ALTERATIONS IN RECEPTORS CONTROLLING DOPAMINE SYNTHESIS AFTER CHRONIC ETHANOL INGESTION

The influence of acute and chronic ethanol treatment and withdrawal on regulation of dopamine synthesis in striatal and mesolimbic areas of mouse brain was evaluated. Tyrosine hydroxylase activity was estimated by measuring in vivo DOPA accumulation after inhibition of aromatic amino acid decarboxylase. Eight hours after a single (3 g/kg) dose of ethanol, DOPA synthesis was increased and pimozide, a dopamine receptor antagonist, stimulated DOPA synthesis to the same degree in ethanol-treated and control animals. On the other hand, 8 h after withdrawal of animals from chronic ethanol treatment, endogenous dopamine synthesis was the same in ethanol-withdrawn and control animals, but the stimulation of dopamine synthesis produced by low doses of pimozide or haloperidol was significantly less in the animals that had consumed ethanol. This effect was even more apparent at 24h after withdrawal; by 3 days after withdrawal the decreased response of ethanol-withdrawn animals to the administration of dopamine receptor blockers was no longer statistically significant. At all time points tested, high doses of pimozide or haloperidol stimulated DOPA synthesis equally in control and ethanol-withdrawn animals. Chronic ethanol treatment and withdrawal may alter the coupling between dopamine receptors which regulate dopamine synthesis and tyrosine hydroxylase.     

CHANGES IN BRAIN ALCOHOL DEHYDROGENASE ACTIVITY DURING CHRONIC ETHANOL INGESTION AND WITHDRAWAL

Abstract— Chronic ethanol ingestion in rats leads to a slow rise in brain alcohol dehydrogenase activity which levels off after 2 weeks at approximately twice the initial activity. The half-time of the rise is approximately 8 days. Abrupt withdrawal of the ethanol is followed by a rapid decline of the brain alcohol dehydrogenase activity to the normal level with a half-time of approximately 15 h. The difference in time constants between the rise in enzyme activity during ethanol-feeding and its decline following withdrawal suggests that the increased enzyme activity is at least in part the result of a reduced rate constant of enzyme degradation in the presence of ethanol. The effect of ethanol on brain alcohol dehydrogenase activity is not altered by supplementation of the diet with carbohydrate or vitamins. The effect is seen only in the cerebral hemispheres and not in the brain-stem. Acquisition of tolerance to ethanol during chronic ethanol ingestion and its extinction following withdrawal follow almost the same time courses as the changes in brain alcohol dehydrogenase activity.     

ALTERATIONS IN GABA METABOLISM AND MET-ENKEPHALIN CONTENT IN RAT BRAIN FOLLOWING REPEATED ELECTROCONVULSIVE SHOCKS

The effects of electroconvulsive shock (ECS; 120 V for 1 s through ear-clip electrodes) or sub-convulsive shocks (70 V for 1 s) on rat brain GABA and met-enkephalin concentration and GABA turnover has been examined 24 h after a single treatment (×1) or once daily for 10 days (×10). ECS × 10 increased GABA concentrations in the N. caudatus and N. accumbens and decreased the synthesis rate of GABA by 40% and 50% respectively in these regions. Sub-convulsive shocks (× 10 × 10) or ECS × 1 had no effect. No consistent changes were seen in the substantia nigra. Met-enkephalin concentrations increased by 50% in the N. caudatus after ECS × 10 but were unchanged in the cortex and pons/medulla. No other shock regimen had any effect on the concentration of this peptide. The results are discussed in relation to the enhanced monoamine-induced responses seen only after ECS × 10.     

REGIONAL BRAIN INDOLEAMINE METABOLISM FOLLOWING CHRONIC PORTACAVAL ANASTOMOSIS IN THE RAT

Abstract— Four weeks after portacaval anastomosis in the rat. a profound change in the pattern of the plasma neutral ammo acids occurred. These changes were accompanied by marked regional changes in brain trvptophan. 5-HT (5-hydroxytryptamine) and 5-HIAA (5-hydroxyindoleacetic acid). Tryptophan was elevated in all the regions studied as was 5-HIAA. 5-Hydroxytryptamine was significantly elevated only in midbrain and medulla pons. A small but significant increase in free trvptophan concentration in plasma was seen in rats following portacaval anastomosis, but this elevation was insufficient in magnitude to account for thc changes in brain trvptophan. Administration of a solution containing equimolar concentrations of the three branched-chain amino acids, leucine. isoleucine and valine. caused a decrease in brain indoles towards normal levels. These results suggest that the altered plasma neutral amino acid pattern which accompanies portacaval anastomosis and its effect on competitive amino acid transport across the blood brain barrier is an important factor contributing to the raised levels of indoles in brain under these circumstances. The relationship of these results to the recently reported use of amino acid infusions in the treatment of hepatic encephalopathy is discussed.     

EFFECTS OF CHRONIC ETHANOL INGESTION ON BRAIN AMINOACYL-tRNA SYNTHETASES AND tRNA

The effects of chronic ethanol ingestion on the in vivo aminoacylation of brain transfer RNA (tRNA) were examined in C57BL/6J mice. A pronounced inhibition in the formation of [¹⁴C]leucy]-tRNA and [¹⁴C]phenylalanyl-tRNA was observed in the ethanol drinking mice. Properties of aminoacyl-tRNA synthetases and tRNA were examined following their separation and isolation on a DEAE-cellulose column. Synthesis of [¹⁴C]leucyl-tRNA was found to have a complete dependence on ATP and Mg²⁺. Incubations were carried out by cross-matching tRNA from control rat brain with synthetases obtained from the brains of control or ethanol-drinking mice. Under these conditions, a decreased ability for aminoacylation could be demonstrated when the source of enzyme was derived from ethanol-treated brain. The data indicate that the major effect of ethanol ingestion on the aminoacylation reaction is exerted on aminoacyl-tRNA synthetases.     

ALTERATIONS IN BRAIN OCTOPAMINE AND BRAIN TYROSINE FOLLOWING PORTACAVAL ANASTOMOSIS IN RATS

Abstract— Following portacaval anastomosis in the rat, plasma and brain tyrosine are markedly elevated, although brain tyrosine is increased to a significantly greater degree than plasma tyrosine. Both plasma and brain tyrosine as well as brain octopamine correlated well with a presumed index of impaired hepatic function, the ratio of liver weight to body weight in shunted rats. A significant positive correlation was observed between brain octopamine and brain tyrosine. The significance of these findings to the etiology and treatment of clinical hepatic encephalopathy is discussed.     

EFFECTS OF ETHANOL ON SEROTONIN METABOLISM IN BRAIN

The effect of ethanol on serotonin metabolism in brains of mice was determined both after a single injection and ‘chronic’ administration of ethanol. Behavioral effects were also monitored.‘Chronic’ administration of ethanol by inhalation to mice resulted in an increased susceptibility to Metrazole induced seizures. This susceptibility was evident for 48 h after ‘withdrawal’ of mice from ethanol chambers. No differences in brain 5-HT levels between control and ethanol treated mice were evident during withdrawal. However, a significant elevation in brain 5-HIAA levels was noted during this period. Short lived increases in brain 5-HIAA levels were also noted after a single injection of ethanol. Ethanol treatment produced no significant changes in the activity of brain MAO, aldehyde dehydrogenase, or aldehyde reductase. Other mechanisms for ethanol induced increases in brain 5-HIAA are discussed.     

PRECONVULSIVE CHANGES IN BRAIN GLUCOSE METABOLISM FOLLOWING DRUGS INHIBITING GLUTAMATE DECARBOXYLASE

—Brain glucose and glycogen concentrations have been studied in mice treated with allylglycine, 4-deoxypyridoxine and isoniazid, and the effects compared with the preconvulsive increase in brain glucose and glycogen concentration that follows d , l -methionine sulphoximine treatment. Allylglycine (180 mg/kg), 4-deoxypyridoxine (250 mg/kg), isoniazid (150 mg/kg) and d ,l -methionine sulphoximine (300 mg/kg) when given to mice at room temperature, cause a fall in rectal temperature which can be prevented by maintaining the mice in an incubator at 33-34°C. An increase in brain glucose concentration is seen after allylglycine (+ 133%), d ,l -methionine sulphoximine (+ 113%) and 4-deoxypyridoxine (+ 70%) treatment when mice are kept at room temperature and killed before convulsions occur. This is associated with a rise in blood glucose concentration after allylglycine, but not after the other drugs. Preventing the fall in rectal temperature reduces, but does not abolish, the rise in brain glucose concentration seen after allylglycine, d ,l -methionine sulphoximine and 4-deoxypyridoxine. Brain glycogen concentration increases at room temperature after D,L-methionine sulphoximine and 4-deoxypyridoxine, but in mice with maintained body temperature only 4-deoxypyridoxine produces an increase in brain glycogen. Isoniazid does not increase brain glucose or glycogen at room temperature, but reduces their concentration in mice kept in the incubator. All four drugs are known to act on amino acid metabolism; d ,l -methionine sulphoximine potently inhibits glutamine synthetase whereas 4-deoxypyridoxine, allylglycine and isoniazid inhibit glutamate decarboxylase. The connection, if any, between a block in the further metabolism of glutamate and an increase in brain glucose and glycogen is unknown.     

THE EFFECTS OF INTOXICATING DOSES OF ETHANOL UPON INTERMEDIARY METABOLISM IN RAT BRAIN

Abstract— The effect of acute (8-min) and prolonged (13-h) exposures to high doses of ethanol upon the intermediary metabolites of rat brain has been studied, with the use of a new freezing technique which minimizes post-mortem changes. Injection of ethanol (80 mmol/kg body wt) produced general anaesthesia within 8 min after administration. At this time there were increases in the brain contents of glucose, glucose-6-phosphate and citrate; there was no change in arterial pCO₂. Rats under ethanol anaesthesia for 13 h showed increases in brain contents of glycogen, glucose and glucose 6-phosphate; and decreases in lactate, pyruvate, α-oxoglutarate and malate. Under similar experimental conditions, arterial pCO₂, increased from 37 to 51 Torr. The changes in levels of metabolites after injection of ethanol were similar to those after administration of many volatile anaesthetic agents or elevation of brain CO₂ by other means. Although brain levels of malate and α-oxoglutarate decreased after prolonged exposure to ethanol, the mitochondrial redox state was maintained. Accordingly, the levels of glutamate and aspartate fell in accordance with the law of mass action. The maintenance of the cytoplasmic and mitochondrial redox states in the brain during ethanol intoxication was in marked contrast to the effects on the liver. We suggest that the different effects observed in brain and liver result from the action of ethanol upon the nerve cell membrane in brain, whereas the primary target in liver is alcohol dehydrogenase.     

EFFECTS OF HYPOPHYSECTOMY ON RNA METABOLISM IN RAT BRAIN STEM

Abstract— Ribosomal aggregates were isolated from rat brain stem and characterized as polysomes by sedimentation analysis and by their sensitivity to RNase and EDTA treatment.
Three weeks following hypophysectomy there was a significant decrease in the content of large polysomes in the rat brain stem. The incorporation of radioactive uridine into RNA was studied using a double-labelling technique with [³H]- and [¹⁴C]uridine and labelling periods of 70 and 180 min. It was found that after hypophysectomy the incorporation of radioactive uridine into total, nuclear and cytoplasmic RNA and in polysomes was decreased after 70 and 180 min. Information on the nature of the rapidly-labelled RNA in the various subcellular fractions was obtained by sucrose gradient sedimentation analysis.
After 70 min of labelling the nucleus contained heterogeneous RNA with a considerable fraction of RNA sedimenting faster than 28 S. In the cytoplasmic fraction heterogeneous 4 to 30 S RNA was found, presumably associated with RNP particles, whereas after 180 min the polyribosomal aggregates were also labelled.
The present results indicate a profound effect of hypophysectomy on the metabolism of all species of brain RNA investigated.     

THE GAMMA-AMINOBUTYRIC ACID (GABA) SYSTEM IN BRAIN DURING ACUTE AND CHRONIC ETHANOL INTOXICATION

Abstract— The effects of acute and chronic ethanol intoxication on the GAGA system of rats have been investigated. Under the terminal conditions provoked. by ethanol (6–8 g/kg, i.p.) the brain GABA content sharply increased. There was a simultaneous decrease of 35–40% in the glutamate decarboxylase (GAD) activity of the cerebellum and cerebral hemispheres. In contrast, the transaminase, GABA-T was either unchanged, or it increased: by 28% only in cerebellum and by 1.5–2.0–fold in liver and kidney. It is suggested that effects of acute ethanol intoxication at different doses (2–8 g/kg) on the brain GABA system is connected with the phases of the functional condition of the CNS and a disturbance of homeostatic function. Chronic ethanol consumption caused a decrease in brain GABA. an increase of GAD activity in cerebellum and cerebral hemispheres, and no change in GABA-T activity. The activity of this last enzyme was increased 1.5–2.0-fold in liver and kidneys of rats consuming a diet containing 10% ethanol daily. A 50-fold purified preparation of GABA-T obtained from pig brain was inhibited by butanol-l and propanol-1 (0.03–0.6m) with no effect of ethanol. It is suggested that the mechanisms involved in the ethanol effect on nervous cells are linked with the GABA system and the phases of the functional condition of the CNS.     

REGIONAL BIOSYNTHESIS OF ACETYLCHOLINE IN BRAIN FOLLOWING FORCED ORAL CHRONIC BARBITONE TREATMENT TO RAT

Rats received a solution of sodium barbitone as their only drinking fluid for 33 and 42–44 weeks. In three groups (A3, A12 and A30) the barbitone solution was withheld and replaced by water 3, 12 and 30 days respectively before death. Two other groups consisted of animals drinking barbitone until death (B) and untreated controls (C). Abstinence convulsions were recorded by jiggle cages. Thirty nmol of tritium-labelled choline ([³H]Ch) were injected i.v. and the rats were killed by decapitation 1 min later. A significantly higher content of tritium-labelled acetylcholine ([³H]ACh) was found in the cerebellum + medulla oblongata + midbrain of rats receiving barbitone until death (group B) (+22%) and abstinent for 3 days (+54%) (group A3) compared with group C. The [³H]ACh content was also significantly increased in the hippocampus + cortex of rats abstinent for 3 days (+23%). In the striatum no significant effect on [³H]ACh content was found in any of the groups. The ratio [³H]ACh/[³H]Ch was significantly increased in the cerebellum + medulla oblongata + midbrain of rats in group B and A3 and in the hippocampus + cortex in group A3. These results might indicate an increased turnover of ACh. The effect of long-term barbitone treatment on the enzyme activities of brain choline acetyltransferase and acetylcholinesterase was also studied but no significant effect was found.     

RNA METABOLISM IN ISOLATED MOUSE BRAIN NUCLEI DURING EARLY POSTNATAL DEVELOPMENT

—RNA metabolism in isolated brain nuclei has been shown to be dramatically altered during early postnatal brain development. The present study involved an examination of the RNA products synthesized by nuclei at various stages of postnatal neural maturation. In all cases, the majority of the RNA appeared to be heterodisperse, non-ribosomal and non-tRNA in nature. In comparison to the RNA isolated from nuclei of neonatal tissue, the RNA from nuclei of 12-day and 30-day-old mouse brain was found to be of smaller molecular weight. Despite the heterodisperse nature of these RNA molecules, the addition of α-amanitin did not completely inhibit nuclear synthesis. An investigation of RNA synthesis in isolated neuronal and glial cell nuclei revealed that nucleic acid metabolism in these respective cell populations had different and distinct developmental patterns. Preparations enriched with glial cell nuclei were found to be most active at birth and then decreased in activity (3–4-fold) during neural maturation. On the other hand, the rate of RNA synthesis in fractions enriched in neuronal cell nuclei was observed to increase dramatically in activity (4–5-fold) until 14 days of age. From 14 days of age until adulthood, RNA synthetic activity remained essentially the same.     

NUCLEOTIDE METABOLISM IN RAT BRAIN

Abstract— The uptake, the conversion to nucleotides, and their incorporation into RNA for labelled glycine, aspartate, the free bases and nucleosides of purines and pyrimidines were investigated with cortical slices of rat cerebrum. At the end of a 1-hr incubation time the slice-to-medium ratio of the radioactivities for labelled aspartate, glycine, adenine and adenosine were 34, 26, 20 and 5, respectively, while the slice-to-medium ratios for hypoxanthine, inosine, guanine, guanosine, xanthine, orotate, cytidine, cytosine, uridine, and uracil ranged from 1.3:1 to 2:1. Over 99 per cent of the total radioactivity taken up by the cortical slices was present in the TCA supernatant and 86, 82, 65, 50, 34, 23, 20 and 1.6 per cent of this radioactivity was in the form of nucleotides at the end of a 1-hr incubation with labelled adenine, adenosine, hypoxanthine, inosine, uridine, orotate, cytidine, and glycine, respectively. The incorporation of various radioactive precursors into RNA of cortical slices suggests that nucleotides originating from either de novo synthesis or preformed purine derivatives enter the same nucleotide pool utilized for RNA synthesis. The supernatant fraction from homogenized cerebrum was investigated for the presence of various anabolic and catabolic enzymes associated with nucleotide metabolism. These results were correlated with the data from the RNA incorporation studies, and a possible role for AMP: pyrophosphate phosphoribosyltransferase (adenine phosphoribosyltransferase, I.U.B. 2.4.2.7) to achieve intercellular transfer of AMP is discussed.     

THE EFFECTS OF CHRONIC REGIMENS OF CLORGYLINE AND PARGYLINE ON MONOAMINE METABOLISM IN THE RAT BRAIN

The effects of chronic administration of clorgyline and pargyline on rat brain monoamine metabolism have been examined. The inhibitory selectivity of these drugs towards serotonin deamina-tion (MAO type A) and phenylethylamine deamination (MAO type B) can be maintained over a 21-day period by proper selection of low doses of these drugs (0.5-1.0 mg/kg/24h). The results are consistent with MAO type A catalyzing the deamination of serotonin and norepinephrine and with MAO type B having little effect on these monoamines. Dopamine appears to be dcaminated in vivo principally by MAO type A. Clorgyline administration during a 3-week period was accompanied by persistent elevations in brain norepinephrine concentrations; serotonin levels were also increased during the first 2 weeks, but returned towards control levels by the third week of treatment. Low doses of pargyline did not increase brain monoamine concentrations, but treatment with higher doses for 3 weeks led to elevations in brain norepinephrine and 5-hydroxytryptamine; at this time significant MAO-A inhibition had developed. The changes in monoamine metabolism seen at the end of the chronic clorgyline regimen are not due to alterations in tryptophan hydroxylase activity. At this time tyrosine hydroxylase activity was also unaffected.     

OBSERVATIONS ON PURINE METABOLISM IN RAT BRAIN

Both de novo and preformed base or salvage pathways are simultaneously operative in the biosynthesis of purine nucleotides in rat brain per se. A preferential utilization of de novo precursors is demonstrated.     

EFFECTS OF CHRONIC CONSUMPTION OF ETHANOL AND SUCROSE ON RAT WHOLE BRAIN 5-HYDROXYTRYPTAMINE

Abstract— Whole brain 5-hydroxytryptamine (5-HT) levels were determined after 3 weeks in rats chronically consuming ethanol, in pair-fed controls, and in pair-fed controls consuming sucrose in quantities isocaloric to the ethanol of the first group. A cryogenic harvesting and storage technique was developed to insure accurate measurement of whole rat brain 5-HT. It was found that whole brain 5-HT is completely stable for at least 16 days after decapitation into liquid N₂ followed by storage of frozen whole heads at – 70°C. Using this technique and spectrophotofluorometric 5-HT assays, no differences were found in whole brain 5-HT among the three groups. Chronic consumption of ethanol and sucrose apparently lead to no chronic change in rat whole brain 5-HT.