Sidney George Shaw, DPhil (1948-2017).

On March 4, 2017 at the age of 68, Sidney George Shaw (Sid) unexpectedly died from complications following surgery, only four years after retiring from the University of Bern. Trained in biochemistry at Oxford University, Sid had quickly moved into molecular pharmacology and became a key investigator in the field of enzyme biochemistry, vasoactive peptide research, and receptor signaling. Sid spent half his life in Switzerland, after moving to the University of Bern in 1984. This article, written by his friends and colleagues who knew him and worked with him during different stages of his career, summarizes his life, his passions, and his achievements in biomedical research. It also includes personal memories relating to a dear friend and outstanding scientist whose intellectual curiosity, humility, and honesty will remain an example to us all.

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.

Nimodipine prior to alcohol withdrawal prevents memory deficits during the abstinence phase.

Effects of the dihydropyridine, nimodipine, an antagonist at L-type calcium channels, on the memory loss in rats caused by long term alcohol consumption were examined. Either a single dose of nimodipine or 2 weeks of repeated administration was given prior to withdrawal from 8 months of alcohol consumption. Memory was measured by the object recognition test and the T maze. Both nimodipine treatments prevented the memory deficits when these were measured between 1 and 2 months after alcohol withdrawal. At the end of the memory testing, 2 months after cessation of chronic alcohol consumption, glucocorticoid concentrations were increased in specific regions of rat brain without changes in plasma concentrations. Both nimodipine treatment schedules substantially reduced these rises in brain glucocorticoid. The data indicate that blockade of L-type calcium channels prior to alcohol withdrawal protects against the memory deficits caused by prolonged alcohol intake. This shows that specific drug treatments, such as nimodipine, given over the acute withdrawal phase, can prevented the neuronal changes responsible for subsequent adverse effects of long term consumption of alcohol. The results also suggest the possibility that regional brain glucocorticoid increases may be involved in the adverse effects of long term alcohol intake on memory. Such local changes in brain glucocorticoid levels would have major effects on neuronal function. The studies indicate that L-type calcium channels and brain glucocorticoid levels could form new targets for the treatment of cognitive deficits in alcoholics.

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.

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

RATIONALE: In humans, social stress over long and short term can increase alcohol consumption, but the mechanisms involved are not understood. OBJECTIVES: This 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. METHODS: Alcohol 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. RESULTS: Five 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. CONCLUSION: The 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.

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.

Interactions between low concentrations of ethanol and nicotine on firing rate of ventral tegmental dopamine neurones.

This study investigated interactions between ethanol and nicotine on dopamine-sensitive neurone firing in the ventral tegmental area (VTA), recorded in midbrain slices. No changes in spontaneous activity of the neurones were seen with nicotine at 10, 25, or 100 nM; at 250 nM there was a small significant increase in firing rate. Ethanol, applied alone, caused a significant increase in firing rate at 40 mM and at 60 nM but not at 20 mM. Combinations of 10 or 25 nM nicotine with 20 or 40 mM ethanol did not result in increased firing rates compared with either drug alone. However, nicotine 100 nM plus ethanol 60 mM significantly increased the rate of spontaneous firing compared with that after either drug alone at these concentrations. In contrast, nicotine at 250 nM plus ethanol at 60 mM did not increase firing rate, compared with each drug alone. Ketanserin, 2 microM, prevented the potentiating effect of nicotine 100 nM plus ethanol 60 mM. The results show synergism between ethanol and nicotine at specific concentrations that are likely to be present in the brain during the behavioural effects of these drugs, but the interaction is complex and may involve multiple drug actions.

Effects of corticosterone on place conditioning to ethanol.

RATIONALE AND OBJECTIVE: Effects of corticosterone on place conditioning to ethanol were investigated in mice using two conditioning schedules; the conventional method and a rapid conditioning schedule in which exposure to the CS+ followed immediately on exposure to the CS-. METHODS: Effects of administration of corticosterone, 10 mg/kg, on the acquisition of place conditioning produced by ethanol, 1-2.5 g/kg, were investigated using the conventional method of conditioning, with exposure to the CS+ and the CS- on alternate days, and also using the rapid conditioning method. Total and free blood corticosterone concentrations were measured after administration of ethanol and corticosterone. RESULTS: In the conventional, alternate day, conditioning schedule, ethanol produced significant place preference at 2 and at 2.5 g/kg, but when these alcohol doses were given with corticosterone 10 mg/kg, significant place conditioning was not seen. In contrast, in the rapid, same day, conditioning schedule corticosterone significantly decreased the dose at which ethanol produced an apparent place preference, with significant place conditioning being seen with ethanol at 1 and 1.5 g/kg in combination with corticosterone, 10 mg/kg. Total and free corticosterone concentrations were increased after ethanol, 1.5 g/kg, compared with controls, and administration of corticosterone, 10 mg/kg, caused a significantly greater increase. There were no significant differences in spontaneous locomotor activity or brain alcohol concentrations between any of the treatment groups. CONCLUSIONS: The effects of corticosterone on ethanol-induced place conditioning are substantially affected by the conditioning schedule used.

Nimodipine prevents scopolamine-induced impairments in object recognition.

The effects of acute administration of the dihydropyridine calcium channel antagonist, nimodipine, were studied on the actions of scopolamine in the object recognition test. Scopolamine at 0.125 mg/kg decreased the difference in the time spent exploring novel and familiar objects when given either 15 min before, or immediately after, exposure to objects. Administration of nimodipine at 10 mg/kg, or 1 mg/kg, at the same time as the scopolamine completely prevented the deleterious effects on memory in this task. This effect was seen when nimodipine and/or scopolamine were given prior to the object exposure and also when the drugs were given after the experience of seeing the objects. Nimodipine had no effects on performance when given in the absence of scopolamine. This lack of change in total time spent exploring the objects indicated that the effects of scopolamine and nimodipine were not due to changes in motor coordination or alertness. The results are discussed in the light of the role of cholinergic transmission in memory and the known actions of dihydropyridines on brain function.

Nimodipine prevents the effects of ethanol in tests of memory.

The effects of acute administration of the dihydropyridine calcium channel antagonist, nimodipine, were studied on the actions of ethanol in the radial arm maze and the object recognition test. In the former test, the effects of the drugs were examined on the performance in finding the four baited arms, after previous training in this task. Ethanol, at 1 g/kg, increased both the number of re-entries into baited arms (counted as errors of working memory) and the total number of arm choices required to complete the task. Administration of nimodipine, 10 mg/kg, with the ethanol, completely prevented the deleterious effects on memory in this task, but had no effects on the performance when given in the absence of ethanol. In the object recognition task, ethanol, 1 g/kg, significantly decreased the differences in the time spent exploring novel and familiar objects. Nimodipine, 10 mg/kg, given with the ethanol, completely prevented this effect, but nimodipine alone had no effects. The lack of changes in total exploration times indicated that the effects of ethanol in these tests were not due to loss of motor co-ordination or of alertness. The results are discussed in the light of the known actions of the drugs on brain function.

Effects of intraperitoneal injections of saline on the alcohol and sucrose consumption of C57/BL10 mice.

RATIONALE: To determine the effects of multiple saline injections on alcohol drinking by male and female C57/BL10 mice with low preference for alcohol. OBJECTIVE: An investigation of the effects of multiple saline injections on alcohol consumption, with a comparison of corresponding effects on sucrose consumption. METHODS: The effects of a range of injection schedules on preference for 8% alcohol, or 1% sucrose, compared with tap water, were measured in two-bottle choice tests. RESULTS: The multiple saline injection schedule significantly increased the alcohol preference, even when no alcohol was available during the injection period. The actual administration of fluid was not necessary for the increase in alcohol preference, since sham injections without fluid administration also increased alcohol preference. A single injection of saline did not alter the alcohol preference 3 weeks later. Daily saline injections for 3 weeks did not alter the consumption of the dilute sucrose solution. In the population of mice used, the preference for sucrose over water was found to follow a biphasic distribution, similar to that reported earlier in these mice for alcohol preference, but there was no correlation between alcohol preference and sucrose preference. CONCLUSIONS: The results suggest that lasting changes in the areas of the brain that specifically control alcohol intake are produced by repetition of a routine laboratory procedure.

Low alcohol preference among the "high alcohol preference" C57/BL10 mice; factors affecting such preference.

The effects of age, ethanol concentration and minor stress on the variation in alcohol preference of C57 strain mice were determined. In two bottle choice tests, an older population of mice contained slightly more low-preference mice than a younger population. A wide range of ethanol preference was consistently seen in young mice for 8% and 6% ethanol, but the previously reported biphasic pattern of distribution was revealed only with 8% ethanol. Very few animals showed high preference for concentrations of 10% or 12% ethanol. Moving low alcohol preference mice to a new location (but not repeated cage changing or ultrasonic noise) significantly increased the alcohol preference. Exploratory locomotor activity did not correlate with the subsequent alcohol consumption. Blood and brain alcohol concentrations showed that the differences in alcohol preference were not due to differences in metabolism of ethanol. The C57 strain mice with low preference for alcohol provides a valuable model for the study of the effects of minor stress on alcohol consumption.

Alterations in mesolimbic dopamine function during the abstinence period following chronic ethanol consumption.

Previous work demonstrated that the locomotor stimulant actions of amphetamine, cocaine and nicotine were increased when these drugs were given during the abstinence phase after chronic ethanol consumption. These changes were seen at 6 days and at 2 months after cessation of alcohol. The present study examined neuronal alterations which might be related to these changes in behaviour. Markedly reduced spontaneous firing rates of dopaminergic cells in the ventral tegmental area (VTA) in midbrain slices were seen 6 days into the abstinence period after cessation of chronic ethanol consumption, but by 2 months the firing rates had returned to control values. Increased affinity of striatal receptors for the D1-like receptor ligand 3H-SCH23390, but no change in the receptor density, was found both at the 6 day and the 2 month intervals. The binding properties of striatal D2-like receptors, of D1-like and D2-like receptors in the frontal cerebral cortex, and the release of tritiated dopamine from slices of striatum or frontal cerebral cortex, were unchanged at 6 days and 2 months. It is suggested that the decreased neuronal firing leads to a persistent increase in sensitivity of D1-like receptors and that these changes could explain the increased effects of the other drugs of abuse.

Chronic infusion of nicotine can increase operant self-administration of alcohol.

Effects of nicotine, administered by continuous infusion via osmotic minipumps, were studied on the operant self-administration of alcohol by rats, using a variable interval (15 s) schedule, and measuring the acquisition, maintenance, extinction and reinstatement of responding for alcohol. Doses of nicotine of 0.25, 1.25 and 7.5 mg/kg/24 h had no significant effects on the maintenance of responding for alcohol, but 5 mg/kg/24 h nicotine resulted in a significant increase in responding on the lever delivering the reward when water was substituted for the alcohol, indicating delayed extinction of responding. During infusion of 2.5 mg/kg/24 h nicotine, responding was significantly greater over the "sucrose-fading" training sessions, during acquisition of responding, when mixtures of alcohol and sucrose were provided as reward. When minipumps infusing 2.5 mg/kg/24 h nicotine were implanted after the alcohol responding had been acquired, the responding for alcohol increase during the first week of nicotine infusion, but corresponding nicotine infusion doses of 0.25, 1.25 and 7.5 had no significant effects. The results indicate that nicotine can increase operant responding for alcohol and this is crucially dependent on the dose of nicotine and the time of testing. The results have implications for the frequently encountered dependence on the combination of alcohol and nicotine.

Studies on a model of long term alcohol drinking.

This study investigated the effects of a dihydropyridine calcium channel antagonist in a free drinking model previously reported to show increased and 'uncontrolled' drinking. The results showed, rather than the previously reported increase in consumption, a gradual decrease in alcohol intake over 9-18 months. When the alcohol was withdrawn from one group of rats after 55 weeks free choice, the animals showed no behavioural signs of physical withdrawal, but they did demonstrate the expected elevated ethanol intake on reintroduction to ethanol after 2 weeks abstinence. A second group of rats were given 62 weeks free choice access to ethanol in groups of four, then transferred to single housing and baseline drinking levels established. Intraperitoneal injections of nimodipine 5 mg/kg, 20 mg/kg or Tween vehicle were then given once daily. Nimodipine had no effect on ethanol intake of animals with continuous access to ethanol, or of those animals withdrawn from ethanol and then reintroduced after 2 weeks of abstinence. However the unexpectedly low alcohol intake may have prevented any effects of nimodipine being seen.

Prolonged changes in neurochemistry of dopamine neurones after chronic ethanol consumption.

The effects of 3 weeks of chronic ethanol consumption in mice on brain concentrations and turnover of monamine transmitters was examined. The measurements were made at 24 h, 6 days and 2 months after cessation of the ethanol intake to examine changes that might be relevant to relapse drinking. Increases in noradrenaline and dopamine concentrations, and decreases in the ratios of dopamine metabolites to dopamine, were seen in ventral tegmental tissue at 24 h after alcohol consumption. Increased noradrenaline was also evident at the 6-day interval, but no other changes were seen at this time. At the 2-month interval, the ventral tegmentum from ethanol-treated animals showed decreases in metabolite/dopamine ratios. No changes were seen in 5-hydroxytryptamine or its metabolite. In striatal tissue, none of these changes were seen, but at 24 h decreases occurred in the content of dopamine and its metabolites and a decrease in 5-hydroxyindoleacetic acid. The results indicate changes occur in monoamine turnover in the VTA as long as 2 months after cessation of chronic ethanol consumption; such changes may be related to the prolonged nature of alcohol dependence.

Differential effects of a dihydropyridine calcium channel antagonist on the components of ethanol tolerance.

The dihydropyridine calcium channel antagonist, nimodipine, was found to decrease the extent of tolerance that developed to the ataxic action of ethanol in experimental designs in which the tolerance was not context-specific, when ethanol was given by liquid diet. When ethanol was given by injection, so that cues were present for the effects of ethanol during the chronic treatment, tolerance to the ataxic actions of ethanol was unaffected. Nimodipine, however, decreased the tolerance to the hypothermic actions of ethanol, when the ethanol was given by injection. When the rats were given practice sessions on the motor task while under the influence of the ethanol, during the chronic treatment, nimodipine did not affect tolerance to the ataxic actions of ethanol. When nimodipine was given before the motor task learning and ethanol after the practice sessions, the tolerance to the ataxic effect of ethanol was increased. A similar schedule of drug treatment with the NMDA antagonist CGP37849 given before the practice sessions, and ethanol afterwards, resulted in decreased tolerance to ethanol. It is suggested that these changes in ethanol tolerance may be explained by dual actions of nimodipine in, firstly, decreasing the form of tolerance to ethanol that is not dependent on contextual cues and, secondarily, in increasing the learning of a motor task.

Investigations into pharmacological antagonism of general anaesthesia.

The effects of convulsant drugs, and of thyrotropin releasing hormone (TRH), were examined on the general anaesthetic actions of ketamine, ethanol, pentobarbitone and propofol in mice. The aim was to investigate the possibility of selective antagonism, which, if seen, would provide information about the mechanism of the anaesthesia. The general anaesthetic effects of ketamine were unaffected by bicuculline; antagonism was seen with 4-aminopyridine and significant potentiation with 300 mg kg(-1) NMDLA (N-methyl-DL-aspartate). The calcium agonist, Bay K 8644, potentiated the anaesthesia produced by ketamine and antagonism of such anaesthesia was seen with TRH. A small, but significant, antagonism of the general anaesthesia produced by ethanol was seen with bicuculline, and a small, significant, potentiation with 4-aminopyridine. There was an antagonist effect of TRH, but no effect of NMDLA. Potentiation of the anaesthetic effects of pentobarbitone was seen with NMDLA and with 4-aminopyridine and the lower dose of bicuculline (2.7 mg kg(-1)) also caused potentiation. There was no significant change in the ED(50) value for pentobarbitone anaesthesia with TRH. Bicuculline did not alter the anaesthetic actions of propofol, while potentiation was seen with NMDLA and 4-aminopyridine. TRH had no significant effect on propofol anaesthetic, but Bay K 8644 at 1 mg kg(-1) significantly potentiated the anaesthesia. These results suggest that potentiation of GABA(A) transmission or inhibition of NMDA receptor-mediated transmission do not appear to play a major role in the production of general anaesthesia by the agents used.

Acamprosate, but not naltrexone, inhibits conditioned abstinence behaviour associated with repeated ethanol administration and exposure to a plus-maze.

RATIONALE: Drugs that reduce relapse in alcoholics are thought to inhibit either positive reinforcement for drinking (e.g. naltrexone) or negative reinforcement (e.g. acamprosate), and may reduce the impact of conditioned stimuli associated with previous alcohol use. We have developed a model for such conditioning by repeatedly pairing ethanol administration with plus-maze exposure. Substitution of saline for ethanol greatly increased stretched-attend postures and time in the central square, conditioned to the environment. OBJECTIVE: To test the hypothesis that if this behaviour indicates a negative affective state caused by the expectation of ethanol, it should be inhibited by drugs that reduce negative, but not positive, reinforcement. METHODS: The effects of naltrexone and acamprosate on alcohol-conditioned abstinence behaviour were compared. RESULTS: Acute administration of either drug alone produced no significant effects on plus-maze behaviour in naive mice. Naltrexone had no significant effect on the alcohol-conditioned abstinence behaviour, but acamprosate reduced the incidence of stretched-attend postures. CONCLUSIONS: The experiments replicated previous findings for alcohol/environment conditioned behaviour, and demonstrated, as predicted, that this was decreased by acamprosate but not by naltrexone. Effects of acamprosate on conditioned negative reinforcement may be the cause of this effect, but more work is required to establish the usefulness of this model in evaluation of anti-relapse drugs.