Yoshiya L. Murashima

Distribution of γ-Aminobutyric Acid and Glutamate Decarboxylase in the Layers of Rat Oviduct

Abstract: An enzymatic microassay method for glutamate decarboxylase (GAD) and γ‐aminobutyric acid (GABA) was improved to yield a high sensitivity and a low blank. The 20‐μm thick freeze‐dried sections (0.2–1.5 μg dry weight) were prepared from the oviduct and ovary of rat. The analysis of these microsamples by the improved method showed that, contrary to the previous observations, the rat ovary is devoid of GAD activity and contains a trace amount of GABA. Both are present abundantly in the oviduct. In the oviduct mucosa, significant GAD activity was found in the estrous phase, whereas the activity was nearly null during other phases of the estrous cycle. GABA concentration in the oviduct mucosa was 10‐fold higher than in the cerebral cortex; its variation during the estrous cycle was not remarkable. In the muscle layer of oviduct, GAD activity had a low peak in the estrous phase and GABA concentration was almost constant during the estrous cycle. The denervation experiment showed that GAD is present in the nerve terminals innervating the oviduct.

Choline acetyltransferase activities in single motor neurons from vertebrate spinal cords.

Abstract: Single cell bodies of spinal motor neurons were isolated from freeze‐dried sections of fresh spinal cords from six species of vertebrates. Single human neurons were also isolated from the spinal cords of three autopsy cases without neurological diseases. Choline acetyltransferase activity of these single neurons was determined by measuring acetyl‐CoA formation from CoASH and acetylcholine by use of the enzymatic amplification reactions, CoA and NADP cyclings. The enzyme activity was unevenly distributed in the cytosol of spinal motor neurons of all species, but not measurable in rabbit dorsal root ganglion cells. The specific activity on a dry weight basis varied widely among the individual neurons from the species studied. The average activity was highest with rat neurons and lowest with yellowtail neurons. The neurons from cold‐blooded animals (bullfrog and yellowtail) had about one‐tenth the activity compared with the warm‐blooded animals (cat, rabbit, rat, and hen). Human neurons, obtained under different morbid and postmortem conditions with three autopsy cases, had very low activities corresponding to those of cold‐blooded animals. Since the choline acetyltransferase activity lost from mouse brain after 11 h at 38°C was 50%, the activity in human neurons was believed to actually be low in vivo.

Glutamate decarboxylase activities in single vertebrate neurons

Abstract: An enzymatic microassay method for glutamate decarboxylase (GAD) and γ‐aminobutyric acid (GABA) was improved to a degree yielding high sensitivity and low blank. Single cell bodies of anterior horn cells and dorsal root ganglion cells were dissected out from the freeze‐dried sections of rabbit and chicken spinal cords and Purkinje cell bodies from those of rabbit cerebellum. A minute amount of GABA, present in single neurons or synthesized by GAD in single neurons, was enzymatically converted to NADPH. The NADPH was amplified 10,000‐350,000‐fold and measured, using an enzymatic amplification reaction (NADP cycling). GAD was contained in all Purkinje cell bodies and its average activity was four‐ to fivefold higher than those of the molecular and granular layers of rabbit cerebellum. The GABA concentration was threefold higher in Purkinje cell bodies than in these layers. GAD activity, at a level similar to that in the cerebellar layers, was found in almost all the cell bodies of anterior horn cells from rabbit and chicken. GABA was detected in 40% of rabbit neurons and not in chicken neurons. Dorsal root ganglion cells from both species contained no measurable GAD or GABA.

Role of immediate early gene expression during establishment of epileptogenesis in the brain of EL mice

Rapid genomic responses to neural stimulation may play a critical role in long-term synaptic plasticity.’ Recent works have strengthened this idea by demonstrating that neurotransmitter-induced long-term synaptic plasticity requires RNA and protein synthesis during critical time window immediately following stimulation.2 These studies prompted the suggestion that genes, such as c-jios, which are rapidly induced by cell-surface-receptor stimulation by either growth factor or neurotransmitters3 might be involved in the early genomic response underlying long-term synaptic pla~ticity.~ Activation of immediate early genes (IEG) occurs by increased transcription and is thought to represent the primary genomic response to stimulations, because it does not require protein synthesis. Several IEG code for transcription factors. These include members of the&s families, which dimerize to form the transcription regulatory complex AM5 as well as zk6 which is a member of a family of proteins characterized in the DNA-binding domains. We observed previously that zfwas expressed maximally 30 min after seizure in both cerebrum and cerebellum under the same condition of the seizure threshold. There were no regional difference^.^ In parallel with the seizure history, the maximum zif expression site in the limbic system shifted from the pyriform cortex and the entorhinal cortex to the dentate gyrus and further to the hippocampal CA3 and CAl .’ On the other hand, during development of the animals as well as through repetitive seizures, the zij expression site shifted gradually from the primary input system of the hippocampus (i.e., the pyriform cortex, the entorhinal cortex and the dentate gyrus), to the hippocampal CAl.7 In the parietal cortex, the expression site was initially localized and then became universal. In general, the initially localized expression site finally became universal and weak all over the cortex.’ To understand the role of IEG response in epileptogenesis in term of the ‘abnormal’ synaptic plasticity epileptogenesis,8 we studied the expression of LEG in the EL mice, a well known mutant model of epilepsy.

GABA Concentration and GAD Activity Levels in Normal and Degenerated Retinas From Mice

Freeze-dried sections (14 μm thick) were prepared from mice with normal (C57BL strain) and degenerated (C3H strain) retinas. GABA concentration and GAD activity were determined in the microsamples (1.8–20 ng dry weight) of retinal layers and sublayers, using an enzymatic amplication reaction, NADP cycling. 1) GABA was distributed over all layers of normal retina with a broad concentration peak covering both inner nuclear and plexiform layers. In contrast, GAD activity was mostly localized in the inner plexiform layer. 2) GABA concentration was similar in one-fourth of the sublayers of each inner nuclear or plexiform layer. GAD activity was highest in the innermost sublayer of the inner nuclear layer. An increasing gradient of GAD activity was present in the inward direction in the inner plexiform layer. 3) In the degenerated retina, lacking in photoreceptors, the inner nuclear and plexiform layers remained, and GABA and GAD levels in these layers were similar to those in normal retina.

Antiepileptic effects of allopurinol involved in hippocampal specific SOD (superoxide dismutase) induction in the mutant El mouse.

Recently the xanthine oxidase inhibitor, allopurinol, has been clinically demonstrated to exert antiepileptic effects: particularly on secondarily generalized seizures.’ The El mouse is an epileptic mutant model of secondarily generalized seizure.6 Several lines of evidence indicate that in the El, the parietal cortex plays an important role in seizure initiation and the hippocampus plays a role in the generalization of se iz~res .~ And the developmental formation of the “Focus Complex” which is mainly constituted from the parietal cortex and hippocampus should be deeply involved in the epileptogene~is.~ Antiepileptic effects of allopurinol were determined in the El. Furthermore, the mode of action of allopurinol was investigated by measuring the activities of superoxide dismutase (SOD) according to the time course after the administration of allopurinol.