Neurobehavioral effects of alcohol in AMPA receptor subunit (GluR1) deficient mice.

Of the ionotropic glutamatergic receptors, the NMDA receptor is clearly implicated in the acute and chronic effects of ethanol; however, the role of the AMPA receptor in mediating the effects of ethanol in vivo is as yet unclear. Using mice deficient in the AMPA receptor subunit GluR1 (GluR1-/- mice), we investigated whether the AMPA receptor had a significant role in mediating the effects of ethanol. GluR1-/- mice showed greater locomotor activity in a novel environment, but by the fifth day of repeated testing their activity was the same as that of wild-type mice. In contrast to their enhanced locomotor activity, on an accelerating rotarod GluR1-/- mice performed consistently worse than wild-types. With regard to the effects of ethanol on motor responses, GluR1-/- mice did not differ significantly from wild-type mice in ethanol's sedative or incoordinating effects. However, the GluR1-/- mice were insensitive to the hypothermic effects of a hypnotic dose of ethanol in contrast to wild-types; this effect was dissociable from the hypnotic effects of ethanol. Further, tolerance to ethanol developed equally for GluR1-/- mice versus wild-type mice. In terms of alcohol drinking behavior, compared to wild-types, GluR1-/- mice differed neither in the acquisition of voluntary ethanol consumption nor in stress-induced ethanol drinking, nor in the expression of an alcohol deprivation effect (ADE) which is used as a model of relapse-like drinking behavior. In summary, although the loss of a hypothermic effect of ethanol in GluR1-/- mice indicates a critical role for the AMPA receptors in this effect, the GluR1 subunit of the AMPA receptor does not seem to play a critical role in the etiology of alcohol dependence. However, changes observed in activity patterns may be related to the putative role of AMPA receptors in attention deficit hyperactivity disorder.

Cyclothiazide and AMPA receptor desensitization: analyses from studies of AMPA-induced release of [3H]-noradrenaline from hippocampal slices.

1. Responses in brain produced by the activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of ionotropic receptor for L-glutamate are often rapidly desensitizing. AMPA-induced desensitization and its characteristics, and the potentiating effect of cyclothiazide were investigated in vitro by analysing AMPA-induced release of [3H]-noradrenaline from prisms of rat hippocampus. 2. AMPA (1-1000 microM) stimulated the release of [3H]-noradrenaline in a concentration-dependent manner that was both calcium-dependent and tetrodotoxin-sensitive, and attenuated by the AMPA-selective antagonists, NBQX (1 and 10 microM), LY 293558 (1 and 10 microM) and GYKI 52466 (10 and 30 microM). 3. By use of an experimental procedure with consecutive applications of AMPA (100 microM, 28 min apart), the second response was reduced, indicative of receptor desensitization, and was reversed by cyclothiazide in a concentration-dependent manner (1-300 microM). The concentration-response curve for AMPA-induced release of [3H]-noradrenaline was shifted leftwards, but the reversal by cyclothiazide of the desensitized response was partial and failed to reach the maximal response of the first stimulus. 4. Observations made with various schedules of cyclothiazide application indicated that the initial AMPA-evoked response was already partially desensitized (150% potentiation by 100 microM cyclothiazide) and that the desensitization was not likely to be due to a time-dependent diminution and was longlasting (second application of cyclothiazide was ineffective). 5. Co-application of a number of drugs with actions on second messenger systems, in association with the second AMPA stimulus, revealed significant potentiation of the AMPA-induced release of [3H]-noradrenaline: forskolin (10 microM, +78%), Rp-cAMPS (100 microM, +65%), Ro 31-8220 (10 microM, +163%) and thapsigargin (100 pM, + 161%). 6. The AMPA receptor-mediated response regulating the release of [3H]-noradrenaline from rat hippocampal slices was desensitized and cyclothiazide acted to reverse partially the desensitization in a concentration-dependent manner. Since the time-course of desensitization was longer lasting than that noted in previous electrophysiological studies, multiple events may be involved in the down-regulation of AMPA receptor activity including receptor phosphorylation and depletion of intracellular Ca2+ stores.

Cholecystokinin-GABA interactions in rodent cortex: analyses of cholecystokinin effects on K(+)- and L-glutamate-induced release of [3H]GABA from rat cortical slices and cultured mouse cortical neurones.

Neurones of the cerebral cortex immunoreactive for the neuropeptide, cholecystokinin (CCK), also invariably contain GABA. Hence CCK is believed to modulate some aspect of GABAergic synaptic activity. The present study therefore investigated the effects of CCK on basal, K(+)- and L-glutamate-induced release of [3H]GABA from slices of rat neocortex and cultured murine neocortical neurones. Rat neocortical prisms loaded with [3H]GABA (10 nM) were superfused with Krebs-Henseleit buffer and stimulated twice (S1 and S2, 2 min) with K+ (30 mM). Release associated with each stimulus was measured and expressed relative to basal release (R1 and R2). The effects of non-selective and CCKB selective agonists, CCK-8S and CCK-4, respectively, on basal and K(+)-induced release of [3H]GABA were subsequently assessed by alternately including the peptides in S2 and comparing R2/R1 and S2/S1 ratios to control experiments. Contrary to previous findings, CCK-8S (30 nM-1 microM) and CCK-4 (0.3 nM-1 microM) failed to influence basal or K(+)-induced release. In similar experiments, murine cortical neurones superfused with HEPES balanced salt buffer, released exogenous [3H]GABA upon stimulation (1 min) with either K+ (55 mM) or L-glutamate (30 microM). However, CCK-8S, CCK-4 (both 300 nM-1 microM) and the CCKB selective antagonist, L365,260 (1 microM), failed to influence basal, K(+)- or L-glutamate-induced release of [3H]GABA from these neurones when included in S2. These data therefore do not support the postulate that CCK acting via CCKA or CCKB receptors modulates release of GABA under the present experimental conditions. GABA-CCK interactions were not specifically studied because only L-glutamate (30 microM) significantly elevated release of CCK-like immunoreactivity (115% above basal) in murine cortical neurones: basal release of CCK was estimated to be 7 and 11 pM from neurones and slices, respectively. Further studies employing more rigorous stimulation and perhaps examining endogenous GABA release are necessary to fully investigate the co-release of CCK and GABA.

Distribution of opioid peptide gene expression in the limbic system of Fawn-Hooded (alcohol-preferring) and Wistar-Kyoto (alcohol-non-preferring) rats.

Preprodynorphin and preproenkephalin mRNA expression was examined in the CNS of two rat strains, the alcohol-preferring Fawn-Hooded (FH) and the alcohol-non-preferring Wistar-Kyoto (WKY), using in situ hybridisation histochemistry. Relative to the WKY, the FH showed significantly lower levels of preproenkephalin mRNA in the striatum and nucleus accumbens (-24% and -17% respectively), but a higher level of preprodynorphin mRNA in the hippocampus (+33%). The depressed level of preproenkephalin mRNA in the nucleus accumbens may be implicated in alcohol-seeking behaviour.

The role of opioid-dopamine interactions in the induction and maintenance of ethanol consumption.

1. Alcohol is one of the most widely used recreational drugs, but also one of the most widely abused, causing vast economic, social and personal damage. 2. Several animal models are available to study the reinforcing mechanisms that are the basis of the abuse liability of ethanol. Innate differences in opioid or dopamine neurotransmission may enhance the abuse liability of ethanol, as indicated by animal and human studies. 3. Opioid antagonists have been shown to be effective, both experimentally and clinically, in decreasing ethanol consumption, presumably since ethanol induces the release of endogenous opioid peptides in vivo. However, ethanol may also stimulate the formation of opiate-like compounds, which could interact with opioid (or dopamine) receptors. Ethanol may cause changes in neurotransmission mediated via opioid receptors that determines whether alcohol abuse is more or less likely. 4. Ethanol appears to facilitate dopamine release by increasing opioidergic activity, disinhibiting dopaminergic neurons (by inhibition of GABAergic neurotransmission) via mu-opioid receptors in the ventral tegmental area (VTA) and delta-opioid receptors in the nucleus accumbens (NAcc). The effects of ethanol would be antagonised by presynaptic kappa-opioid receptors present on dopaminergic terminals in the NAcc. 5. Mesolimbic dopamine release induced by ethanol consumption seems to indicate ethanol-related stimuli are important, focussing attention on and enabling learning of the stimuli. However, studies indicate that there are redundant pathways, and neural pathways 'downstream' of the mesolimbic dopamine system, which also enable the reinforcing properties of ethanol to be mediated.

Alcoholism and neuropeptides: an update.

As with other addictions, human alcoholism is characterised as a chronically relapsing condition. Consequently, the "holy grail" from a therapeutic viewpoint is the development of clinically effective, safe drugs that promote high compliance rates and prevent relapse. Here we discuss the potential of therapeutics targeting neuropeptide systems implicated in aberrant alcohol-seeking behaviour. Clearly, much of the data so far available comes from pre-clinical studies; however, one of the first effective therapeutic strategies for alcoholism (still in use today) was the use of non-selective opioid receptor antagonists, such as naltrexone (Revia). In addition to opioid receptors, other neuropeptide receptors including those for corticotrophin releasing factor (CRF), neuropeptide Y and nociceptin may represent valid therapeutic targets to regulate alcohol consumption and the affective consequences of alcohol withdrawal.

Alterations in central preproenkephalin mRNA expression after chronic free-choice ethanol consumption by fawn-hooded rats.

BACKGROUND: Neurotransmission mediated via opioid and dopamine receptors is believed to be involved in the reinforcing and/or rewarding effects of ethanol consumption. We previously examined the effect of ethanol consumption (and naltrexone treatment, used clinically to treat alcoholism) on micro-opioid receptor density. We describe here the effect of free-choice ethanol consumption and naltrexone treatment on preproenkephalin, preprodynorphin, and dopamine D1 and D2 receptor mRNA expression in the central nervous system. METHODS: Fawn-hooded rats were given continual free-choice access to a 5% ethanol solution or water (4 weeks) followed by 2 weeks of water alone. At the end of this abstinence period, osmotic minipumps were implanted subcutaneously to deliver saline (n = 4) or naltrexone (n = 4; 8.4 mg/kg/day for 4 weeks). After recovery from surgery, the rats again were given access to 5% ethanol under the same free-choice conditions (4 weeks). A third group of age-matched controls drank only water during the behavioral trial. At the end of the behavioral trial, the rats were decapitated, and a quantitative examination of peptide precursor mRNAs was made by using in situ hybridization histochemistry. RESULTS: Naltrexone treatment significantly decreased preprodynorphin expression in the nucleus accumbens, but neither naltrexone treatment nor ethanol consumption significantly affected dopamine D1 and D2 receptor mRNA expression. In contrast, ethanol consumption increased preproenkephalin mRNA in the central and intercalated nuclei of the amygdala but decreased preproenkephalin mRNA in the nucleus accumbens and olfactory tubercle. The decreased level of preproenkephalin mRNA in the nucleus accumbens may reflect a neuroadaptive response to increased release of dopamine, whereas the increased level of preproenkephalin mRNA in the central nucleus of the amygdala may be associated with an anxiolytic effect of ethanol consumption. CONCLUSIONS: The data support the putative role of opioid peptides in the effects of ethanol and suggest that the nucleus accumbens and central nucleus of the amygdala are loci for the reinforcing effects of ethanol.

Ethanol consumption by Fawn-Hooded rats following abstinence: effect of naltrexone and changes in mu-opioid receptor density.

BACKGROUND: Relapse after abstinence can be modelled in rats using an alcohol deprivation effect (ADE) of enhanced ethanol consumption after a period of enforced abstinence from ethanol; however, not all rat strains display such an effect. We wanted to examine the effect of naltrexone on ethanol consumption by ethanol-preferring Fawn-Hooded (FH) rats using such a model. METHODS: FH rats were given continual free-choice access to a 5% ethanol solution or water (4 weeks) followed by 2 weeks of water alone. At the end of this abstinence period, osmotic minipumps were implanted subcutaneously to deliver saline (n = 4) or naltrexone (n = 4; 8.4 mg/kg/day for 4 weeks). After recovery from surgery, the rats were again given access to 5% ethanol under the same free-choice conditions (4 weeks). A third group of age-matched controls drank only water during the behavioral trial. At the end of the behavioral trial, the rats were decapitated and an autoradiographic examination was made of micro-opioid receptor density through the forebrain using the ligand [125I]FK-33824. RESULTS: First, a period of enforced abstinence from ethanol consumption caused a significant (p < 0.05) and prolonged increase in ethanol preference (+18%) and decrease in water consumption (-53%), although the volume of ethanol consumed (ml/day) did not vary, indicating an atypical ADE in this rat strain. Second, naltrexone significantly (p < 0.05) decreased ethanol consumption by the FH rats in terms of absolute amount of ethanol consumed and preference for ethanol solution, but this effect of naltrexone diminished over time, concurrent with a robust and significant elevation in micro-opioid receptor density in all brain regions examined (p < 0.05). Finally, ethanol consumption alone also upregulated micro-opioid receptor density relative to nondrinking controls in a number of brain regions, which included the nucleus accumbens (+29%) and caudate-putamen (+15%,p < 0.05), but decreased micro-opioid receptor density in other regions including the substantia nigra pars reticulata, which was suggestive of an indirect effect on micro-opioid receptors. CONCLUSIONS: The data suggest that continual long-term naltrexone treatment may not be effective in the treatment of alcoholism, possibly because of the induced increase in micro-opioid receptor density.