Targeted Inhibition of Upregulated Sodium-Calcium Exchanger in Rat Inferior Colliculus Suppresses Alcohol Withdrawal Seizures.

The inferior colliculus (IC) is critical in initiating acoustically evoked alcohol withdrawal-induced seizures (AWSs). Recently, we reported that systemic inhibition of Ca2+ entry via the reverse mode activity of the Na+/Ca2+ exchanger (NCXrev) suppressed AWSs, suggesting remodeling of NCX expression and function, at least in the IC, the site of AWS initiation. Here, we probe putative changes in protein expression in the IC of NCX isoforms, including NCX type 1 (NCX1), 2 (NCX2), and 3 (NCX3). We also evaluated the efficacy of targeted inhibition of NCX1rev and NCX3rev activity in the IC on the occurrence and severity of AWSs using SN-6 and KB-R943, respectively. We used our well-characterized alcohol intoxication/withdrawal model associated with enhanced AWS susceptibility. IC tissues from the alcohol-treated group were collected 3 h (before the onset of AWS susceptibility), 24 h (when AWS susceptibility is maximal), and 48 h (when AWS susceptibility is resolved) following alcohol withdrawal; in comparison, IC tissues from the control-treated group were collected at 24 h after the last gavage. Analysis shows that NCX1 protein levels were markedly higher 3 and 24 h following alcohol withdrawal. However, NCX3 protein levels were only higher 3 h following alcohol withdrawal. The analysis also reveals that bilateral microinjections of SN-6 (but not KB-R7943) within the IC markedly suppressed the occurrence and severity of AWSs. Together, these findings indicate that NCX1 is a novel molecular target that may play an essential role in the pathogenesis and pathophysiology of AWSs.

Activity of hippocampal adult-born neurons regulates alcohol withdrawal seizures.

Alcohol withdrawal (AW) after chronic alcohol exposure produces a series of symptoms, with AW-associated seizures being among the most serious and dangerous. However, the mechanism underlying AW seizures has yet to be established. In our mouse model, a sudden AW produced 2 waves of seizures: the first wave includes a surge of multiple seizures that occurs within hours to days of AW, and the second wave consists of sustained expression of epileptiform spikes and wave discharges (SWDs) during a protracted period of abstinence. We revealed that the structural and functional adaptations in newborn dentate granule cells (DGCs) in the hippocampus underlie the second wave of seizures but not the first wave. While the general morphology of newborn DGCs remained unchanged, AW increased the dendritic spine density of newborn DGCs, suggesting that AW induced synaptic connectivity of newborn DGCs with excitatory afferent neurons and enhanced excitability of newborn DGCs. Indeed, specific activation and suppression of newborn DGCs by the chemogenetic DREADD method increased and decreased the expression of epileptiform SWDs, respectively, during abstinence. Thus, our study unveiled that the pathological plasticity of hippocampal newborn DGCs underlies AW seizures during a protracted period of abstinence, providing critical insight into hippocampal neural circuits as a foundation to understand and treat AW seizures.

Alcohol withdrawal upregulates mRNA encoding for CaV2.1-α1 subunit in the rat inferior colliculus.

We previously reported increased current density through P-type voltage-gated Ca2+ channels in inferior colliculus (IC) neurons during alcohol withdrawal. However, the molecular correlate of this increased P-type channel current is currently unknown. Here, we probe changes in mRNA and protein expression of the pore-forming CaV2.1-α1 (P/Q-type) subunits in IC neurons during the course of alcohol withdrawal-induced seizures (AWSs). Rats received three daily doses of ethanol or the vehicle every 8 h for 4 consecutive days. The IC was dissected at various time intervals following alcohol withdrawal, and the mRNA and protein levels of the CaV2.1-α1 subunits were measured. In separate experiments, rats were tested for acoustically evoked seizure susceptibility 3, 24, and 48 h after alcohol withdrawal. AWSs were observed 24 h after withdrawal; no seizures were observed at 3 or 48 h or in the control-treated rats. Compared to control-treated rats, the mRNA levels of the CaV2.1-α1 subunit were increased 1.9-fold and 2.1-fold at 3 and 24 h, respectively; change in mRNA expression was nonsignificant at 48 h following alcohol withdrawal. Western blot analyses revealed that protein levels of the CaV2.1-α1 subunits were not altered in IC neurons following alcohol withdrawal. We conclude that expression of the Cacna1a mRNA increased before the onset of AWS susceptibility, suggesting that altered CaV2.1 channel expression may play a role in AWS pathogenesis.

Prefrontal cortex expression of chromatin modifier genes in male WSP and WSR mice changes across ethanol dependence, withdrawal, and abstinence.

Alcohol-use disorder (AUD) is a relapsing disorder associated with excessive ethanol consumption. Recent studies support the involvement of epigenetic mechanisms in the development of AUD. Studies carried out so far have focused on a few specific epigenetic modifications. The goal of this project was to investigate gene expression changes of epigenetic regulators that mediate a broad array of chromatin modifications after chronic alcohol exposure, chronic alcohol exposure followed by 8 h withdrawal, and chronic alcohol exposure followed by 21 days of abstinence in Withdrawal-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected mouse lines. We found that chronic vapor exposure to highly intoxicating levels of ethanol alters the expression of several chromatin remodeling genes measured by quantitative PCR array analyses. The identified effects were independent of selected lines, which, however, displayed baseline differences in epigenetic gene expression. We reported dysregulation in the expression of genes involved in histone acetylation, deacetylation, lysine and arginine methylation and ubiquitinationhylation during chronic ethanol exposure and withdrawal, but not after 21 days of abstinence. Ethanol-induced changes are consistent with decreased histone acetylation and with decreased deposition of the permissive ubiquitination mark H2BK120ub, associated with reduced transcription. On the other hand, ethanol-induced changes in the expression of genes involved in histone lysine methylation are consistent with increased transcription. The net result of these modifications on gene expression is likely to depend on the combination of the specific histone tail modifications present at a given time on a given promoter. Since alcohol does not modulate gene expression unidirectionally, it is not surprising that alcohol does not unidirectionally alter chromatin structure toward a closed or open state, as suggested by the results of this study.

Alcohol Withdrawal-Induced Seizure Susceptibility is Associated with an Upregulation of CaV1.3 Channels in the Rat Inferior Colliculus.

BACKGROUND: We previously reported increased current density through L-type voltage-gated Ca(2+) (CaV1) channels in inferior colliculus (IC) neurons during alcohol withdrawal. However, the molecular correlate of this increased CaV1 current is currently unknown. METHODS: Rats received three daily doses of ethanol every 8 hours for 4 consecutive days; control rats received vehicle. The IC was dissected at various time intervals following alcohol withdrawal, and the mRNA and protein levels of the CaV1.3 and CaV1.2 α1 subunits were measured. In separate experiments, rats were tested for their susceptibility to alcohol withdrawal-induced seizures (AWS) 3, 24, and 48 hours after alcohol withdrawal. RESULTS: In the alcohol-treated group, AWS were observed 24 hours after withdrawal; no seizures were observed at 3 or 48 hours. No seizures were observed at any time in the control-treated rats. Compared to control-treated rats, the mRNA level of the CaV1.3 α1 subunit was increased 1.4-fold, 1.9-fold, and 1.3-fold at 3, 24, and 48 hours, respectively. In contrast, the mRNA level of the CaV1.2 α1 subunit increased 1.5-fold and 1.4-fold at 24 and 48 hours, respectively. At 24 hours, Western blot analyses revealed that the levels of the CaV1.3 and CaV1.2 α1 subunits increased by 52% and 32%, respectively, 24 hours after alcohol withdrawal. In contrast, the CaV1.2 and CaV1.3 α1 subunits were not altered at either 3 or 48 hours during alcohol withdrawal. CONCLUSIONS: Expression of the CaV1.3 α1 subunit increased in parallel with AWS development, suggesting that altered L-type CaV1.3 channel expression is an important feature of AWS pathogenesis.

Alcohol withdrawal is associated with a downregulation of large-conductance Ca²âº-activated K⁺ channels in rat inferior colliculus neurons.

RATIONALE: Large conductance calcium-activated potassium (BK(Ca) or K(Ca)1.1) channels are well-known molecular targets for the action of alcohol and therefore may play an important role in the pathogenesis of alcohol withdrawal syndrome. OBJECTIVES: We evaluate the modifications of total outward K⁺ currents and protein expression of BK(Ca) channels α-subunit in inferior colliculus (IC) neurons obtained from controls and rats subjected to alcohol withdrawal associated with enhanced susceptibility to seizures. METHODS: Outward K⁺ currents and BK(Ca) channel proteins were measured using the whole cell configuration of patch clamp techniques and Western blot analysis, respectively. RESULTS: Total outward K⁺ current density was significantly reduced in IC neurons at 24 and 48 h during the alcohol withdrawal period when the susceptibility to seizures was maximal and absent, respectively. The iberiotoxin-sensitive (BK(Ca)) current density and conductance also were significantly reduced at 24 h following alcohol withdrawal. Consistent with functional data, the levels of protein expression of α-subunit associated with BK(Ca) channels also was significantly reduced in IC neurons at 24 and 48 h following alcohol withdrawal. CONCLUSIONS: The downregulation of BK(Ca) channels outlasts the finite period of elevated susceptibility to alcohol withdrawal seizures. These findings indicate that BK(Ca) channels, per se, may not be fundamentally important for the generation of alcohol withdrawal seizures.

Actin dynamics in development of behavioral sensitization after withdrawal from long-term ethanol administration to mice.

BACKGROUND AND METHODS: The present study investigated the role of actin depolymerizing factor (ADF) in the brain of mice after withdrawal from continuous ethanol (EtOH) vapor inhalation for 9 days using C57BL/6J and ADF mutant mice. RESULTS: C57BL/6J mice with withdrawal signs 10 hours after withdrawal from EtOH vapor inhalation showed transient and significant enhancement of locomotor activity by a single injection of EtOH (2 g/kg, i.p.) and of EtOH-induced place preference 3 days after withdrawal from EtOH vapor inhalation, suggesting the development of sensitization of locomotion activity to EtOH and of place preference 3 days after withdrawal from EtOH in C57BL/6J mice with EtOH physical dependence. The levels of ADF and G-actin in the ventral tegmental area, including a little bit of surrounding tissues, increased immediately (0 hours), 10 hours, and 3 days after withdrawal from EtOH vapor. F-actin, synaptic vesicle-associated protein 38, and postsynaptic density 95 increased 0 hours and 3 days after withdrawal with their decreases 10 hours after withdrawal from EtOH vapor. An F-actin stabilizing agent phalloidin (3 nmol/mouse/d, i.c.v., once a day) administered daily for 3 days after withdrawal from continuous EtOH vapor inhalation for 9 days significantly suppressed the increase in both EtOH-induced place preference and locomotor activity by a single injection of EtOH 3 days after withdrawal from long-term EtOH vapor inhalation for 9 days. In addition, the changes in behavioral sensitization in ADF mutant mice were significantly weaker than those observed in C57BL/6J mice (wild-type mice for ADF mutant mice). CONCLUSIONS: The findings presented here suggest that withdrawal from EtOH physical dependence causes behavioral sensitization to EtOH, which may be, at least in part, mediated by alternation of actin dynamics.

Sex differences in acoustic startle responses and seizure thresholds between ethanol-withdrawn male and female rats.

AIMS: We have found consistent and significant sex differences in recovery from the increased seizure susceptibility observed during ethanol withdrawal (EW) in our rat model system. The main objective of the present study was to determine if sex differences in EW generalized to an additional behavioral measure startle reactivity. METHODS: Acoustic startle or seizure threshold responses were measured in separate groups of rats at 1 day or 3 days of EW. RESULTS: Both pair-fed control and EW males showed greater increases in acoustic startle responses than either the female or ovariectomized female (OVX) counterparts. There was a selective effect of pregnanolone on acoustic startle in that it reduced peak force of response only at 3 days EW in male rats. Unexpectedly, it modestly increased startle reactivity in control female and OVX rats. Acute treatment with low-dose ethanol trended toward reducing startle responses in control animals, as expected, while generally enhancing startle responses during EW. All sex conditions showed an enhanced startle response during EW following administration of the higher dose of estradiol compared to control animals. Estradiol did not alter seizure thresholds in control animals. However, it was anticonvulsant for males at 3 days EW, females and OVX at 1 day EW. CONCLUSIONS: Observed sex differences in the startle reactivity during EW were consistent with earlier findings comparing EW seizure risk in male and female rats. Responses of OVX suggested that both hormones and differences in brain structures between males and females have a role in these sex differences. Our findings add weight to recommendations that treatment of alcohol withdrawal in humans should consider hormonal status as well as withdrawal time.

Homocysteine, alcoholism and its molecular networks.

The emerging research on biomarkers in alcohol dependence has lead to a deeper understanding of the neurobiological mechanisms in alcoholism. The molecular networks and the pathophysiological circuits are complex and not completely unrevealed up to now. One of the most interesting biomarkers described to play an important role in alcohol dependence is the amino-acid homocysteine, which has particularly been linked with brain atrophy and withdrawal seizures. However, the molecular networks of homocysteine are complex and include an important impact on epigenetic regulation via homocysteine's action as a methyl-group donator in human metabolism. So, alterations in human homocysteine levels can influence DNA-methylation of specific gene areas which may change expression and synthesis of proteins possibly important for the genesis and maintenance of alcohol dependence.

Alcohol withdrawal seizures.

The topic of alcohol withdrawal syndrome (AWS), including delirium tremens and especially seizures, is reviewed. From mice and rat studies, it is known that both N-methyl-d-aspartate (NMDA) and gamma-aminobutyric acid (GABA) receptors are involved in AWS. During alcohol intoxication chronic adaptations of NMDA and GABA receptors occur, and during alcohol withdrawal a hyperexcitable state develops. In studies on humans, during intoxication the NMDA receptors are activated and mediate tonic inhibition. In withdrawal, a rebound activation of these receptors occurs. Both GABA-A and GABA-B receptors, especially the alpha2 subunit of GABA-A receptors, are also likely involved. Homocysteine increases with active drinking, and in withdrawal, excitotoxicity likely is induced by a further increase in homocysteine, viewed as a risk factor for AWS and also as a screening tool. The dopamine transporter gene is also associated with AWS. Characteristics involves changes in the ECG, especially an increase in QT interval, and EEG changes, including abnormal quantified EEG, at times periodic lateralized epileptiform discharges, and especially seizures, usually occurring 6-48h after the cessation of drinking. Therapy has emphasized benzodiazepines, mainly diazepam and lorazepam, but more standard antiepileptic drugs, like carbamazepine and topiramate, are also effective and safe.

Selected line difference in sensitivity to a GABAergic neurosteroid during ethanol withdrawal.

The neurosteroid allopregnanolone (ALLO) is a potent positive modulator of gamma-aminobutyric acid(A) (GABA(A)) receptors. Earlier work indicates that sensitivity to the anticonvulsant effect of ALLO was enhanced during ethanol (EtOH) withdrawal in rats and in C57BL/6 mice, an inbred strain with mild EtOH withdrawal. In contrast, ALLO sensitivity was reduced during EtOH withdrawal in DBA/2 mice, an inbred strain with severe EtOH withdrawal. Thus, the present studies examined ALLO sensitivity during EtOH withdrawal in another animal model of EtOH withdrawal severity, the Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) selected lines. Male mice were exposed to EtOH vapor or air for 72 h. During peak withdrawal, animals were injected with ALLO [0, 3.2, 5, 10 or 17 mg/kg, intraperitoneally (i.p.)] and tested for their sensitivity to the anticonvulsant effect. In separate studies, potentiation of GABA-stimulated chloride uptake by ALLO (10 nm to 10 microm) was assessed in microsacs prepared from mouse brain mice during peak withdrawal. Notably, WSP mice were cross-tolerant to the anticonvulsant effect of ALLO during EtOH withdrawal (i.e. significant decrease in the efficacy of ALLO) when compared with values in air-exposed mice. In contrast, sensitivity to the anticonvulsant effect of ALLO was unchanged during EtOH withdrawal in the WSR line. Functional sensitivity of GABA(A) receptors to ALLO was significantly decreased during EtOH withdrawal in WSP mice in a manner consistent with the change in behavioral sensitivity to ALLO. These findings suggest that mice selectively bred for differences in EtOH withdrawal severity are differentially sensitive to ALLO during EtOH withdrawal.

Alteration of kappa-opioid receptor system expression in distinct brain regions of a genetic model of enhanced ethanol withdrawal severity.

Abrupt withdrawal from chronic alcohol exposure can produce convulsions that are likely due to ethanol (EtOH) neuroadaptations. While significant efforts have focused on elucidating dependence mechanisms, the alterations contributing to EtOH withdrawal severity are less well characterized. The present studies examined the kappa-opioid receptor (KOP-R) system in Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice, selected lines that display severe and mild convulsions upon removal from chronic EtOH exposure. Previous data demonstrated significant increases in whole brain prodynorphin (Pdyn) mRNA in WSP mice only during EtOH withdrawal. No significant effects of EtOH exposure or withdrawal were observed in WSR mice. The present study characterized Pdyn mRNA and the KOP-R in WSP and WSR mice during EtOH withdrawal using in situ hybridization (ISH) and KOP-R autoradiography. Analyses were performed in brain regions that express Pdyn mRNA and/or KOP-R and that might participate in seizure circuitry: the piriform cortex, olfactory tubercle, nucleus accumbens, caudate-putamen, claustrum, dorsal endopiriform nucleus, and cingulate cortex. ISH analyses confirmed previous findings; EtOH withdrawal increased Pdyn mRNA in multiple brain regions of WSP mice, but not WSR. Basal KOP-R binding was higher in WSR mice than in WSP mice, suggesting an anti-convulsant role for receptor activation. Finally, increased KOP-R density was present during EtOH withdrawal in WSP mice. These data suggest that differences in the KOP-R system among the lines might contribute to their selected difference in EtOH withdrawal severity.

Ethanol withdrawal seizure susceptibility is associated with upregulation of L- and P-type Ca2+ channel currents in rat inferior colliculus neurons.

Calcium currents in the inferior colliculus (IC) are thought to play an important role in ethanol withdrawal hyperexcitability. Here, we report on the modulation of Ca(2+) channel currents in acutely dissociated IC neurons of rats, exhibiting higher incidence of audiogenic seizures when subjected to ethanol withdrawal. Whole cell Ca(2+) channel currents were activated by depolarizing pulses from a holding potential of -90 mV, in 10 mV increments, using barium (Ba(2+)) as the charge carrier. The high threshold voltage-activated (HVA) Ca(2+) channel current density increased significantly in IC neurons following ethanol withdrawal. The gating parameters of HVA Ca(2+) channel currents were only slightly altered, while the fraction of current that did not fully inactivate at positive potentials increased significantly following ethanol withdrawal. Pharmacological dissection of HVA Ca(2+) channel currents suggested that the enhanced current, associated with increased incidence of audiogenic seizures following ethanol withdrawal, was carried by L- and P-type Ca(2+) channels. The upregulation of L- and P-type currents may be responsible for IC neuronal hyperexcitability associated with increased susceptibility to ethanol withdrawal seizures.

The relationship between hippocampal acetylcholine release and cholinergic convulsant sensitivity in withdrawal seizure-prone and withdrawal seizure-resistant selected mouse lines.

BACKGROUND: The septo-hippocampal cholinergic pathway has been implicated in epileptogenesis, and genetic factors influence the response to cholinergic agents, but limited data are available on cholinergic involvement in alcohol withdrawal severity. Thus, the relationship between cholinergic activity and responsiveness and alcohol withdrawal was investigated in a genetic animal model of ethanol withdrawal severity. METHODS: Cholinergic convulsant sensitivity was examined in alcohol-naïve Withdrawal Seizure-Prone (WSP) and-Resistant (WSR) mice. Animals were administered nicotine, carbachol, or neostigmine via timed tail vein infusion, and the latencies to onset of tremor and clonus were recorded and converted to threshold dose. We also used microdialysis to measure basal and potassium-stimulated acetylcholine (ACh) release in the CA1 region of the hippocampus. Potassium was applied by reverse dialysis twice, separated by 75 min. Hippocampal ACh also was measured during testing for handling-induced convulsions. RESULTS: Sensitivity to several convulsion endpoints induced by nicotine, carbachol, and neostigmine were significantly greater in WSR versus WSP mice. In microdialysis experiments, the lines did not differ in basal release of ACh, and 50 mM KCl increased ACh output in both lines of mice. However, the increase in release of ACh produced by the first application of KCl was 2-fold higher in WSP versus WSR mice. When hippocampal ACh was measured during testing for handling-induced convulsions, extracellular ACh was significantly elevated (192%) in WSP mice, but was nonsignificantly elevated (59%) in WSR mice. CONCLUSIONS: These results suggest that differences in cholinergic activity and postsynaptic sensitivity to cholinergic convulsants may be associated with ethanol withdrawal severity and implicate cholinergic mechanisms in alcohol withdrawal. Specifically, WSP mice may have lower sensitivity to cholinergic convulsants compared with WSR because of postsynaptic receptor desensitization brought on by higher activity of cholinergic neurons.

No association between metabotropic glutamate receptors 7 and 8 (mGlur7 and mGlur8) gene polymorphisms and withdrawal seizures and delirium tremens in alcohol-dependent individuals.

- Up-regulation of the glutamatergic neurotransmission from chronic ethanol intoxication may cause a hyperexcitable state during alcohol withdrawal that may lead to seizures and delirium tremens. The aim of our study was to evaluate the association between a history of alcohol withdrawal-induced seizures and delirium tremens and a mGlurR7 (Tyr433Phe); and a mGlurR8 (C2756T) metabotropic glutamate receptor polymorphism in alcoholics compared to controls. A total of 182 patients meeting DSM-IV alcohol dependence criteria and 117 controls, both groups being of German descent, were investigated. mGluR7 and mGluR8 polymorphisms were determined using polymerase chain reaction of lymphocyte DNA. History of alcohol withdrawal-induced delirium tremens and seizures were obtained using the Semi-Structured Assessment of Genetics in Alcoholism (SSAGA). Data were cross-checked with inpatients' clinical files. No significant associations were obtained between both receptor polymorphisms and alcohol withdrawal-induced seizures and delirium tremens. The negative results in this study question the role of these polymorphisms in the pathogenesis of alcohol withdrawal-induced seizures and delirium tremens.

NMDA receptor subunit mRNA and protein expression in ethanol-withdrawal seizure-prone and -resistant mice.

BACKGROUND: Ethanol withdrawal seizure-prone (WSP) and -resistant (WSR) mice have been genetically selected for differences in handling-induced convulsion severity during withdrawal from chronic ethanol administration. Importantly, drug-naïve mice from these selected lines also differ in handling-induced convulsion severity. Different N-methyl-D-aspartate (NMDA) receptor subunit and splice variant associations confer varying sensitivities to ethanol, and may play a role in the different behavioral responses of the WSP and WSR mice. METHODS: In situ hybridization of riboprobes was used to characterize NMDA receptor subunit and splice variant mRNA expression in cortex and hippocampus from WSP and WSR mice. In addition, immunoblotting and immunohistochemistry were used to examine the expression of specific NMDA receptor subunits and splice variants in hippocampus and cortex from the selected mouse lines. RESULTS: In situ hybridization of riboprobes indicated that, in brain sections from both WSP and WSR mice, there was a differential regional distribution of mRNA for the mouse NR1, NR2A, NR2B, and NR2C NMDA receptor subunits. However, there were no differences between the selected lines in the hybridization of riboprobes to hippocampal subfields or cortical layers. In addition, hybridization of the probe for a 63-base N1-terminal cassette of ethanol-sensitive NR1 splice variants labeled both cortex and hippocampus. The level of hybridization did not differ across subfields of the hippocampus. Results from Western blot and immunohistochemical experiments also indicated that there were no differences between selected lines in NMDA receptor subunit protein expression. However, there was a correlation between mRNA and protein expression in hippocampus and cortex for each NMDA receptor subunit that was examined. CONCLUSIONS: The data suggest that at the level of both mRNA and protein, NMDA receptor subunit and splice variant expression can be uncoupled from convulsion severity in mice that have been selectively bred for symptoms of ethanol withdrawal.

Reduced ethanol withdrawal severity and altered withdrawal-induced c-fos expression in various brain regions of mice lacking protein kinase C-epsilon.

Withdrawal from chronic ethanol consumption can be accompanied by motor seizures, which may be a result of altered GABA(A) receptor function. Recently, we have generated and characterized mice lacking the epsilon isoform of protein kinase C as being supersensitive to the behavioral and biochemical effects of positive GABA(A) receptor allosteric modulators, including ethanol. The aim of the present study was to determine whether protein kinase C-epsilon null mutant mice display altered seizure severity during alcohol withdrawal. In addition, we used c-fos immunohistochemistry immediately following seizure assessment to identify potential brain regions involved in any observed differences in withdrawal severity. Mice were allowed to consume an ethanol-containing or control liquid diet as the sole source of food for 14 days. During the 7-h period following removal of the diet, both ethanol-fed wild-type and protein kinase C-epsilon null mutant mice displayed an overall increase in Handling-Induced Convulsion score versus control-fed mice. However, at 6 and 7h following diet removal, the Handling-Induced Convulsion score was reduced in ethanol-fed protein kinase C-epsilon null mutant mice compared to ethanol-fed wild-type mice. Ethanol-fed protein kinase C-epsilon null mutant mice also exhibited a decrease in the number of Fos-positive cells in the lateral septum, and an increase in the number of Fos-positive cells in the dentate gyrus, mediodorsal thalamus, paraventricular nuclei of the thalamus and hypothalamus, and substantia nigra compared to ethanol-fed wild-type mice. These data demonstrate that deletion of protein kinase C-epsilon results in diminished progression of ethanol withdrawal-associated seizure severity, suggesting that selective pharmacological inhibitors of protein kinase C-epsilon may be useful in the treatment of seizures during alcohol withdrawal. These data also provide insight into potential brain regions involved in generation or suppression of ethanol withdrawal seizures.

Modulation of audiogenic seizures by histamine and adenosine receptors in the inferior colliculus.

Susceptibility to behaviorally similar audiogenic seizures (AGS) occurs genetically and is inducible during ethanol withdrawal (ETX). Comparisons between AGS mechanisms of genetically epilepsy-prone rats (GEPR-9s) and ethanol-withdrawn rats (ETX-Rs) are yielding information about general pathophysiological mechanisms of epileptogenesis. The inferior colliculus (IC) is the AGS initiation site. Excitatory amino acid (EAA) abnormalities in the IC are implicated in AGS, and histamine and adenosine receptor activation each reduce EAA release and inhibit several seizure types. Previous studies indicate that focal infusion of an adenosine receptor agonist into the IC blocked AGS in GEPR-9s, but the effects of adenosine receptor activation in the IC on AGS in ETX-Rs are unknown. The effects of histamine receptor activation on either form of AGS are also unexamined. The present study evaluated effects of histamine or a nonselective adenosine A(1) agonist, 2-chloroadenosine, on AGS by focal microinjection into the IC. Ethanol dependence and AGS susceptibility were induced in normal rats by intragastric ethanol. Histamine (40 or 60 nmol/side) significantly reduced AGS in GEPR-9s, but histamine in doses up to 120 nmol/side did not affect AGS in ETX-Rs. 2-Chloroadenosine (5 or 10 nmol/side) did not affect AGS in ETX-Rs, despite the effectiveness of lower doses of this agent in GEPR-9s reported previously. Thus, histamine and adenosine receptors in the IC modulate AGS of GEPR-9s, but do not modulate ETX-induced AGS. The reasons for this difference may involve the chronicity of AGS susceptibility in GEPR-9s, which may lead to more extensive neuromodulation as compensatory mechanisms to limit the seizures compared to the acute AGS of ETX-Rs.