Akira Mitani

Critical levels of extracellular glutamate mediating gerbil hippocampal delayed neuronal death during hypothermia: Brain microdialysis study

When the brain temperature was lowered by 2 degrees C from normothermic temperature, a protective effect on postischemic neuronal death was exhibited and levels of extracellular glutamate were attenuated to about half of those at normothermic brain temperature in the gerbil hippocampus. Hypothermia has been reported to confer a protective effect on ischemia-induced delayed neuronal death. The present study was carried out to quantify this protective effect of hypothermia on the degree of alteration in extracellular release of glutamate during ischemia and the final histopathological outcome in the hippocampus. Extracellular glutamate levels were measured by microdialysis. In gerbils whose brain temperature was maintained at normothermia (37 degrees C), glutamate increased during ischemia and the early period of recirculation (by 15-fold), and CA1 neurons were consistently damaged. In animals whose brain temperature was maintained at 35 or 33 degrees C during ischemia, the release of glutamate was significantly attenuated to half or a quarter, respectively, at 37 degrees C. In animals whose brain temperature was maintained at 31 degrees C during ischemia, the release of glutamate was slightly lower than that at 33 degrees C. No CA1 ischemic neuronal damage was seen in 60% of gerbils at 35 degrees C and none was seen in any gerbils at 33 and 31 degrees C. In animals whose brain temperature was maintained at 39 degrees C during ischemia, the release of glutamate was slightly higher than that at 37 degrees C, and a high mortality rate of animals (75%) was observed. Our results reinforce other recent evidence suggesting that one of the mechanisms by which lowering of the brain temperature by only a few degrees during ischemia exerts a protective effect in the hippocampus, involves the reduction of ischemia-induced glutamate release.

Cholinergic projections from the laterodorsal and pedunculopontine tegmental nuclei to the pontine gigantocellular tegmental field in the cat

This study demonstrates that the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) are sources of cholinergic projections to the cat pontine reticular formation gigantocellular tegmental field (PFTG). Neurons of the LDT and PPT were double-labeled utilizing choline acetyltransferase immunohistochemistry combined with retrograde transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). In the LDT the percentage of cholinergic neurons retrogradely labeled from PFTG was 10.2% ipsilaterally and 3.7% contralaterally, while in the PPT the percentages were 5.2% ipsilaterally and 1.3% contralaterally. These projections from the LDT and PPT to the PFTG were confirmed and their course delineated with anterograde labeling utilizing Phaseolus vulgaris leucoagglutinin (PHA-L) anterograde transport.

Direct projections from the dorsal column nuclei and the spinal trigeminal nuclei to the cochlear nuclei in the cat

A retrograde and anterograde wheat germ agglutinated horseradish peroxidase WGA-HRP study in the cat indicated that some neurons in the dorsal column nuclei and the interpolar and caudal spinal trigeminal nuclei send fibers to the dorsal and ventral cochlear nuclei; to the pyramidal cell layer of the dorsal cochlear nucleus and to the cochlear granule cell domain, bilaterally with an ipsilateral dominance.

The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma

Glaucoma, a progressive optic neuropathy due to retinal ganglion cell (RGC) degeneration, is one of the leading causes of irreversible blindness. Although glaucoma is often associated with elevated intraocular pressure (IOP), IOP elevation is not detected in a significant subset of glaucomas, such as normal tension glaucoma (NTG). Moreover, in some glaucoma patients, significant IOP reduction does not prevent progression of the disease. Thus, understanding IOP-independent mechanisms of RGC loss is important. Here, we show that mice deficient in the glutamate transporters GLAST or EAAC1 demonstrate spontaneous RGC and optic nerve degeneration without elevated IOP. In GLAST-deficient mice, the glutathione level in Müller glia was decreased; administration of glutamate receptor blocker prevented RGC loss. In EAAC1-deficient mice, RGCs were more vulnerable to oxidative stress. These findings suggest that glutamate transporters are necessary both to prevent excitotoxic retinal damage and to synthesize glutathione, a major cellular antioxidant and tripeptide of glutamate, cysteine, and glycine. We believe these mice are the first animal models of NTG that offer a powerful system for investigating mechanisms of neurodegeneration in NTG and developing therapies directed at IOP-independent mechanisms of RGC loss.

Origin of intracellular Ca2+ elevation induced by in vitro ischemia-like condition in hippocampal slices

Microfluorometry was used to investigate the origin of intracellular Ca2+ ([Ca2+]i) elevation in field CA1 of gerbil hippocampal slices perfused with a glucose-free physiological medium equilibrated with a 95% N2/5% CO2 gas mixture (standard in vitro ischemia-like condition). Large [Ca2+]i elevation was detected about 4 min after the beginning of standard in vitro ischemia-like condition, which was accompanied with a negative shift of extracellular DC potential. When slices were perfused with Ca(2+)-free in vitro ischemia-like medium, large [Ca2+]i elevation was observed about 3.5 min after the beginning of Ca(2+)-free in vitro ischemia-like condition, however, the increase in [Ca2+]i was more gradual and of a lesser extent compared with that detected in the slices perfused with the standard in vitro ischemia-like medium that contained Ca2+. When slices were perfused with the Ca(2+)-free in vitro ischemia-like medium that contained dantrolene (50 microM) which is known to prevent Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores, the increase in [Ca2+]i was more gradual and of a lesser extent compared with that detected in the slices perfused with the Ca(2+)-free in vitro ischemia-like medium that did not contain dantrolene. These results indicate that large [Ca2+]i elevation induced by in vitro ischemia-like condition in field CA1 of the hippocampus was caused by both Ca2+ influx from extracellular space and Ca2+ release from intracellular Ca2+ stores, and that a part of the Ca2+ release was due to Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores.

Changes in Intracellular Ca2+ and Energy Levels During In Vitro Ischemia in the Gerbil Hippocampal Slice

Abstract: The time course of the decline in energy levels during an in vitro ischemia‐like condition was compared with changes in intracellular Ca2+ concentration ([Ca2+]i) in subregions of the gerbil hippocampal slice [CA1, CA3, and the inner and outer portions of the dentate gyrus (DG)]. Hippocampal transverse slices were loaded with a fluorescent indicator, rhod‐2. During the on‐line monitoring of [Ca2+]i, the slices were perfused with an in vitro ischemia‐like medium (33°C). The slices were collected at several experimental time points, frozen, dried, and dissected into subregions. The contents of adenine nucleotides (ATP, ADP, and AMP) and phosphocreatine (PCr) were measured by HPLC methods. Region‐specific and acute [Ca2+]i elevations were observed in CA1 ∼4 min after onset of the in vitro ischemia‐like condition and also in the inner portion of the DG with a delay of 10–40 s. The change in ATP levels was related to the increase in [Ca2+]i. ATP levels in all subregions gradually decreased before the acute [Ca2+]i elevation. Concomitant with the acute [Ca2+]i elevation in CA1 and the inner portion of the DG, ATP levels in the subregions rapidly decreased, whereas declines in levels of high‐energy‐charge phosphates were gradual in CA3 and the outer portion of the DG, in which the remarkable [Ca2+]i elevation was not observed. These results suggest that ATP depletion observed in CA1 and the inner portion of the DG is due to the region‐specific increase in [Ca2+]i, which activates a Ca2+‐ATP‐driven pump and produces a subsequent fall in neuronal ATP content.

Selective vulnerability of hippocampal CA1 neurons cannot be explained in terms of an increase in glutamate concentration during ischemia in the gerbil: Brain microdialysis study

Ischemia-induced selective neuronal injury to field CA1 is not attributable to selective glutamate release in field CA1 during ischemia. Excessive release of glutamate has been proposed to play a major role in ischemia-induced selective neuronal death in field CA1 of the hippocampus. It is well known that, following carotid arterial occlusion of 5 min duration in the gerbil, the pyramidal neurons in field CA1 show delayed neuronal death, whereas the neurons in field CA3 do not show any neuronal degeneration. In the present study, we measured the levels of released glutamate during ischemia in field CA1 and field CA3, separately, and evaluated whether there are subregional differences in the concentration of released glutamate which could be a satisfactory explanation for the selective vulnerability of hippocampal neurons to ischemia. Extracellular glutamate levels were significantly increased during ischemia in both field CA1 and field CA3. No significant differences were detected in the time-course of change in glutamate release and the levels of glutamate between field CA1 and field CA3. This result indicates that the increased glutamate levels do not play a pivotal part in the detrimental effect of glutamate during 5-min ischemia. Some differentiated post-synaptic organization may act as a crucial factor in the development of ischemia-induced selective neuronal death in the gerbil hippocampus.

Temperature dependence of hypoxia-induced calcium accumulation in gerbil hippocampal slices

Microfluorometry was used to investigate temperature dependence of hypoxia-induced intracellular calcium accumulation in gerbil hippocampal slice. When slices were superfused with hypoxic medium at 37 degrees C, 35 degrees C, 33 degrees C or 31 degrees C, latencies of acute increase of calcium accumulation, which was accompanied by a large negative shift of extracellular DC potentials, were delayed in a dose-dependent manner: mean latencies in field CA1 were 130 s, 182 s, 232 s and 277 s after hypoxia, respectively. This retardation in calcium accumulation may be involved in the mechanisms by which hypothermia diminishes ischemic injury.

Dantrolene protects against ischemic, delayed neuronal death in gerbil brain.

The effect was examined of dantrolene, a drug for malignant hyperthermia acting through preventing release of Ca from the ryanodine-type intracellular stores in muscle cells, on ischemic delayed neuronal death in field CA1 of gerbil hippocampus. Dantrolene (1.6 mM in concentration, 3 microliters each in volume), when administered bilaterally in the lateral ventricles 30 min after reperfusion after transient forebrain ischemia for 3 min at 37 degrees C, significantly protected against the neuronal death. It is proposed that the dantrolene-sensitive (most likely, the ryanodine-type) intracellular Ca stores in CA1 pyramidal cells play a pivotal role in the development of ischemic neuronal damage.

Distribution of premotor neurons for the hypoglossal nucleus in the cat.

After injecting horseradish peroxidase into the hypoglossal nucleus, labeled neuronal cell bodies were constantly seen bilaterally with a slight ipsilateral dominance in the parvocellular reticular formation and reticular regions around the hypoglossal nucleus, ipsilaterally in the nucleus of Kölliker-Fuse, and contralaterally within the hypoglossal nucleus. A few labeled neurons were often found bilaterally with an ipsilateral dominance in the inter- and supratrigeminal regions around the motor trigeminal nucleus, parabrachial nucleus, ventral portions of the medial reticular formation of the pons and medulla oblongata, and dorsal tegmental regions and central gray of the midbrain.