In vivo gene transfer to the brain cortex using a single injection of HSV-1 vector into the medial septum.

This study shows that an ICP4-replication-deficient herpes simplex virus containing the Moloney murine leukaemia virus LTR fused with the coding sequence for the beta-galactosidase gene can be used as a very effective vector for delivering the beta-galactosidase reporter gene into the rat brain septum. F344 rats received bilateral stereotaxic injections into the nucleus of the diagonal band and into the medial septum. The X-gal stain was used to detect the activity of the expressed beta-galactosidase enzyme. The delivered reporter gene was expressed successfully not only in the neuronal cells of the injected areas but also in cells that project to the injection area such as cortex cells about 6 mm away from the injection sites. Expression was visible at 1, 3 and 9 weeks following injection. We conclude that this vector can effectively deliver genes into different regions of the mature mammalian brain and also to areas distant from the injection site.

Brain neuronal degeneration caused by episodic alcohol intoxication in rats: effects of nimodipine, 6,7-dinitro-quinoxaline-2,3-dione, and MK-801.

Rats repeatedly intoxicated with alcohol (ethanol, three times daily) over a 4-day period display neuronal degeneration in the dentate gyrus; entorhinal, piriform, insular, orbital, and perirhinal cortices; and in the olfactory nerve fibers and terminals in the olfactory bulb. Postulating a role for excitotoxicity, we have attempted to prevent the degeneration using antagonists that are neuroprotective in this type of brain damage. In an initial study, continuous subcutaneous infusion of a high dose of the glutamate/NMDA receptor antagonist MK-801 (2 mg/kg/day) by itself caused extensive neuronal degeneration in several brain regions and severe behavioral intoxication that precluded survival if combined with high blood alcohol levels (approximately 300 mg/dl). Moreover, the lower, nonneurotoxic blood alcohol levels (approximately 150 mg/dl) that were compatible with survival worsened the MK-801-induced brain damage. In a subsequent experiment, daily intraperitoneal injections of a lower dose of MK-801 (1 mg/kg/day) resulted in no MK-801 toxicity and, when combined with neurotoxic levels of alcohol, no reduction in alcohol-induced neurotoxicity. Nimodipine, a voltage-gated Ca2+ channel blocker, reduced the neuronal damage in the dentate gyrus, but greatly increased it in the piriform cortex when administered intragastrically at 600 mg/kg/day; it provided no protection from alcohol-dependent degeneration when given intragastrically at 100 mg/kg/day. Continuous intracerebroventricular delivery of 0.24 to 0.29 mg/day of 6,7-dinitro-quinoxaline-2,3-dione, a glutamate/alpha-amino-3-hydroxy-5-methyl-4-isoxazole receptor antagonist, failed to diminish alcohol-dependent neuronal damage in any brain region. We conclude that brain damage from episodic "binge" alcohol intoxication is not primarily mediated by excitotoxic mechanisms, implying that other, nonexcitotoxic pathophysiological mechanisms, are involved. Furthermore, MK-801, far from protecting from the alcohol-induced damage, at high doses causes widespread neuropathology that is significantly potentiated by alcohol.

Disseminated corticolimbic neuronal degeneration induced in rat brain by MK-801: potential relevance to Alzheimer's disease.

Blockade of N-methyl-D-aspartate (NMDA) glutamate receptors by MK-801 induces neuronal degeneration in the posterior cingulate/retrosplenial cortex and other corticolimbic regions although damage in the latter has not been adequately characterized. This disseminated corticolimbic damage is of interest since NMDA hypofunction, the mechanism that triggers this neurodegenerative syndrome, has been postulated to play a role in the pathophysiology of Alzheimer's disease (AD). Several histological methods, including electron microscopy, were used to evaluate the neurotoxic changes in various corticolimbic regions of rat brain following MK-801 or a combination of MK-801 plus pilocarpine. We found that MK-801 triggers neuronal degeneration in a widespread pattern similar to that induced by phencyclidine and that females showed more damage than males. The neurotoxic reaction involved additional brain regions when muscarinic receptors were hyperactivated by administering pilocarpine with MK-801. Ultrastructural evaluation revealed that a major feature of the neurotoxic action involves degeneration of dendritic spines which entails loss of synaptic complexes. The ultrastructural appearance of degenerating neurons was generally inconsistent with an apoptotic mechanism, although evidence equivocally consistent with apoptosis was observed in some instances. The cell death process evolved relatively slowly and was still ongoing 7 days posttreatment. Relevance of these results to AD is discussed.

Multifocal brain damage induced by phencyclidine is augmented by pilocarpine.

Phencyclidine and other antagonists of the N-methyl-D-aspartate subtype of glutamate receptor cause psychosis in humans. In low doses these agents induce a reversible neurotoxic reaction in the rat brain that is limited to the retrosplenial granular cortex. Some investigators have reported that phencyclidine at higher doses or by more prolonged treatment causes a more disseminated pattern of damage. However, it has not been clearly demonstrated whether the disseminated damage is reversible or irreversible and whether it is consistently reproducible, nor is it known how many and which neurons are at risk. In the present study we addressed these questions using several histological approaches (plastic-embedded thin sections for light microscopy and ultrathin plastic sections for electron microscopy, paraffin-embedded haematoxylin and eosin sections, 72 kDa heat shock protein immunocytochemistry and de Olmos silver impregnation) to study the lesions induced in rat brain by phencyclidine (alone or when augmented with pilocarpine). We found that phencyclidine can kill a relatively large number of neurons distributed over many cerebrocortical and limbic brain regions, but the multifocal pattern of damage occurred in only a small percentage of treated rats. The addition of a low dose of pilocarpine to phencyclidine caused the widespread pattern of damage to manifest on a much more consistent basis. Available evidence suggests that disinhibition of multiple converging excitatory pathways is the mechanism by which phencyclidine triggers widespread neuronal degeneration; however, the specific combination of excitatory inputs that contributes to the pathological process may differ from region to region.

Neuronal degeneration in rat cerebrocortical and olfactory regions during subchronic "binge" intoxication with ethanol: possible explanation for olfactory deficits in alcoholics.

Severe, repetitive ("binge") ethanol intoxication in adult rats (intragastric delivery 3 times daily for 4 days in a modification of the Majchrowicz method) precipitates neuronal degeneration in selected cerebral cortical regions involved in memory and olfaction, confirming the results of Switzer and colleagues (Anat. Rec. 202: 186a, 1982). Neuronal damage was visualized with the de Olmos cupric silver technique for degenerating neurons and processes (argyrophilia), and was quantitated by total counts and densities of argyrophilic cells/fields. The specificity of the degeneration provides a neuropathological basis for the olfactory memory deficits in chronic alcoholics. In highly intoxicated rats, argyrophilia was most extensive among hippocampal dentate gyrus granule cells, pyramidal neurons in layer 3 of the entorhinal cortex, and olfactory nerve terminals in the olfactory bulb. Degenerating pyramidal neurons were also consistently seen in the insular cortex and olfactory cortical regions, such as the piriform and perirhinal cortices. There were few argyrophilic neurons in the CA regions of the hippocampus and none in the cerebellum--regions generally shown to have cell loss in long-term ethanol feeding models--but degenerating mossy fibers in the CA2 region were observed. Degeneration was maximal before the peak period of abstinence symptoms in this model, because argyrophilic densities were no greater 36 hr, compared with 8 hr after the last ethanol dose. High blood ethanol levels were required, because argyrophilia, absent from isocaloric controls, also was only evident in ethanol-intoxicated rats with mean blood ethanol levels for days 2 to 4 above 300 mg/dl; however, it increased substantially between 350 and 550 mg/dl. The resemblance of the argyrophilic distribution to the regional neuropathology that occurs in experimental seizures indicates that the ethanol-induced degeneration may have an excitotoxic basis. Progressive reductions in the seizure threshold (e.g., kindling phenomena that have been documented during binge ethanol intoxication) might be associated with excitotoxic hyperactivity during the repetitive nadirs between high blood and brain ethanol peaks. However, direct toxic actions of ethanol or its metabolites could also be involved. Overall, the model should be useful for studying mechanisms of ethanol-induced selective cortical and olfactory brain damage.

MK-801 neurotoxicity in male mice: histologic effects and chronic impairment in spatial learning.

Several histological and behavioral experiments were conducted to investigate the neurotoxic effects of MK-801 in male mice. Moderate subcutaneous (s.c.) doses of MK-801 (0.5 and 1.0 mg/kg) induced the formation of intracytoplasmic vacuoles in pyramidal neurons in layers III and IV of the posterior cingulate/retrosplenial (PC/RS) cortex in 50% and 100% of the mice from the two respective treatment groups. Electron microscopic analysis of the vacuoles indicated that mitochondria and endoplasmic reticulum are the cellular organelles most prominently involved in this pathomorphological change. Treating mice with a high systemic dose of MK-801 (10 mg/kg s.c. or intraperitoneal (i.p.)) caused selective, irreversible degeneration of a small number of PC/RS cortical neurons. Compared to saline controls, the acquisition performance of mice treated i.p. with 10 mg/kg MK-801 was chronically impaired on a spatial learning task (modified hole board food search task) when tested at several posttreatment intervals (up to at least 5 months), although the groups did not differ on activity or sensorimotor tests conducted 2 weeks posttreatment. In summary, MK-801 caused histopathological changes in the mouse brain similar to those observed in the rat. Furthermore, high dose MK-801 treatment that killed a small number of mouse PC/RS cortical neurons resulted in a chronic acquisition impairment in spatial learning, an effect not previously demonstrated in any species.