Hilary J. Little

Anticonvulsant profile of the dihydropyridine calcium channel antagonists, nitredipine and nimodipine

The effects of the dihydropyridine calcium channel antagonists, nitrendipine and nimodipine, on convulsions produced by different mechanisms have been studied in rats. Nitrendipine and nimodipine significantly raised the thresholds to pentylenetetrazol for up to six hours after their injection. The calcium channel agonist, BAY K 8644, lowered the convulsion threshold to pentylenetetrazol and antagonised the effects of nitrendipine. In contrast, the severity of seizures produced by N-methyl-dl-aspartate (NMA) was increased by nitrendipine. BAY K 8644 also slightly increased the effects of NMA. Nimodipine and nitrendipine caused small, but significant, increases in the threshold pressures for the convulsions caused by raising the atmospheric pressure with helium gas. The compounds had no effect on strychnine convulsions. The conclusion is that the calcium channel antagonists are anticonvulsant against only certain types of convulsions, such as pentylenetetrazol and high pressure (and ethanol withdrawal, reported previously). Others may be increased, such as NMA seizures, or unaffected, such as strychnine-induced convulsions.

Enduring Effects of Chronic Ethanol in the CNS: Basis for Alcoholism

This symposium focused on functional alterations in the mesolimbic dopamine system during the abstinence phase after chronic alcohol intake. Mark Brodie first described his recordings from midbrain slices prepared after chronic alcohol treatment in vivo by daily injection in C57BL/6J mice. No changes were found in the baseline firing frequency of dopaminergic neurones in the VTA (ventral tegmental area), but the excitation produced in these neurones by an acute ethanol challenge was significantly increased in neurons from ethanol-treated mice compared with those from the saline-treated controls. There was also a significant decrease in the inhibitory response to GABA by the dopamine neurones following the chronic ethanol treatment. These data suggest that the timing pattern and mode of ethanol administration may determine the types of changes observed in dopaminergic reward area neurons. Annalisa Muntoni lectured on the relationship between electrophysiological and biochemical in vivo evidence supporting a reduction in tonic activity of dopamine neurons projecting to the nucleus accumbens at various times after suspension of chronic ethanol treatment and morphological changes affecting dopamine neurons in rat VTA. Hilary J. Little then described changes in dopaminergic neurone function in the VTA during the abstinence phase. Decreases in baseline firing were seen at 6 days after withdrawal of mice from chronic ethanol treatment but were not apparent after 2 months abstinence. Increases in the affinity of D1 receptors in the striatum, but not in the cerebral cortex, were seen however up to 2 months after withdrawal. Scott Steffensen then described his studies recording in vivo from GABA containing neurones in the VTA in freely moving rats. Chronic ethanol administration enhanced the baseline activity of these neurones and resulted in tolerance to the inhibition by ethanol of these neurones. His results demonstrated selective adaptive circuit responses within the VTA or in extrategmental structures that regulate VTA-GABA neurone activity.

Social defeat increases alcohol preference of C57BL/10 strain mice; effect prevented by a CCKB antagonist

RationaleIn humans, social stress over long and short term can increase alcohol consumption, but the mechanisms involved are not understood.ObjectivesThis study was conducted to examine the effects of social defeat, using the resident/intruder paradigm, on the alcohol preference of “low alcohol drinking” individuals in a colony of C57BL/10 strain mice and the effects of two anxiolytic drugs.MethodsAlcohol preference, in a two-bottle choice (8% v/v alcohol or water), was measured, in separate experiments, after either a single experience of social defeat by a resident male mouse, five consecutive daily defeat experiences or one experience per week for 4 weeks. Comparison was made with effects of repeated social defeat on the preference for dilute sucrose. In addition, the actions of the CCKB receptor antagonist, CAM1028, and of diazepam were examined on the effects of repeated defeat experiences.ResultsFive consecutive daily defeat experiences had a slow onset effect in increasing alcohol preference and consumption, compared with five daily exposures to a novel environment. A single defeat, or one defeat per week, did not significantly alter alcohol preference or intake. There were no effects of five daily defeat experiences on sucrose preference or consumption. The effect of repeated defeats on alcohol preference was significantly decreased by administration of the CCKB receptor antagonist, CAM1028, prior to each experience, but not by corresponding administration of diazepam.ConclusionThe results show that social stress increases alcohol intake in low alcohol preference C57BL/10 mice and suggest that CCK transmission may be involved in this effect.

Selective increases in regional brain glucocorticoid: A novel effect of chronic alcohol

The hypothalamo-pituitary-adrenal axis shows functional changes in alcoholics, with raised glucocorticoid release during alcohol intake and during the initial phase of alcohol withdrawal. Raised glucocorticoid concentrations are known to cause neuronal damage after withdrawal from chronic alcohol consumption and in other conditions. The hypothesis for these studies was that chronic alcohol treatment would have differential effects on corticosterone concentrations in plasma and in brain regions. Effects of chronic alcohol and withdrawal on regional brain corticosterone concentrations were examined using a range of standard chronic alcohol treatments in two strains of mice and in rats. Corticosterone was measured by radioimmunoassay and the identity of the corticosterone extracted from brain was verified by high performance liquid chromatography and mass spectrometry. Withdrawal from long term (3 weeks to 8 months) alcohol consumption induced prolonged increases in glucocorticoid concentrations in specific regions of rodent brain, while plasma concentrations remained unchanged. This effect was seen after alcohol administration via drinking fluid or by liquid diet, in both mice and rats and in both genders. Shorter alcohol treatments did not show the selective effect on brain glucocorticoid levels. During the alcohol consumption the regional brain corticosterone concentrations paralleled the plasma concentrations. Type II glucocorticoid receptor availability in prefrontal cortex was decreased after withdrawal from chronic alcohol consumption and nuclear localization of glucocorticoid receptors was increased, a pattern that would be predicted from enhanced glucocorticoid type II receptor activation. This novel observation of prolonged selective increases in brain glucocorticoid activity could explain important consequences of long term alcohol consumption, including memory loss, dependence and lack of hypothalamo-pituitary responsiveness. Local changes in brain glucocorticoid levels may also need to be considered in the genesis of other mental disorders and could form a potential new therapeutic target.

Effects of minor laboratory procedures, adrenalectomy, social defeat or acute alcohol on regional brain concentrations of corticosterone

Concentrations of corticosterone in brain areas of TO strain mice were measured by radioimmunoassay. The studies examined the effects of routine laboratory maneuvers, variation during the circadian peak, adrenalectomy, social defeat and acute injections of alcohol on these concentrations. Brief handling of mice increased corticosterone levels in plasma but not in striatum and reduced those in the hippocampus. Single injections of isotonic saline raised the plasma concentrations to a similar extent as the handling, but markedly elevated concentrations in the three brain regions. Five minutes exposure to a novel environment increased hippocampal and cerebral cortical corticosterone levels and striatal concentrations showed a larger rise. However, by 30 min in the novel environment, plasma concentrations rose further while those in striatum and cerebral cortex fell to control levels and hippocampal corticosterone remained elevated. Over the period of the circadian peak the hippocampal and striatal concentrations paralleled the plasma concentrations but cerebral cortical concentrations showed only small changes. Adrenalectomy reduced plasma corticosterone concentrations to below detectable levels after 48 h but corticosterone levels were only partially reduced in the hippocampus and striatum and remained unchanged in the cerebral cortex. Single or repeated social defeat increased both brain and plasma concentrations after 1 h. Acute injections of alcohol raised the regional brain levels in parallel with plasma concentrations. The results show that measurements of plasma concentrations do not necessarily reflect the levels in brain. The data also demonstrate that corticosterone levels can change differentially in specific brain regions. These results, and the residual hormone seen in the brain after adrenalectomy, are suggestive evidence for a local origin of central corticosterone.

Kindling and withdrawal changes at the benzodiazepine receptor.

Drugs acting at benzodiazepine receptors can have two types of pharmacological profile: benzodiazepine agonists are anxiolytic, anticonvulsant and sedative, whilst benzo diazepine inverse agonists cause anxiety and convulsions. In 1982 we showed that a benzo diazepine antagonist, Ro 15-1788, prevented the effects of both types of compound at doses without intrinsic activity in the tests used. We put forward the hypothesis that the benzo diazepine receptor complex could undergo two possible conformational changes, resulting in increases (benzodiazepine agonists) or decreases (benzodiazepine inverse agonists) in the effects of the inhibitory transmitter γ-aminobutyric acid (GABA). This concept has been widely accepted. We have now studied the effects of inverse agonists after chronic treatment with inverse agonists themselves and with benzodiazepine agonists, in order to see if tolerance develops (as seen with the agonists) or whether an opposite change occurs.

Corticosterone Increases Damage and Cytosolic Calcium Accumulation Associated With Ethanol Withdrawal in Rat Hippocampal Slice Cultures

BACKGROUND Evidence suggests that stress hormones (i.e., glucocorticoids) may be increased during acute or chronic consumption of ethanol and during withdrawal from ethanol consumption, effects that may contribute to the development of cognitive impairment. The goal of the current studies was to examine the hypothesis that increased glucocorticoid levels in conjunction with ethanol exposure and withdrawal may cause hippocampal damage. METHODS Organotypic hippocampal slice cultures were exposed to 50 mM ethanol for 10 days and withdrawn for 1 day. After withdrawal, cytotoxicity and cytosolic Ca2+ accumulation were measured using the nucleic acid stain propidium iodide and Calcium Orange, AM, respectively. Cultures were also treated with nontoxic concentrations of corticosterone (0.001-1 microM) during ethanol exposure and withdrawal or only during withdrawal. Additional cultures were coexposed to corticosterone and RU486 (0.1-10.0 microM), spironolactone (0.1-10.0 microM), or MK-801 (20 microM) during ethanol exposure and/or withdrawal. RESULTS Ethanol withdrawal did not increase propidium iodide fluorescence and cytosolic Ca2+ levels. However, significant increases in propidium iodide fluorescence and in cytosolic Ca2+ accumulation were observed in cultures when corticosterone (> or = 100 nM) was exposed during ethanol treatment and/or withdrawal. These effects of corticosterone on ethanol withdrawal were attenuated by RU486 and MK-801 but not by spironolactone coexposure. CONCLUSIONS This report demonstrated that corticosterone exposure during ethanol treatment and/or withdrawal resulted in significant hippocampal damage, possibly via activation of glucocorticoid receptors and enhancement of the glutamatergic cascade. The findings from these studies suggest that glucocorticoids contribute to the neuropathological consequences of alcohol dependence in humans.

Effects of the glucocorticoid antagonist, mifepristone, on the consequences of withdrawal from long term alcohol consumption

BACKGROUND Studies were carried out to test the hypothesis that administration of a glucocorticoid Type II receptor antagonist, mifepristone (RU38486), just prior to withdrawal from chronic alcohol treatment, would prevent the consequences of the alcohol consumption and withdrawal in mice. MATERIALS AND METHODS The effects of administration of a single intraperitoneal dose of mifepristone were examined on alcohol withdrawal hyperexcitability. Memory deficits during the abstinence phase were measured using repeat exposure to the elevated plus maze, the object recognition test, and the odor habituation/discrimination test. Neurotoxicity in the hippocampus and prefrontal cortex was examined using NeuN staining. RESULTS Mifepristone reduced, though did not prevent, the behavioral hyperexcitability seen in TO strain mice during the acute phase of alcohol withdrawal (4 hours to 8 hours after cessation of alcohol consumption) following chronic alcohol treatment via liquid diet. There were no alterations in anxiety-related behavior in these mice at 1 week into withdrawal, as measured using the elevated plus maze. However, changes in behavior during a second exposure to the elevated plus maze 1 week later were significantly reduced by the administration of mifepristone prior to withdrawal, indicating a reduction in the memory deficits caused by the chronic alcohol treatment and withdrawal. The object recognition test and the odor habituation and discrimination test were then used to measure memory deficits in more detail, at between 1 and 2 weeks after alcohol withdrawal in C57/BL10 strain mice given alcohol chronically via the drinking fluid. A single dose of mifepristone given at the time of alcohol withdrawal significantly reduced the memory deficits in both tests. NeuN staining showed no evidence of neuronal loss in either prefrontal cortex or hippocampus after withdrawal from chronic alcohol treatment. CONCLUSIONS The results suggest mifepristone may be of value in the treatment of alcoholics to reduce their cognitive deficits.

The Importance of Glucocorticoids in Alcohol Dependence and Neurotoxicity

Alterations in hypothalamo-pituitary adrenal (HPA) function have been described in alcoholics and in rodents after chronic alcohol consumption but the role of glucocorticoids in alcohol consumption, and the mechanisms involved, has received little attention until recently. Both alcohol consumption and withdrawal from chronic alcohol intake raise circulating glucocorticoid levels, and prolonged high concentrations of glucocorticoids are known to have detrimental effects on neuronal function and cognition. This minireview covers the ways in which glucocorticoids may be involved in drinking behavior, from social drinking to dependence, and the negative consequences of alcohol consumption seen during withdrawal which may have a detrimental effect on treatment outcome. Research shows prolonged increases in brain glucocorticoid concentrations and decreased brain glucocorticoid receptor availability (consistent with increased levels of endogenous ligand) after withdrawal from chronic alcohol treatment. Evidence suggests that increased glucocorticoid levels in the brain after chronic alcohol treatment are associated with the cognitive deficits seen during abstinence which impact on treatment efficacy and quality of life. Studies on organotypic cultures also demonstrate the importance of glucocorticoids in the neuropathological consequences of alcohol dependence.

Thiamine deficiency in the pathogenesis of chronic ethanol-associated cerebellar damage in vitro

Nutritional deficiencies associated with long-term ethanol consumption may cause neuronal damage in ethanol-dependent individuals. Thiamine deficiency, in particular, is thought to contribute to ethanol-associated cerebellar degeneration, although damage may occur in adequately nourished alcoholics. Thus, the present study examined the effects of thiamine depletion and ethanol exposure on cytotoxicity in rat cerebellum. Organotypic cerebellar slice cultures were treated starting at 25 days in vitro with 100 mM ethanol for 11 days or 10 days followed by a 24-h withdrawal period. This exposure paradigm has previously been shown in hippocampal slice cultures to result in spontaneous cytotoxicity upon ethanol withdrawal. Additional cerebellar cultures were exposed to the thiamine depleting agent pyrithiamine (10-500 microM) for 10 or 11 days, some in the presence of ethanol exposure or withdrawal. Other cultures were co-exposed to thiamine (1-100 microM), 500 microM pyrithiamine, and ethanol for 10 or 11 days. The results demonstrated that neither 11-day ethanol treatment nor withdrawal from 10-day exposure significantly increased cerebellar cytotoxicity, as measured by propidium iodide fluorescence. The 11-day treatment with 100 or 500 microM pyrithiamine significantly increased propidium iodide fluorescence approximately 21% above levels observed in control tissue. Cultures treated with both ethanol (11 days or 10 days plus withdrawal) and 500 microM pyrithiamine displayed a marked increase in cytotoxicity approximately 60-90% above levels observed in control cultures. Pyrithiamine and ethanol-induced cytotoxicity was prevented in cultures co-exposed to thiamine (10-100 microM) for the duration of pyrithiamine treatment. Findings from this report suggest that the cerebellum may be more sensitive to the toxic effects of thiamine deficiency, as compared with alcohol withdrawal, associated with alcohol dependence.