Involvement of cholinergic and GABAergic systems in the reversal of memory disruption by NS-105, a cognition enhancer.

The effects of (+)-5-oxo-D-prolinepiperidinamide monohydrate (NS-105) on the scopolamine-, electrolytic lesion of the nucleus basalis magnocellularis (NBM)-, AF64A-, baclofen-, cerebral ischemia- and electroconvulsive shock (ECS)-induced memory disruption in the passive avoidance response or radial arm maze tasks were investigated in rats. The effects of NS-105 were compared with those of aniracetam, bifemelane, idebenone, and indeloxazine in two tasks of the passive avoidance response. Furthermore, effects of NS-105 on in vivo release of acetylcholine (ACh) in the cerebral cortex, high-affinity choline uptake (HACU) of the cerebral cortex in rats with lesion of NBM, HACU of the hippocampus in rats treated with pentobarbital and activity of choline acetyltransferase (ChAT) of the cerebral cortex in rats with lesion of NBM were examined. NS-105 showed antiamnestic actions in a variety of animal models of cholinergic dysfunction employed in this study. Aniracetam improved memory disruption caused by scopolamine, but bifemelane, idebenone, and indeloxazine did not. NS-105 (10 mg/kg) showed the increase of ACh release from the cerebral cortex and the enhancement of HACU both in the cerebral cortex and hippocampus, but showed no change in activity of ChAT. NS-105 also reversed memory disruption induced by baclofen, a potent GABA(B) receptor agonist, but all of reference drugs did not. These results suggest that antiamnestic action of NS-105 is due to the facilitation of cholinergic neuronal activity and the suppression of GABA(B) receptor-mediated responses.

Behavioral and electroencephalographic studies of beagles with an Eck's fistula: suitability as a model of hepatic encephalopathy.

Behavioral manifestations, electroencephalograms (EEGs) and visually evoked potentials (VEPs) were studied in beagles with Eck's fistula (portacaval shunt [PCS]), an established model of hyperammonemia, to determine whether they developed CNS disorders characteristic of hepatic encephalopathy. After PCS, behavioral changes occurred in the form of listlessness, sluggishness (altered gait, snapping and transient catatonia-like symptoms) and apparent blindness, which appeared in that order and progressed to coma and death in some animals. The EEGs from the frontal cortex showed a gradual decrease in voltage and frequency. Development of snapping and catatonia-like symptoms coincided with the occurrence of high voltage fast waves in the EEGs from the occipital cortex. In comatose Eck's fistula dogs. flattening of the EEGs was recorded from the frontal cortex and a lowered voltage was noted in the EEGs from the occipital cortex. After PCS, the latencies and amplitudes of the components of VEP were increased. The snapping and catatonia-like symptoms were markedly ameliorated by carbamazepine and the coma by flumazenil and thyrotropin-releasing hormone. These findings indicate that Eck's fistula dogs provide a useful model of hepatic encephalopathy.

[EEG studies on the analeptic effect of montirelin hydrate (NS-3), a TRH analog, in posterior hypothalamic area-lesioned rats].

Effects of a novel TRH analog, montirelin hydrate (NS-3), on the EEG were compared with those of TRH in intact and posterior hypothalamic area (PHA)-lesioned rats. In the intact rats, NS-3 at 0.01 mg/kg and TRH at 1 mg/kg produced no changes in the sleep-waking cycle. NS-3 at 0.1 mg/kg caused an increase in the time spent in the arousal stage and delayed the onset of the slow wave sleep (SS). On the other hand, TRH at 10 mg/kg increased the time spent in the SS stage and decreased the time in the A1 stage during 2 to 4 hr after injection without increasing the arousal stage, although it delayed the onset of SS. No abnormal EEG was observed after NS-3 or TRH administration in the intact rats. Electrolytic ablation of the PHA elicited continuous behavioral resting and caused increases in the slow-wave components in the cortical EEG and loss of generation of the hippocampal rhythmic slow activity. NS-3 at 0.01 mg/kg and TRH at 1 mg/kg caused the reappearance of the hippocampal rhythmic slow activity. In addition, NS-3 at 0.1 mg/kg and TRH at 10 mg/kg decreased the slow-wave components and increased the fast-wave components in the cortical EEG. In conclusion, NS-3 showed a distinct analeptic effect with a 100-fold higher potency than that of TRH in both the intact and PHA-lesioned rats.

[Montirelin hydrate (NS-3), a TRH analog, improved the disturbance of consciousness caused by head concussion and pentobarbital in mice].

Effects of a novel TRH analog, montirelin hydrate (NS-3), on the coma caused by head concussion and narcosis induced by pentobarbital were compared with those of TRH in mice. Head concussion caused a behavioral comatose state with loss of the righting reflex and spontaneous motor activity. NS-3 shortened the latent periods to the recovery of the righting reflex (0.03-0.1 mg/kg, i.v.) and spontaneous motor activity (0.1 mg/kg, i.v.) following the head concussion. In the case of TRH, higher doses were needed to induce such effects. NS-3 (0.1-0.3 mg/kg, i.v.) reversed the pentobarbital-induced narcosis in a dose-dependent manner. A similar effect was elicited by 30- to 100-fold higher doses of TRH than NS-3. The analeptic effect of NS-3 in the pentobarbital-narcotized mice was antagonized by SCH23390, a dopamine D1 antagonist or by the combined treatment with prazosin and scopolamine, while neither prazosin nor scopolamine alone antagonized the analeptic effect of NS-3. Taken together with the finding that NS-3 did not bind to dopamine, adrenaline or muscarine receptors, it is suggested that NS-3 may restore the disturbance of consciousness by activating the brain dopamine, noradrenaline and acetylcholine neurons without stimulating these receptors directly.

[Ameliorating effect of lactitol on experimental hepatic encephalopathy in Eck fistula dogs].

Eck fistula (portacaval shunted) dogs were prepared for use as an experimental model of chronic hepatic encephalopathy. The effect of lactitol on hepatic encephalopathy was investigated by observing the behavior, electroencephalograms (EEGs) and visually evoked potentials (VEPs) of the experimental dogs. Lactitol was administered intragastrically once a day for 12 weeks from the third week after the portacaval-shunt operation and the behavior, EEGs and VEPs of the dogs were observed every two weeks. Dogs not given lactitol became sluggish, then apparently blind, and eventually fell into a coma, over a period of several weeks after the operation. Some dogs died. The EEGs revealed low-voltage slow waves and, at a later stage, displayed flattening in some dogs. The VEPs displayed prolonged latency of both the positive and the negative component as well as an increased amplitude. Lactitol at 1 or 3 g/kg/day suppressed the behavioral symptoms and the changes in the EEGs and the VEPs. These results suggest that lactitol may be useful for the treatment of various nervous symptoms in patients with hepatic encephalopathy accompanied by hyperammonemia.

[Lactitol suppresses the disturbance of consciousness caused by experimentally induced hepatic encephalopathy in rats: an EEG study].

Electroencephalographic (EEG) studies were conducted to demonstrate the ameliorating effects of lactitol and its reference drug lactulose on the disturbance of consciousness caused by severe hepatic encephalopathy in rats permanently implanted with cortical electrodes. A novel experimental animal model of combined-type human hepatic encephalopathy was prepared by portacaval shunting followed by a single treatment with dimethylnitrosamine (30 mg/kg, i.p.). Lactitol or lactulose was orally administered twice a day for seven days and once on the morning of the eighth day. Ammonium acetate (500 mg/kg) was injected into the cecum 4 hr after the final administration of the drug. In control animals not treated with either drug, but in which hepatic encephalopathy had been induced, ammonium acetate induced a comatose state defined by a loss of the righting reflex accompanied by slowing or flattening of the cortical EEG. In control animals, significant increases in delta (1-3 Hz)-activity and significant decreases in beta (13-25 Hz)-activity during coma were detected by means of EEG power spectral analysis. Lactitol at doses of 3 g/kg/day or higher or lactulose at 6 g/kg/day significantly suppressed these EEG changes. Both drugs also suppressed in a dose-dependent manner the loss of the righting reflex. Lactitol may therefore be useful for ameliorating the disturbance of consciousness in patients with hepatic encephalopathy.

Effect of NS-3, a thyrotropin-releasing hormone analog, on in vivo acetylcholine release in rat brain: regional differences and its sites of action.

The effects of thyrotropin-releasing hormone (TRH) and its analog, NS-3 [CG-3703: (3R,6R)-6-methyl-5-oxo-3-thiomorpholinylcarbonyl-L-histidyl- prolinamide tetrahydrate], on acetylcholine (ACh) release were examined in the rat brain by using in vivo microdialysis, and possible sites of action of NS-3 were subsequently examined. Both NS-3 (0.1 and 0.3 mg/kg i.v.) and TRH (3 mg/kg i.v.) produced an increase in the ACh release from the cerebral cortex and hippocampus but not from the striatum, although the action of NS-3 was much longer in duration than that of TRH. The ACh-releasing action of NS-3 was more marked in the hippocampus than in the cerebral cortex. Application of NS-3 (0.1-10 microM) via a dialysis probe into the nucleus basalis Meynert but not into the cortex caused a rapid and significant increase in cortical ACh release. Similarly, hippocampal ACh release was elevated by NS-3 (1 microM) perfused into the medial septum-diagonal band but not into the hippocampus. These findings suggest that NS-3 produces regionally specific enhancement of ACh release in the rat brain, and such effects of NS-3 are far more potent and longer in duration than those of TRH. In addition, the nucleus basalis Meynert and medial septum-diagonal band may be the respective sites of action of NS-3 in enhancing cortical and hippocampal ACh release.

Cholinergic deafferentation prevents delayed neuronal death of the hippocampal CA1 pyramidal neurons after transient forebrain ischaemia.

The relation between CA1 neurons, fimbria-fornix and cholinergic neurons of the basal forebrain was examined with the aid of Acetylcholine esterase (AChE) staining, Woelcke's staining and immunohistochemistry of Choline-acetyl transferase (ChAT). The transected side of the hippocampus was poorly stained by AChE two weeks after the transection, when the ipsilateral medial septum ChAT-positive neurons were reduced, but showed good recovery with AChE six weeks later. Nerve growth factor (NGF) was added at a dose of 10 micrograms/100 microliters immediately after the aspiration, and after that once per week with cisternal puncture. As a result, ipsilateral medial septum ChAT-positive neurons were preserved, but cross innervation with relation to hypertrophy of the cholinergic neurons was not detectable even six weeks after the transection. Furthermore, delayed CA1 neuronal death on the transected side of the hippocampus following occlusion of four vessels for 30 minutes was not detectable two weeks after the operation, although neuronal density was reduced after six weeks. The density of neurons on the transected side of the hippocampus in the CA1 subfield with treated NGF had not decreased significantly six weeks later. Therefore, we suspect that the input from cholinergic fibres must be transported to the hippocampal pyramidal neurons responding to NGF, and it was confirmed that cholinergic deafferentation prevents the delayed neuronal death of CA1 pyramidal neurons during transient ischaemia.

Changes in somatosensory circuits after subcortical infarct in rats.

We compared the functional and anatomical alterations of somatosensory circuits in the acute (1-3 days after infarct) and chronic (3 months after infarct) stage after subcortical striatal infarct in Wistar rats. Occlusion of the left middle cerebral artery produced subcortical striatal infarct in approximately 69% of the rats. The others developed cortical infarct. The function of the somatosensory circuits was evaluated by [14C]2-deoxyglucose autoradiography during physiological stimulation of the right vibrissae and face. In rats with subcortical infarct, the areas activated by sensory stimulation of the right vibrissae and face, applied 1 and 3 days after occlusion, were reduced compared to sham-operated controls (P < 0.05). In the chronic stage of subcortical infarct, the areas of metabolic activation of the left anterior vibrissal and facial sensory area were increased compared to rats with acute subcortical infarct (P < 0.05). To evaluate the anatomical changes in the somatosensory pathway, at 1 day and 3 months after occlusion, we injected wheat germ agglutinin-horseradish peroxidase solution as an axonal transport substance bilaterally into the anterior vibrissal and facial sensory area. Tract tracing studies in both the acute and chronic stage of subcortical infarct showed a reduction in the peroxidase-positive area in the left thalamus compared to the control hemispheres (P < 0.01). The functional disturbance and recovery of the somatosensory circuits after subcortical infarct are discussed.

Modification of anoxic neuronal injury by human recombinant epidermal growth factor and its possible mechanism.

A primary culture of cortical neurons was established from a 17-day rat embryo. Twenty-four hours after plating, the culture medium was changed to a chemically defined one, and human recombinant EGF (rEGF) was added to the medium at concentrations of 1, 10, and 100 ng/ml. Anoxic stress was exerted on the cultured neurons for 4 hr. Without anoxia, rEGF at a concentration of 10 ng/ml supported neuronal survival. When rEGF was not present in the culture medium, anoxic stress reduced neuronal density to one-tenth that of the nonanoxic group. The rEGF improved neuronal recovery from anoxia significantly, with survival dependent on rEGF dosage. Monoclonal antibody for EGF receptors canceled the effect of rEGF on survival of neurons in a dose-dependent manner. Therefore, rEGF functioned to support neuronal survival from anoxia through EGF receptors. Ontogeny of the EGF receptors was detected immunocytochemically with anti-EGF receptor antibody. About 70% of the cultured neurons were stained by the anti-EGF receptor antibody 3 days after change of medium, and immunoreactivity for EGF receptors was found to be located on the soma of neurons. Binding activity of EGF receptors was detected 5 days after change of medium with receptor binding assay and Scatchard analysis. High-affinity binding sites were detected only in neurons cocultured with rEGF (10 ng/ml) for 5 days. The data indicate that rEGF in sufficient concentration induces high-affinity sites of the EGF receptor and that EGF may have an antianoxic effect through the EGF receptors.

Neuronal network disturbance after focal ischemia in rats.

We studied functional disturbances following left middle cerebral artery occlusion in rats. Neuronal function was evaluated by [14C]2-deoxyglucose autoradiography 1 day after occlusion. We analyzed the mechanisms of change in glucose utilization outside the infarct using Fink-Heimer silver impregnation, axonal transport of wheat germ agglutinin-conjugated-horseradish peroxidase, and succinate dehydrogenase histochemistry. One day after occlusion, glucose utilization was remarkably reduced in the areas surrounding the infarct. There were many silver grains indicating degeneration of the synaptic terminals in the cortical areas surrounding the infarct and the ipsilateral cingulate cortex. Moreover, in the left thalamus where the left middle cerebral artery supplied no blood, glucose utilization significantly decreased compared with sham-operated rats. In the left thalamus, massive silver staining of degenerated synaptic terminals and decreases in succinate dehydrogenase activity were observed 4 and 5 days after occlusion. The absence of succinate dehydrogenase staining may reflect early changes in retrograde degeneration of thalamic neurons after ischemic injury of the thalamocortical pathway. Terminal degeneration even affected areas remote from the infarct: there were silver grains in the contralateral hemisphere transcallosally connected to the infarct and in the ipsilateral substantia nigra. Axonal transport study showed disruption of the corticospinal tract by subcortical ischemia; the transcallosal pathways in the cortex surrounding the infarct were preserved. The relation between neural function and the neuronal network in the area surrounding the focal cerebral infarct is discussed with regard to ischemic penumbra and diaschisis.

[Immunohistochemical study of c-myc oncogene product in human brain tumors].

C-myc oncogene is widely distributed in eukaryotic cells and is supposed to play an important role in the cellular proliferation and differentiation. Enhanced expression of this oncogene is reported in many kind of tumors, which is often associated with increased malignancy. It seems, therefore, important to study the expression of this oncogene in analyzing the cell biologic features of brain tumors. In the present paper we investigated the distribution of this oncogene product in paraffin-embedded tissue of various kind of brain tumors with a monoclonal antibody to synthetic c-myc peptide. The results demonstrated that c-myc product was detectable in most of the astrocytoma lineage. The immunoreaction within the cell nuclei was more intense in grade 3 and grade 4 astrocytomas than in grade 2 tumors. The expression in grade 4 tumors was, however, rather weaker that in grade 3 tumors. In benign, non-glial tumors like meningiomas and neurinomas, the nuclear immunoreaction was usually absent or only weak, although it was enhanced in a case of acoustic and spinal neurinomas associated with von Recklinghausen's disease.

[Ischemia-induced neurotrophic activity detected in the peri-infarcted brain tissue].

In the experimental ischemia induced by occlusion of the rat middle cerebral artery, axons of both cortical and thalamic neurons were affected. However, cortical neurons survived thereafter and thalamic neurons died because of retrograde degeneration. The fate in these two groups of neurons was remarkably different and may be related to neurotrophic activity induced by ischemia. To detect ischemia-induced neurotrophic activity, fetal cortical neuron was cultured and neurotrophic activity was detected by applying tissue extract to the culture system. Fetal neurons obtained from 17 days rat embryo were cultured for 24 hours at 3.75 x 10(4) cells per 15 mm well in modified Eagle's minimum essential (MEM) supplemented by 10% fetal bovine serum (FBS), then for next 3 to 7 days with Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (DME/F-12) supplemented with insulin 5 mg/l, transferrin 10 mg/l, progesterone 6.3 micrograms/l, Na2SeO3 5.2 micrograms/l. The tissue extract was obtained from the rat subjected to ischemia and homogenized with DME/F-12. The homogenate was centrifuged at 100,000 g for 90 minutes and the supernatant was obtained. Application of peri-ischemic cortical extract improved neurons' survival by 50% as compared to extract of the contralateral side. However, the thalamic extract of the ischemic side had no neurotrophic activity as compared to the contralateral side. The activity was detected in the extract obtained at 8 days after ischemia but not detected in the extract obtained at 4 days after ischemia. The neurotrophic activity was disappeared by heating the extract at 90 degrees C for 10 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)