Makoto Yoshikawa

Immunocytochemical localization of the striatal enriched protein tyrosine phosphatase in the rat striatum: A light and electron microscopic study with a complementary DNA-generated polyclonal antibody

The present study concerns the immunocytochemical localization of the striatal enriched protein tyrosine phosphatase in the rat striatum. A novel molecular biology technique allowed us to produce a complementary DNA-generated polyclonal antibody raised against the non-catalytic domain of the striatal enriched protein tyrosine phosphatase, which selectively recognized the striatal enriched protein tyrosine phosphatase protein with 46,000 mol. wt on western blots. Immunocytochemical analysis with the specific antibody revealed strong striatal enriched protein tyrosine phosphatase immunoreactivity in the striatum. Light microscopy showed striatal striatal enriched protein tyrosine phosphatase-immunopositive neurons to be of medium size (mean diameter of 14.4 microns), and to comprise approximately 80% of the total neuronal population in the striatum. These cells had round, triangular or polygonal cell bodies with relatively little cytoplasm. Nerve fibers stained positively for striatal enriched protein tyrosine phosphatase were also present in the globus pallidus and the substantia nigra, and the nigral labeling on the ipsilateral side almost disappeared subsequent to cerebral hemitransection, suggesting these immunolabeled structures to be striatal projections. Double-immunofluorescence analysis demonstrated separate populations of striatal enriched protein tyrosine phosphatase-positive cells and neurons stained for parvalbumin. Also, ultrastructural study showed that the striatal enriched protein tyrosine phosphatase-positive neurons (n = 50) possessed no nuclear indentations or intranuclear inclusions. Thus, most striatal striatal enriched protein tyrosine phosphatase-positive neurons were thought to be of the medium-sized spinous type. At the light microscopic level, stained striatal neurons exhibited striatal enriched protein tyrosine phosphatase immunolabeling in their somata, dendrites and axonal processes, but not in their nuclei. Electron microscopic observation showed strong striatal enriched protein tyrosine phosphatase-immunoreactivity on the inner surface of the plasmalemma, on the outer surfaces of mitochondria and on microtubules, particularly of dendrites. A heavy deposit of immunoreaction product was also present on postsynaptic densities in labeled dendrites, while a light deposit was seen on the synaptic vesicles of nerve terminals. The characteristic distribution profile of striatal enriched protein tyrosine phosphatase suggested that the enzyme may play a role in a variety of functional properties of striatal neurons, especially in postsynaptic signaling processes and in regulation of microtubular functions. On the basis of the present findings, we propose the following conclusions: (i) a protein tyrosine phosphorylation system regulated by striatal enriched protein tyrosine phosphatase is involved in certain specialized cellular processes (e.g. signal transduction cascades) of medium-sized spinous neurons distinct from those of other neuronal subsets in the striatum; (ii) a striatal medium spiny neuron is characterized by its expression of striatal enriched protein tyrosine phosphatase and, therefore, the enzyme is useful for detection of the distinct subset of striatal cells or for tracing their axonal projection fibers in the basal ganglia.

GABA Receptor Agonist Promotes Reformation of the Striatonigral Pathway by Transplant Derived from Fetal Striatal Primordia in the Lesioned Striatum

Striatal lesions are known to cause the anterograde transneuronal degeneration of the substantia nigra pars reticulata (SNr) neurons in consequence to loss of GABAergic inhibitory striatonigral efferents. The present study was undertaken to examine whether long-term intraventricular administration of the GABA agonist muscimol could promote reformation of the striatonigral pathway arising from transplants by rescuing host SNr neurons from transneuronal death in rats with striatal ischemic lesions. Compared to nongrafted rats with striatal lesions, (i) a prominent axonal projection from the transplants to the ipsilateral substantia nigra, (ii) a significant increase in number of survived neurons in the ipsilateral SNr, and (iii) a significant reduction in number of apomorphine-induced turning behaviors were found in grafted animals with muscimol infusion, but not in those without muscimol administration. These findings suggest that preservation of the host target neurons for grafted cells may increase an efficacy of cerebral implants in establishment of the host-graft fiber connections, possibly, leading to functional restoration.

GABAergic transmission and tyrosine hydroxylase expression in the nigral dopaminergic neurons: An in vivo study using a reversible ischemia model of rats

The authors conducted an in vivo study, using a rat striatal ischemic model, of the effect of GABAergic transmission upon the dopamine synthesizing enzyme tyrosine hydroxylase in the neurons of the substantia nigra pars compacta. Two hours transient middle cerebral artery occlusion produced massive striatal ischemic damage resulting in a marked decrease of GABAergic projection to the ipsilateral substantia nigra. Histological examinations were conducted in rats killed at three, seven, 15, 30 and 94 days after ischemia. The immunoreactivity for tyrosine hydroxylase in the ipsilateral pars compacta was unaltered up to three days after the ischemic insult, but it was markedly decreased at seven days post-ischemia. At this stage, the number of neurons positive for tyrosine hydroxylase was significantly decreased in the ipsilateral pars compacta, whereas there was no significant reduction in the number of pars compacta neurons containing Nissl substance. By 30 days post-ischemia, the tyrosine hydroxylase-positive cell number in the ipsilateral pars compacta appeared to be equivalent to that of the contralateral side. It was also noted that continuous intraventricular administration of a GABAA receptor agonist muscimol, initiated from 24 h post-ischemia, effectively prevented the transient reduction of immunoreactivity for tyrosine hydroxylase in the ipsilateral pars compacta at seven and 15 days after ischemic insult. The present study revealed that the striatal ischemic lesion induced a transient down-regulation of tyrosine hydroxylase synthesis in the pars compacta neurons, which could be prevented by administration of GABA agonist, suggesting that GABAergic transmission greatly affects dopamine metabolism in these cells.

Posteroventral pallidotomy in a patient with parkinsonism caused by hypoxic encephalopathy

We report a patient with a hypokinetic-rigid form of parkinsonism caused by hypoxic encephalopathy, in whom parkinsonian symptoms were markedly alleviated by staged bilateral posteroventral pallidotomy.

Neurotoxicity evoked by N-methyl-D-aspartate in the organotypic static slice cultures of rat cerebral cortices: Effect of GABA(A) receptor activation

Abstract We investigated the neurotoxicity evoked by N-methyl-d-aspartate (NMDA) receptor stimulation in the organotypic static slice cultures of rat cerebral cortices. We also examined whether the γ-aminobutyric acid (GABA)A receptor agonist muscimol has a protective effect on the NMDA-mediated neurotoxicity in this culture system. NMDA-mediated cytotoxicity was evaluated histologically and quantified by the measurement of lactate dehydrogenase (LDH) release into the culture medium. There was an NMDA-induced, dose-dependent leakage of LDH release and neuronal cell death, which were not attenuated by muscimol treatment. The results suggested that NMDA neurotoxicity is reproduced in the organotypic culture, and that GABAA receptor activation exerted no protective action against the NMDA cytotoxicity.

Involvement of N-methyl-d-aspartate receptor in the delayed transneuronal regression of substantia nigra neurons in rats

The substantia nigra pars reticulata (SNr) receives both inhibitory GABAergic and excitatory glutamatergic afferents from diverse origins. Ischemic injury to the striatum and/or the globus pallidus causes delayed transneuronal death of the SNr neurons, in the course of which neuronal disinhibition induced by loss of GABAergic inputs is supposed to trigger a lethal hypermetabolic process. In the in vivo experiment presented herein, we clarified the role of glutamatergic action via the N-methyl-D-aspartate receptor in this cell death process. Continuous intraventricular infusion (0.5 microliter/h) of the N-methyl-D-aspartate receptor antagonist MK-801 (1000 micrograms/ml), or of saline (control group) was initiated 24 h after 2 h of transient middle cerebral artery (MCA) occlusion in rats, by which massive ischemic injury was produced in the striatopallidal regions. The measured rectal temperature was not significantly altered in the MK-801-infused and in the control rats throughout the time period examined. The rats were killed at 15 days after MCA occlusion. The volume of the focal ischemic infarction of the MK-801-infused group did not significantly differ from that of controls. Also, MK-801-infusion did not significantly ameliorate the nigral atrophy subsequent to MCA occlusion. In association with a marked depletion of GABAergic afferent fibers, neuronal cell number in the ipsilateral SNr was significantly decreased in the control group. In contrast, the neuronal cell loss in the nucleus was completely prevented in the MK-801-infusion group. The data suggested that withdrawal of GABAergic inputs may cause a severe imbalance between excitation and inhibition of the SNr neurons and may eventually result in neurotoxicity mediated by the N-methyl-D-aspartate receptor. Suppression of glutamatergic excitatory effects by suitable drugs may be a reasonable therapy for the transneuronal death of the SNr neurons.

Striatal cells containing the Ca2+-binding protein calretinin (protein 10) in ischemia-induced neuronal injury

The present study concerns the vulnerability of striatal interneurons immunopositive for the Ca2+-binding protein calretinin to ischemic neuronal injury. An immunohistochemical study was carried out on the striata of rats which had undergone transient middle cerebral artery occlusion. Two weeks after the ischemia, there was a marked reduction in the number of calretinin-positive neurons in the ipsilateral ischemic lesion, although the striatal interneurons positive for parvalbumin, which are a neuronal population distinct from the calretinin-immunoreactive cells in the striatum, were spared in the insulted areas. The present data indicate that the striatal calretinin-positive neurons are less resistant to transient ischemia, suggesting that there may exist vulnerability differences among the striatal interneurons in ischemia-induced neuronal injury.

Temporal sequence of response to unilateral GPi pallidotomy of motor symptoms in Parkinson's disease

The present study was performed to determine the temporal sequence of the response to unilateral MRI/microelectrode-guided pallidotomy of each cardinal symptom in Parkinson’s disease (PD). For this purpose, we performed a quantitative assessment of motor functions in 19 patients with PD at several time points up to 6 months following surgery. We here report that although all the motor signs were significantly improved 6 months after pallidotomy, the temporal sequence of tremor response was different from those of other symptoms.

Continuous intraventricular drug infusion for the in vivo study of transneuronal degeneration in the striatonigral system of the rat

Injuries to certain parts of the brain may induce neuronal death in distant areas innervated by the sites of the primary lesion. Such characteristic pathological changes, known as anterograde transneuronal degeneration, may occur at the next and more distant synaptic levels and play a part in the slow progression of some types of system degeneration. Delayed transneuronal degeneration of the substantia nigra pars reticulata (SNr) is one example of this form of cell death, and it occurs as a consequence of a neostriatal lesion caused by focal ischemia, Huntingtons disease, or experimental axon-sparing injections of neurotoxin. Ever since the demonstration by Saji and Reis that the administration of GABA receptor agonist effectively prevented delayed transneuronal degeneration of the SNr, the degeneration of nigral reticulata cells has been attributed to the loss of striatal inhibition (Fig. 1A). The latter process severely upset the balance of membrane potential of nigral reticulata cells, producing an effect resembling excitotoxicity. In this report, we describe a continuous intraventricular MK-801 infusion technique that is useful in clarifying the role of glutamatergic action via N-methyl-D-aspartate (NMDA) receptor subclasses involved in exo-focal postischemic death of the SNr.

Mosaic organization of calcineurin immunoreactivity in the adult cat striatum

Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase which is thought to play an essential role in the intracellular Ca(2+)-signal transduction. The present study showed that calcineurin immunolabeling was differentially concentrated in the two distinct compartments showing a mosaic-like pattern in the adult cat striatum. The compartment of heightened calcineurin immunolabeling corresponded to the extrastriosomal matrix visualized by calbindin-D28K immunostaining. Under the light microscope, striatal neurons appeared to be less strongly immunoreactive for calcineurin in the striosomes than in the matrix compartment. These findings suggest that the novel striatal compartments may differ in the intracellular Ca(2+)-signaling cascade associated with protein dephosphorylation.