Effect of chronic ethanol exposure and its withdrawal on the endocannabinoid system

The present study investigated the effect of ethanol (EtOH) exposure and its withdrawal on the central endocannabinoid system utilizing an EtOH vapor inhalation model, which is known to produce functional tolerance and dependence to EtOH. Swiss Webster mice (n=24) were exposed to EtOH vapors for 72h. Mice were sacrificed after 72h following EtOH exposure (n=12) and 24h after its withdrawal (n=12). Radioligand binding assays were performed to measure the density of CB(1) receptor and CB(1) receptor agonist-stimulated [(35)S]GTPgammaS binding in crude synaptic membranes isolated from the cortex, hippocampus, striatum and cerebellum. The density of CB(1) receptor was significantly decreased (31-39%) in all the brain regions when compared to the control group. The CB(1) receptor-stimulated G(i/o) protein activation was also found to be decreased (29-40%) in these brain regions of EtOH exposed mice. Recovery of the CB(1) receptor density, in addition to, the CB(1) receptor-mediated G-protein activation was observed after 24h withdrawal from EtOH. The levels of cortical anandamide, which was significantly increased (147%) by EtOH exposure, returned to basal levels after 24h of withdrawal from EtOH exposure. A significant reduction (21%) in the activity of fatty acid amide hydrolase was found in the cortex of EtOH administered mice. Taken together, the neuroadaptation in the EC system may have a potential role in development of tolerance and dependence to EtOH.     

Glutamyl transferase and acetylcholinesterase activity in various areas of the rat brain in alcoholic intoxication

Albino mongrel rats were used for the determination of the gamma-glutamyl transferase (gamma-GTF) and acetylcholine esterase (AChE) activities in various brain areas (cerebral hemispheres, cerebellum, hippocampus, brain stem) during acute (1.5; 4 and 6 g/kg i. p.) and chronic (15 months) alcoholic intoxication and alcohol withdrawal (24-48 h, 4 and 8 days). An increase or a decrease in the activity of these two enzymes in the various rat brain areas depends on the dose of ethanol and the time of its action. The activity of gamma-GTF grew in all brain areas during chronic ethanol intoxication; the activity of AChE was also enhanced in three brain areas but it was diminished in cerebral hemispheres. Alcohol withdrawal caused diverse changes in the activities of these two enzymes in various areas of the brain. A tendency to normalization of the gamma-GTF and AChE activities is manifested 4-8 days after alcohol withdrawal.     

Aspartate Aminotransferase for Synthesis of Transmitter Glutamate in the Medulla Oblongata: Effect of Aminooxyacetic Acid and 2-Oxoglutarate

The effects of aminooxyacetic acid (AOAA), a transaminase inhibitor, and 2-oxoglutarate, a precursor to glutamate by the activity of aspartate aminotransferase (AAT), on slices of rat medulla oblongata, cerebellum, cerebral cortex, and hippocampus were studied. The slices were superfused and electrically stimulated. There was a Ca2+-dependent stimulus-evoked release of endogenous glutamate, gamma-aminobutyric acid (GABA), and beta-alanine in all regions examined. AOAA (10(-4) and 10(-3) M) decreased the release of glutamate in the medulla oblongata and cerebellum but not in the hippocampus. L-Canaline, a specific inhibitor of ornithine aminotransferase, did not affect the glutamate release in the medulla. 2-Oxoglutarate (10(-3) M) increased the release of glutamate in the medulla oblongata and cerebellum but not in the cerebral cortex and hippocampus. Treatment with AOAA (10(-4) M) almost abolished the activities of AAT in all regions studied. AOAA (10(-4) and 10(-3) M) increased the stimulus-evoked release of GABA in the cerebellum, cerebral cortex, and hippocampus, whereas the stimulus-evoked release of beta-alanine was decreased by this agent in all regions studied. These results suggest the participation of AAT in the synthesis of the transmitter glutamate in the medulla oblongata and cerebellum of the rat.     

The Posttranslational Arginylation of Proteins in Different Regions of the Rat Brain

The posttranslational incorporation of arginine into proteins catalyzed by arginyl-tRNA protein transferase was determined in vitro in different rat brain regions. The incorporation was found in all the regions studied, although with different specific activities (pmol [14C]arginine incorporated/mg protein). Of the regions studied, hippocampus had the highest specific activity followed by striatum, medulla oblongata, cerebellum, and cerebral cortex. Electrophoretic analysis of the [14C]arginyl proteins from the different regions followed by autoradiography and scanner densitometry showed at least 13 polypeptide bands that were labeled with [14C]arginine. The radioactive bands were qualitatively coincident with protein bands revealed by Coomassie Blue. There were peaks that showed different proportions of labeling in comparison with peaks of similar molecular mass from total brain. Most notable because of their high proportions were those of molecular mass 125 kDa in hippocampus, striatum, and cerebral cortex; 112 and 98 kDa in striatum and cerebellum; and 33 kDa in hippocampus and striatum. In lower proportions than in total brain were the peaks of 33 kDa in medulla oblongata and cerebral cortex and of 125 kDa in medulla oblongata.     

Differential Effect of Cerebral Ischemia on Monoamine Content of Discrete Brain Regions of the Mongolian Gerbil (Meriones unguiculatus)

The effect of bilateral cerebral ischemia on noradrenaline, dopamine, and serotonin concentrations in six brain regions (i.e., the cerebral cortex, striatum, hippocampus, midbrain-diencephalon, cerebellum, and pons-medulla oblongata) was examined in the gerbil stroke model. The relative changes in regional cerebral blood flow after bilateral common carotid occlusion were also assessed using the radioactive microsphere technique. At 1 h after bilateral carotid occlusion, a significant decrease of monoamine concentration was observed in the cerebral cortex, striatum, hippocampus, and midbrain-diencephalon whereas no significant change was detected in the cerebellum and pons-medulla oblongata. The fall in NA content was most prominent in the cerebral cortex and hippocampus and percentage reductions of dopamine and serotonin were greatest in the striatum and cerebral cortex, respectively. These results suggest that the monoamine neurons in various brain regions might have different vulnerabilities to ischemic insult and show no evidence of transtentorial diaschisis.     

Effect of thyroidectomy on phosphorylative oxidation and RNA synthesis in various regions of the brain in the adult monkey

The subcellular effects of thyroidectomy in selected brain regions of Cynomolgus monkey were analyzed. 20 days after operation the respiratory rates, the activities of succinate cytochrome c reductase, glycerol-3-phosphate dehydrogenase and of oligomycin-sensitive ATPase were decreased in mitochondria isolated from all brain structures. The highest reduction (30%) was found in cerebral cortex and hippocampus. Cerebellar and striatal activities were reduced by about 20%. A smaller decrease (15%) was observed in thalamus. The effects of thyroidectomy on in vitro RNA synthesis were followed in cerebral cortex, cerebellum and thalamus. In the three analyzed regions, the activities of nucleolar and nucleoplasmic RNA polymerases dropped by 40%. Replacement therapy with T4 (2.5 micrograms/kg/day) or T3 (1 microgram/kg/day) administered immediately after thyroidectomy for 20 days, maintained mitochondrial and nuclear activities at normal level.     

RNA polymerase I and II activities in different brain regions of young, adult, and old rats

The activities of RNA polymerase I and II were assayed in nuclei isolated from different regions (cerebral cortex, cerebellum, hypothalamus, hippocampus, corpus striatum and pituitary) of brains from young (10 days), adult (6 months), and old (2 years) rats. The RNA polymerases I and II activities generally increased during maturation, i.e., from 10 days to 6 months of postnatal age and then showed a decrease from 6 months to 2 years of age in all the regions except in cerebral cortex where the RNA polymerase II activity was highest at 10 days but showed a gradual decrease through the lifespan up to 2 years.     

Poster Sessions CP13: Hypoxia,Ischemia and Oxidative Stress. Changes of brain nitric oxide production in experimental cerebral ischemia

In order to study the role of nitric oxide (NO) in ischemic brain injury. Global cerebral ischemia was established in SD rats by modified Pulsinelli's method. The activities of constitutive nitric oxide synthase (cNOS), inducible NOS (iNOS), neuronal NOS (nNOS), nitrite (NO₂) and cyclic GMP in cerebral cortex, hippocampus, striatum and cerebellum at different time intervals were measured by radioimmunoassy, NADPH‐d histochemistry and fluorometry methods. The results showed that the activities of cNOS increased at 5 min in four regions and decreased in cortex, hippocampus and striatum at 60 min, in cerebellum at 15 min iNOS increased in cortex and striatum at 15 min, in hippocampus and cerebellum at 10 min, and persisted to 60 min. The expression of nNOS increased after 5 min ischemia in cortex, striatum and hippocampus, and return to normal at 30–60 min. The NO₂ and cGMP also increased after 5–15 min ischemia and returned to normal after 30–60 min ischemia. These results indicated that the NO participated in the pathogenesis of cerebral ischemia injury and different types of NOS play different role in the cerebral ischemia injuries. Selected specific NOS inhibitors to decreased the excessive production of NO at early stage may help to decrease the ischemic injury.     

Changes in the cannabinoid receptor binding, G protein coupling, and cyclic AMP cascade in the CNS of rats tolerant to and dependent on the synthetic cannabinoid compound CP55,940

Chronic exposure to CP55,940 produced a significant down-regulation of cannabinoid receptors in the striatum, cortex, hippocampus, and cerebellum of rat brain. At 24 h after SR141716-precipitated withdrawal, we observed a tendency to return to basal levels in the striatum and cortex, whereas the specific binding remained lower in the hippocampus and cerebellum. When we surveyed cannabinoid receptor-activated G proteins, in chronic CP55,940-treated rats the guanosine 5'-O:-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding assay revealed a decrease of activated G proteins in the striatum, cortex, and hippocampus, whereas no significant changes were seen in the cerebellum. At 24 h after the SR141716-precipitated withdrawal, [(35)S]GTPgammaS binding increased compared with that of rats chronically exposed to CP55,940, attaining the control level except for cerebellum, where we observed a trend to overcome the control amounts. Concerning the cyclic AMP (cAMP) cascade, which represents the major intracellular signaling pathway activated by cannabinoid receptors, in the cerebral areas from rats chronically exposed to CP55,940 we found alteration in neither cAMP levels nor protein kinase A activity. In the brain regions taken from CP55, 940-withdrawn rats, we only observed a significant up-regulation in the cerebellum. Our findings suggest that receptor desensitization and down-regulation are strictly involved in the development of cannabinoid tolerance, whereas alterations in the cAMP cascade in the cerebellum could be relevant in the mediation of the motor component of cannabinoid abstinence.     

Protective Role of Cynodon dactylon in Ameliorating the Aluminium-Induced Neurotoxicity in Rat Brain Regions

Cynodon dactylon (Poaceae) is a creeping grass used as a traditional ayurvedic medicine in India. Aluminium-induced neurotoxicity is well known and different salts of aluminium have been reported to accelerate damage to biomolecules like lipids, proteins and nucleic acids. The objective of the present study was to investigate whether the aqueous extract of C. dactylon (AECD) could potentially prevent aluminium-induced neurotoxicity in the cerebral cortex, hippocampus and cerebellum of the rat brain. Male albino rats were administered with AlCl(3) at a dose of 4.2?mg/kg/day i.p. for 4?weeks. Experimental rats were given C. dactylon extract in two different doses of 300?mg and 750?mg/keg/day orally 1?h prior to the AlCl(3) administration for 4?weeks. At the end of the experiments, antioxidant status and activities of ATPases in cerebral cortex, hippocampus and cerebellum of rat brain were measured. Aluminium administration significantly decreased the level of GSH and the activities of SOD, GPx, GST, Na(+)/K(+) ATPase, and Mg(2+) ATPase and increased the level of lipid peroxidation (LPO) in all the brain regions when compared with control rats. Pre-treatment with AECD at a dose of 750?mg/kg b.w increased the antioxidant status and activities of membrane-bound enzymes (Na(+)/K(+) ATPase and Mg(2+) ATPase) and also decreased the level of LPO significantly, when compared with aluminium-induced rats. The results of this study indicated that AECD has potential to protect the various brain regions from aluminium-induced neurotoxicity.     

CYCLIC NUCLEOTIDES and PROTEIN KINASE ACTIVITY IN THE RAT BRAIN POSTMORTEM

Abstract— The components of the cyclic nucleotide system were studied in rat brain at various times after death to determine the stability of the system postmortem. The concentration of cyclic AMP in 4 brain regions was highest 10 min after death and declined to stable levels within 6 h after death. At this time concentrations approximated those normally seen in vitro. The concentration of cyclic GMP in brain regions fell markedly postmortem and was undetectable 6 or 16 h after death. Nor-epinephrine-stimulated cyclic AMP accumulation measured in vitro in slices of the cerebral cortex was the same 10 min and 16 h postmortem. In the cerebellum, however, accumulated levels of cyclic AMP were lower 16 h postmortem, although the degree of stimulation elicited by norepinephrine was the same 10 min and 16 h after death. Cyclic AMP-dependent and independent protein kinase activities were detected in the rat brain at all times after death. There was no change in the cyclic AMP-dependent enzyme activity postmortem, but activity in the absence of cyclic AMP was significantly higher immediately after death compared to 6 or 16 h postmortem. These studies indicate that it should be possible to study the cyclic AMP system in human brain tissue obtained at autopsy.     

Brain benzodiazepine binding sites in ethanol dependent and withdrawal states

The brain benzodiazepine system has been implicated to be important in both the mechanism, and treatment of ethanol related syndromes. In this report evidence is presented which indicates that "peripheral type" benzodiazepine binding sites are probably more relevant than "central type" receptors for the neurochemical consequences of ethanol dependence and withdrawal states. Utilizing radioreceptor binding techniques 20-50% increases in the binding of [3H]RO-5-4864 (a "peripheral type" ligand) to brain membranes derived from rat cerebral cortex, cerebellum and hippocampus are observed in ethanol dependent rats. These increases persist for 3 days after cessation of ethanol. The number of [3H]RO-5-4864 binding sites in cerebellum returns to normal during 4-7 days after ethanol withdrawal. In all brain areas examined no changes were observed in the "central type" benzodiazepine receptor as judged by [3H]-ethyl-Beta-carboline-3-carboxylate, BCCE binding. Scatchard analysis revealed that the number of [3H]RO-5-4864 binding sites is increased in each brain area while the affinity was unchanged.     

Developmental Changes in the NGF Content in the Brain of Young,Growing, Low-Birth-Weight Rats

The NGF content in each region of the brain of four-week-old rats was ranked in the decreasing order of cerebral cortex, hippocampus, cerebellum, midbrain/diencephalon, and pons/medulla ob-longata, and the NGF concentration, in the decreasing order of hippocampus, cerebral cortex, cerebellum, midbrain/diencephalon, and pons/medulla oblongata in both AFD and SFD groups. The NGF content and concentration in the cerebral cortex were about the same value at each age between those in the AFD and SFD groups. Those in the hippocampus were a little higher in the SFD group than in the AFD group at the ages of three and four weeks, unlike those in the other regions, where the values for the cerebellum, midbrain/diencephalon and pons/medulla oblongata tended to be somewhat higher in the AFD group than in the SFD group. The NGF concentrations in the hippocampus and cerebral cortex increased with growth: the concentration in the hippocampus at four weeks of age was about 4-fold of that at one week in the AFD group and about 5.7-fold of that at one week in the SFD group; and likewise the concentration in the cerebral cortex at four weeks of age was about 5.3-fold in the AFD group and about 7-fold in the SFD group. The NGF concentrations in the cerebellum decreased, and those in midbrain/diencephalon and pons/medulla oblongata hardly changed with growth in either AFD or SFD group. From these results NGF may have stronger implications for the neuronal growth in the hippocampus compared with those in the lower brain regions of the SFD rats.     

Modulation of NMDA Receptor Subunit mRNA in Butorphanol-Tolerant and —Withdrawing Rats

The NMDA receptor has been implicated in opioid tolerance and withdrawal. The effects of continuous infusion of butorphanol on the modulation of NMDA receptor subunit NR1, NR2A, NR2B, and NR2C gene expression were investigated by using in situ hybridization technique. Continuous intracerebroventricular (i.c.v.) infusion with butorphanol (26 nmol/l/h) resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels. The level of NR1 mRNA was significantly decreased in the cerebral cortex, thalamus, and CA1 area of hippocampus in butorphanol tolerant and withdrawal (7 h after stopping the infusion) rats. The NR2A mRNA was significantly decreased in the CA1 and CA3 of hippocampus in tolerant rats and increased in the cerebral cortex and dentate gyrus in butorphanol withdrawal rats. NR2B subunit mRNA was decreased in the cerebral cortex, caudate putamen, thalamus, CA3 of hippocampus in butorphanol withdrawal rats. No changes of NR1, NR2A, NR2C subunit mRNA in the cerebellar granule cell layer were observed in either butorphanol tolerant or withdrawal rats. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased significantly in all brain regions except in the thalamus and hippocampus, at the 7 hr after stopping the butorphanol infusion. These results suggest that region-specific changes of NMDA receptor subunit mRNA (NR 1 and NR2) as well as NMDA receptor binding ([³H]MK-801) are involved in the development of tolerance to and withdrawal from butorphanol.     

Acidification-induced changes in Cx43 protein–protein interactions

In order to study the role of nitric oxide (NO) in ischemic brain injury. Global cerebral ischemia was established in SD rats by modified Pulsinelli's method. The activities of constitutive nitric oxide synthase (cNOS), inducible NOS (iNOS), neuronal NOS (nNOS), nitrite (NO₂) and cyclic GMP in cerebral cortex, hippocampus, striatum and cerebellum at different time intervals were measured by radioimmunoassy, NADPH-d histochemistry and fluorometry methods. The results showed that the activities of cNOS increased at 5 min in four regions and decreased in cortex, hippocampus and striatum at 60 min, in cerebellum at 15 min iNOS increased in cortex and striatum at 15 min, in hippocampus and cerebellum at 10 min, and persisted to 60 min. The expression of nNOS increased after 5 min ischemia in cortex, striatum and hippocampus, and return to normal at 30–60 min. The NO₂ and cGMP also increased after 5–15 min ischemia and returned to normal after 30–60 min ischemia. These results indicated that the NO participated in the pathogenesis of cerebral ischemia injury and different types of NOS play different role in the cerebral ischemia injuries. Selected specific NOS inhibitors to decreased the excessive production of NO at early stage may help to decrease the ischemic injury.     

Cholinotoxicity induced by ethylcholine aziridinium ion after intracarotid and intracerebroventricular administration

The effect of ethylcholine aziridinium ion (AF64A) after an intracerebroventricular (icv) injection was compared to that obtained after an intravascular administration. Reductions in choline acetyltransferase (ChAT) and acetylcholinesterase activities in the hippocampus but not in the cerebral cortex or the corpus striatum were observed 10 days after bilateral injection of AF64A into the rat cerebroventricles (3 nmol/side). However, when AF64A was injected into the carotid artery (1 mumol/kg) following a unilateral opening of the blood-brain barrier by a hypertonic treatment, a significant decrease in ChAT activity was observed in the ipsilateral side of the cerebral cortex but not in hippocampus, corpus striatum, or cerebellum. High-affinity choline transport was reduced significantly 11 days after an icv injection of AF64A in all the above mentioned brain regions, and recovered 60 days post injection in the cerebral cortex and in the corpus striatum but not in the hippocampus. Our results suggest that in various brain regions, AF64A causes various degrees of damage to cholinergic neurons, depending on the quantity of the toxin that reaches the target tissue.