Makoto Saji

Prevention of transneuronal degeneration of neurons in the substantia nigra reticulata by ablation of the subthalamic nucleus.

Transneuronal degeneration (TND) of neurons in the substantia nigra reticulata (SNR) occurs after initial ischemic or neurotoxin damage to the striatum. The mechanism is incompletely understood. In rats ibotenic acid (IBO) lesion of the caudate nucleus (CN) and the globus pallidus (GP) caused, 3 weeks later, a 47% loss of neurons (P < 0.001) in the SNR. Rats with IBO lesion confined to either the CN or the GP had SNR neuron numbers comparable to control. The volume of the SNR was decreased, as expected, in all groups with striatal lesions. To test whether the subthalamic nucleus (STN) played a role in the demise of SNR neurons, the STN was lesioned 1 week before animals were exposed to CN and GP injury. STN ablation prevented the expected SNR neuron loss. Based on the current information about basal ganglia anatomy, an imbalance between GABAergic and glutamatergic afferents may have caused TND in the SNR. These results suggest that the potential for the release of intrinsic excitotoxicity exists within certain anatomic networks.

Progressive neuronal loss in the ventral posterior lateral and medial nuclei of thalamus in Niemann–Pick disease type C mouse brain

Niemann-Pick disease type C (NP-C) disease is a progressive and fatal neurological disorder characterized by accumulation of cholesterol and glycosphingolipids in peripheral tissues and that of glycosphingolipids in the brain. A C57BL/KsJ-npc1(spm) mutant strain is a genetically authentic model of NP-C. This study investigated neuronal cell loss and lipid accumulation in the npc1(spm) mouse brain. Nissl-staining revealed abundant swollen neurons in the neocortex, piriform cortex, hippocampus and basal ganglia at 3-4 wk of age. In addition to loss of the Purkinje cells, we found a conspicuous cell loss in the ventral posterial lateral (VPL) and medial (VPM) nuclei of thalamus, which became apparent after 4-5 wk. Biochemical analyses revealed no increase of cholesterol in the lipid extracts whereas a substantial accumulation of cholesterol was detectable in most of the large neurons by filipin staining in the brain of homozygous mice. In contrast to the diffuse staining pattern in normal brains, the neuropils of the neurons in the brain of homozygous mice were stained in a punctate pattern. The ubiquitous accumulation excludes a direct role of cholesterol in the progressive neuronal loss in the Purkinje cell layer and in the VPL and VPM of the thalamus.

Histological and temporal characteristics of nigral transneuronal degeneration after striatal injury.

Neurotoxic injury of the caudate-putamen and lateral globus pallidus unilaterally initiated transneuronal degeneration of neurons in the ipsilateral substantia nigra reticulata (SNR). Quantification of SNR neurons using unbiased stereology demonstrated that neuron loss began 4 days after the initial striatal lesion, followed by significant loss (50%) at 6 days and a plateau at 8 days. Analysis at the light and ultrastructural levels revealed morphological changes consistent with a type of programmed cell death. These temporal and histological results refine an in vivo model in which to explore mechanisms of delayed neuronal degeneration.

Repetitive intermittent hypoxia-ischemia and brain damage in neonatal rats.

OBJECTIVE To know the effect of brief-repetitive intermittent hypoxia-ischemia on the development of perinatal brain damage. STUDY DESIGN Seven-day-old Wistar rats underwent ligation of the unilateral common carotid artery. The animals were allocated to three groups (n=12 in each group) and exposed to 8% oxygen as follows: group A: continuous exposure for 180 min; group B: continuous exposure for 90 min; and group C: 10 min of exposure repeated at 10-min intervals over a period of 180 min (total exposure time, 90 min). Seventy-two hours after exposure to hypoxia, the cerebral cortex was examined to assess the degree of neuronal necrosis and brain damage was classified into four grades of severity, 0-3. To evaluate the extent of brain damage, we used immunohistochemical staining with TIB-128 antibody, which reacts to MAC-1 antigen specific to microglia, and observed the glial reaction in the cerebral cortex, hippocampus, thalamus, and striatum. RESULTS All the brain damage observed in groups A-C occurred on the side where the ligation was performed. The most severe damage was found in group A animals, of which seven showed significant neuronal necrosis, having a grade 2 or more advanced lesion. In group B, neuronal necrosis was modest, with only one animal having a grade 2 lesion. In group C, a significant neuronal necrosis was found in six animals despite having the same period of hypoxic exposure as those in group B. MAC-1 positive cells appeared in the cerebral cortex of histologically damaged animals and extended to the hippocampus, thalamus, and striatum in severely damaged animals from groups A, B, and C. CONCLUSION Examination of the neonatal rat model suggested that repetitive and intermittent, rather than continuous hypoxia-ischemia, causes pronounced damage in the immature brain.

Deafferentation-induced c-fos gene expression in subthalamic nucleus and substantia nigra reticulata is reduced by non-NMDA receptor antagonist

Molecular events underlying the mechanism by which brain injury elicits delayed transneuronal degeneration of neurons remote from the site of initial injury are not well understood. In rats, acute injury of the caudate nucleus (CN) and globus pallidus (GP) by local injection of excitotoxic ibotenic acid (IA) or by transient forebrain ischemia resulted in delayed cell death of neurons in the substantia nigra reticulata (SNr). To elucidate the involvement of glutamate receptor mediated hyperactivity of neurons produced by loss of inhibitory inputs in this delayed degeneration of SNr neurons, the region-specific expression of an immediate early gene, c-fos, and the effect of glutamate receptor antagonists on the c-fos expression were examined by using immunocytochemical and in situ hybridization analysis. Following unilateral IA-injection into the CN and GP, a robust expression of c-fos mRNA and Fos protein was induced specifically in neurons of both subthalamic nucleus (STN) and SNr deafferented by the IA-lesions 36 h after IA-injection. The delayed expression of Fos-protein in SNr neurons lasted for 48 h longer than that in STN neurons. Following unilateral IA-injection confined to the CN, an intense but short-term expression of Fos-protein was exhibited only in neurons of the deafferented SNr. c-fos mRNA and Fos protein were not expressed in neurons of the substantia nigra compacta at any time points examined. The induction of c-fos mRNA and Fos protein in neurons of the STN and SNr following IA-lesions of the CN and GP was reduced markedly by non-NMDA receptor antagonist (GYKI52466), but not by NMDA receptor antagonist (MK-801). The region-specific c-fos expression implies that deprivation of inhibitory afferents (disinhibition) due to destruction of presynaptic neurons can induce increased activity of postsynaptic neurons. The effect of GYKI52466 on the c-fos gene expression in neurons of the deafferented STN and SNr suggests that activation of non-NMDA receptors may be involved in a pathophysiological cascade for the transneuronal degeneration of SNr neurons.

Behavioral correlates of transneuronal degeneration of substantia nigra reticulata neurons are reversed by ablation of the subthalamic nucleus

In rats, acute injury of neurons in the caudate nucleus (CN) and globus pallidus (GP) by local injection of ibotenic acid (IA) or by transient forebrain ischemia has caused transneuronal cell death of neurons in the substantia nigra reticulata (SNr) weeks after the initial injury. Recently transient expression of an immediate early gene c-fos was induced specifically in neurons of the subthalamic nucleus (STN) and SNr at 36-48 h after the IA-lesions, prior to the delayed degeneration of SNr neurons. These cellular and molecular events may alter the level of inhibitory output from the basal ganglia and lead to movement disorders. To test (i) whether movement disorders occur in the early period after unilateral lesions of the CN and GP by IA-injection, and (ii) whether ablation of the STN reverses the early movement disorders, we used a modified version of Porsolt forced swim test in which the lesion-induced asymmetry of motor function becomes apparent as rotation when the animals are forced to swim. Following unilateral IA-lesions of the right CN and GP in rats, rapid contraversive rotation appeared transiently 36-48 h after the lesions, and, in turn, slow ipsiversive rotation appeared at 3-5 days postlesion. Prior ablation of the ipsilateral STN reversed these early movement disorders produced by the unilateral IA-lesions of the CN and GP and instead created persistent contraversive rotation 7-10 days after the lesions. Each phase of the dominant rotation behavior was dependent on asymmetrical limb motor activity; decreased left limb activity caused contraversive rotation, and increased left limb activity caused ipsiversive rotation. Reversal of these early movement disorders suggests that ablation of the STN prevents the transneuronal degeneration of the SNr.

Protective effect of a low dose of colchicine on the delayed cell death of hippocampal CA1 neurons following transient forebrain ischemia.

Transient forebrain ischemia in rats preferentially causes neuron death in the striatum and hippocampal CA1 area. Prior injection of a low dose (1 microl) of colchicine (1 microM) into the unilateral hippocampus prevented ischemic damage of CA1 neurons in both hippocampal hemispheres, whereas no protection against ischemic damage was seen in the striatum. These results suggest that disconnection of hippocampal neurons by blockade of axoplasmic transport with colchicine specifically protects ischemic damage of CA1 neurons.

Transsynaptic cell death of neurons following striatopallidal lesions does not occur in substantia nigra pars reticulata in developing rats

In adult rats, combined lesions of the striatum and globus pallidus (GP) cause transsynaptic cell death of neurons in the substantia nigra pars reticulata (SNr) which becomes apparent 1-2 weeks after the lesions. This delayed cell death of SNr neurons has been explained to be caused by over-excitation of SNr neurons which results from an imbalance between excitatory and inhibitory inputs due to two simultaneous events: acceleration of the excitatory input from the disinhibited subthalamic nucleus (STN) and deprivation of the inhibitory input from the striatum. To examine whether the transsynaptic neuronal death in SNr is caused by the same lesions in developing rats, we destroyed the striatum and GP in rats on postnatal days 10 (P10), P15, P20, P25, P30, P35 and P60 by injecting ibotenic acid. We found that cell death did not occur in SNr neurons in rats younger than P20 and that Fos expression induced in STN neurons after these striatopallidal lesions in P10 and P20 rats was lower than that in P30 or P60 rats. These findings suggest that excitation of STN neurons is not enough to cause cell death of SNr neurons in rats younger than P20. Immature functional connection between the cerebral cortex and STN in the early developing animals may contribute to the resistivity of SNr neurons to transsynaptic delayed cell death.

A tyrosine kinase-like molecule is localized in the nuclear membrane of neurons: hippocampal behavior under stress.

Summry— Protein tyrosine kinases play important roles in the development of the mammalian nervous system during embryogenesis and in the maintenance of function of the adult brain. Using a semi‐nested PCR technique based on a short amino acid motif of protein tyrosine kinases, we isolated a human genomic DNA encoding a peptide whose sequence was related to known mammalian protein tyrosine kinases. The expression was examined by Northern blot analysis, and transcripts were detected almost exclusively in the brain. The corresponding cDNA was sequenced, and it was revealed that the gene designated as byk coded for a receptor‐like molecule with a motif of protein tyrosine kinase. Immunohistochemical analysis demonstrated that the Byk protein was expressed in neurons and was located in the nuclear envelope. To understand the physiological significance of the Byk protein, we investigated the behavior of this molecule in the hippocampus after ischemia. Byk‐like immunoreactivity disappeared from the neurons in the fields CA1 through CA3 and the dentate gurus of the hippocampus following 20 min of ischemia. After recirculation of blood flow, neurons in the CA3 field and the dentate gyrus re‐expressed Byk‐like antigen but CA1 neurons did not. Interestingly, Byk‐like immunoreactivity was detected in microglial cells and astrocytes in the CA1 field that were activated after ischemia. Byk could be a new tool to study the neuron‐glia and glia‐glia interactions.