be reversed by 4-aminopyridine, it is suggested that the effect on release was mediated via the binding of Pb2+ to voltage-gated Ca2+ channels. Thus, it is most likely that the neurotoxic effects of Pb2+ in the mammalian brain involve a decrease of the TTX-sensitive, Ca2+-dependent release of neurotransmitters.     

Elevation of Na+-K+ ATPase immunoreactivity in GABAergic neurons in gerbil CA1 region following transient forebrain ischemia

In a previous study, we suggested that GABAergic neurons might be resistant to ischemic insult, because of the maintenance of the GABA shunt, which is one of the ATP synthetic pathways in neurons. In the present study, we identified Na(+)-K(+) ATPase immunoreactivity in the gerbil hippocampus in order to determine whether changes in Na(+)-K(+) ATPase immunoreactivity correlate with GABA shunt following ischemic insult. At 12 h after ischemia-reperfusion, Na(+)-K(+) ATPase immunoreactivity accumulated in some neurons in the CA1 region. However, the protein content of Na(+)-K(+) ATPase was not altered. Interestingly, the density of Na(+)-K(+) ATPase immunoreactivity in neurons and the protein content in the CA1 region was intensified in the 24 h post-ischemic group. As a result of double immunofluorescence study, Na(+)-K(+) ATPase immunoreactive neurons were identified with GABAergic neurons. Therefore, our findings suggest that the increase of Na(+)-K(+) ATPase in GABAergic neurons may be able to explain the resistance of these cells to ischemic insult, and support our previous hypothesis that GABA may play an important role as a metabolite in the survival of GABAergic neurons after ischemic insult.     

Adenosine receptor antagonists cancelled the ischemic tolerance phenomenon in gerbil

Pretreatment of the brain with sublethal ischemia has been reported to induce neuronal resistance to otherwise lethal ischemia, a phenomenon designated as ischemic tolerance. The protective mechanisms of the phenomenon are not known yet, however, recent experimental data suggest the involvement of adenosine receptor activation in the acquisition of tolerance. In this study, the effect of theophylline, a non-selective adenosine receptor antagonist, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1 receptor antagonist, were investigated to ascertain if these drugs could cancel the protective effect of ischemic tolerance in the gerbil. DPCPX or theophylline was administered at 3 h after a short preconditioning ischemia, and 21 h later animals were subjected to lethal ischemia of 5 min duration. DPCPX at a dose of 1.0 mg/kg (i.p) and theophylline at a dose of 20 mg/kg (i.p) significantly reduced the protective effect of preconditioning in the CA1 hippocampal neurons. These findings suggest the involvement of adenosine receptor activation for the development of ischemic tolerance phenomenon.     

Occlusion of hippocampal electrical junctions by intracellular calcium injection

Low-molecular weight dyes such as Lucifer yellow or carboxyfluorescein have been used to investigate the electrical connectivity of neurons via gap junctions. The interpretation that such dye passage is mediated through intercellular channels has been controversial and difficult to corroborate with direct techniques in mammalian brain. We report here that elevated intracellular free Ca2+, a treatment shown to cause gap junction occlusion in other tissues, significantly blocks dye transfer between hippocampal cells. Furthermore, intracellular injection of FITC-dextran (which is too large to cross gap junctions) never resulted in multiple hippocampal cell fills. These data lend strong support to the argument that the extent of dye-coupling provides a good estimate of the number of intercellular communication channels, and raises the possibility that these channels may be physiologically modulated.     

Lead increases tetrodotoxin-insensitive spontaneous release of glutamate and GABA from hippocampal neurons

This study was aimed at investigating the effects of the environmental pollutant lead (Pb2+) on the tetrodotoxin (TTX)-insensitive release of neurotransmitters from hippocampal neurons. Evidence is provided that Pb2+ (>/=100 nM) increases the frequency of gamma-aminobutyric acid (GABA)- and glutamate-mediated miniature postsynaptic currents (MPSCs) recorded by means of the patch-clamp technique from cultured hippocampal neurons. Because Pb2+ changed neither the amplitude nor the decay-time constant of the MPSCs, Pb2+-induced changes in MPSC frequency are exclusively due to a presynaptic action of this heavy metal. Increase by Pb2+ of the action potential-independent release of GABA and glutamate was concentration dependent and was only partially reversible upon washing of the neurons with nominally Pb2+-free external solution. This effect was also Ca2+ independent and began approximately after 1-2-min exposure of the neurons to Pb2+. The latency for the onset of the Pb2+'s effect on the MPSC frequency and the inability of the chelator ethylenediaminetetraacetic acid (100 microM) to reverse the effect that remained after washing of the neurons with external solution suggested that Pb2+ acted via an intracellular mechanism. Of interest also was the finding that Pb2+ simultaneously increased the release of GABA and glutamate, overriding the ability of these neurotransmitters to decrease the release of one another. Given that synaptic activity is a key mechanism for the establishment of stable synaptic connections early in the development, it is possible that, by interfering with spontaneous transmitter release, Pb2+ has lasting effects on neuronal maturation and plasticity.     

Effect of nicardipine, a Ca channel blocker, on pyruvate dehydrogenase activity and energy metabolites during cerebral ischemia and reperfusion in gerbil brain

The purpose of this study was to determine if nicardipine, a calcium ion channel blocker, affects pyruvate dehydrogenase (PDH) activity and improves energy metabolism during cerebral ischemia and reperfusion. Cerebral ischemia was induced, using the bilateral carotid artery occlusion method, for 60 min followed by reperfusion up to 120 min in gerbils. Nicardipine (1 mg/kg) or saline (vehicle-treated) was given to gerbils 30 min prior to the occlusion of the common carotid arteries. PDH activity and metabolites (ATP, PCr, and lactate) were measured in cortex prior to ischemia, immediately following ischemia, and after each reperfusion period. After 60 min ischemia, PDH activity increased in both groups, and was significantly higher in the nicardipine-treated group. After 20 min reperfusion, PDH activity in the nicardipine-treated group recovered to control levels, whereas, the PDH activity in the vehicle-treated group remained elevated, and was higher than the nicardipine-treated animals. At 60 and 120 min reperfusion, the activities in the vehicle-treated group were significantly below control levels, there were no differences, however, between the two groups. ATP and PCr concentrations were markedly depleted immediately after ischemia in both groups. ATP levels at 20 min reperfusion and PCr levels at 60 min reperfusion were significantly higher in the nicardipine-treated group. Lactate concentrations in both groups increased 7–8 fold, similarly, immediately after ischemia. During reperfusion, the lactate remained elevated in both groups, though the levels in the nicardipine-treated group were lower than those in the vehicle-treated group, but not significantly. Nicardipine treatment normalized PDH activity quickly and improved energy metabolism after reperfusion.     

Histaminergic H(2) blockade facilitates ischemic release of dopamine in gerbil striatum

The blockade of central histaminergic H(2) receptors has been reported to aggravate ischemic neuronal damage. Since excess release of excitatory neurotransmitters is closely related to ischemic neuronal damage, the effects of ranitidine on ischemic release of dopamine were investigated in gerbil striatum. Changes in the extracellular concentration of dopamine produced by transient forebrain ischemia for 4 min were investigated by a microdialysis procedure, and the effect of intracerebroventricular administration of ranitidine (10 nmol) was evaluated. The histologic outcome was examined 7 days after ischemia by light microscopy. Forebrain ischemia produced a marked increase in the dopamine concentration in dialysates, and the level returned to the basal level after reperfusion. The preischemic administration of ranitidine enhanced the increase in the dopamine level during ischemia, and the peak value in the ranitidine group was 203% of that in the saline group. The histologic outcome was aggravated by the ranitidine treatment in the striatum, although aggravation was not observed in the cerebral cortex. The facilitation of the ischemic release of dopamine may be a contributing factor in the aggravation of ischemic damage by H(2) blockade.     

Late expression of Na+/H+ exchanger 1 (NHE1) and neuroprotective effects of NHE inhibitor in the gerbil hippocampal CA1 region induced by transient ischemia

Although acidosis may be involved in neuronal death, the participation of Na⁺/H⁺ exchanger (NHE) in delayed neuronal death in the hippocampal CA1 region induced by transient forebrain ischemia has not been well established. In the present study, we investigated the chronological alterations of NHE1 in the hippocampal CA1 region using a gerbil model after ischemia/reperfusion. In the sham-operated group, NHE1 immunoreactivity was weakly detected in the CA1 region. Two and 3 days after ischemia/reperfusion, NHE1 immunoreactivity was observed in glial components, not in neurons, in the CA1 region. Four days after ischemia/reperfusion, NHE1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as glial cells. These glial cells were identified as astrocytes based on double immunofluorescence staining. Western blot analysis also showed that NHE protein level in the CA1 region began to increase 2 days after ischemia/reperfusion. The treatment of 10 mg/kg 5-(N-ethyl-N-isopropyl) amiloride, a NHE inhibitor, significantly reduced the ischemia-induced hyperactivity 1day after ischemia/reperfusion. In addition, NHE inhibitor potently protected CA1 pyramidal neurons from ischemic damage, and NHE inhibitor attenuated the activation of astrocytes and microglia in the ischemic CA1 region. In addition, NHE inhibitor treatment blocked Na⁺/Ca²⁺ exchanger 1 immunoreactivity in the CA1 region after transient forebrain ischemia. These results suggest that NHE1 may play a role in the delayed death, and the treatment with NHE inhibitor protects neurons from ischemic damage.     

Effects of GABA on presumed presynaptic Ca entry in hippocampal slices

Evoked field potentials and changes in [Ca²⁺]_o were measured in the ‘in vitro’ hippocampal slice of the rat. When [Ca] in the perfusion medium was lowered to 0.2 mM synaptic transmission from Schaffer collateral/comissural fibers was blocked. Nevertheless, repetitive stimulation of afferent fibers still resulted in detectable decreases of [Ca²⁺]_o. In contrast to findings in normal medium these decreases in [Ca²⁺]_o could be larger in stratum radiatum than in stratum pyramidale, so mimicking the spatial distribution of activated afferent fibers. These findings suggest, that the loss of extracellular Ca²⁺ in low Ca²⁺ media is predominantly due to entry into presynaptic terminals. This permits to study effects of drugs on presynaptic endings. We found that iontophoretic application of GABA is capable to block this presumed presynaptic Ca²⁺ entry without affecting the electrical activity of the afferent fibers. This suggests, that presynaptic GABA receptors occur also in the Schaffer collateral/commissural fiber system.     

Neuronal damage following non-lethal but repeated cerebral ischemia in the gerbil

Summary Brief, non-lethal transient forebrain ischemia in the gerbil can injure selectively vulnerable neurons when such ischemia is induced repeatedly. The influence of the number and interval of the ischemic insults on neuronal damage, as well as the time course of damage, following repeated 2-min forebrain ischemia were examined. A single 2-min forebrain ischemia were examined. A single 2-min ischemic insult caused no morphological neuronal damage. A moderate number of hippocampal CA1 neurons were destroyed following two ischemic insults with a 1-h interval, and destruction of almost all CA1 neurons resulted from three or five insults at 1-h intervals. Three and five insults also resulted in moderate to severe damage to the striatum and thalamus, depending on the number of episodes. Although three ischemic insults at 1-h intervals caused severe neuronal damage, this number of insults at 5-min and 4-h intervals caused destruction of relatively few neurons, and non neurons were destroyed at 12-h intervals. Following three ischemic insults at 1-h intervals, damage to the striatum, neocortex, hippocampal CA4 subfield and thalamus was observed at 6–24 h of survival, whereas damage to the hippocampal CA1 subfield appeared at 2–4 days. The results indicate that even a brief non-lethal ischemic insult can produce severe neuronal damage in selectively vulnerable regions when it is induced repeatedly at a certain interval. The severity of neuronal damage was dependent on the number and interval of ischemic episodes.