Background: Chronic ethanol use is known to disrupt normal sleep rhythms, but the cellular basis for this disruption is unknown. An important contributor to normal sleep patterns is a low-threshold calcium current mediated by T-type calcium channels. The T-type calcium current underlies burst responses in thalamic nuclei that are important to spindle propagation, and we recently observed that this current is sensitive to acute low doses of ethanol. Methods: We used a combination of current clamp and voltage clamp recordings in an in vitro brain slice preparation of the dorsal lateral geniculate nucleus (LGN) of macaque monkeys that have chronically self-administered ethanol to determine whether chronic ethanol exposure may affect T-type currents. Results: Current clamp recordings from the LGN of ethanol naive macaques showed characteristic burst responses. However, recordings from the LGN in macaques that self-administered ethanol revealed a significant attenuation of bursts across a range of voltages (n = 5). Voltage clamp recordings from control LGN neurons (n = 16) and neurons (n = 29) from brain slices from chronically drinking macaques showed no significant differences (P > 0.05) in T-type current kinetics or in the membrane resistance of the thalamic cells between the two cohorts. However, mean T-type current amplitude measured in the chronically drinking animals was reduced by 31% (P < 0.01). Conclusions: We conclude that chronic ethanol self-administration reduces calcium currents in thalamic relay cells without altering underlying current kinetics, which may provide a mechanistic framework for the well-documented disruptions in sleep/wake behavior in subjects with chronic ethanol exposure. (c) 2006 Elsevier B.V. All rights reserved.

Withdrawal anxiety following chronic ethanol exposure is often associated with relapse in recovering alcoholics. It is likely that brain regions regulating anxiety-like behaviors adapt during chronic ethanol exposure to ultimately regulate such behaviors. The central amygdala contains numerous neurotransmitter systems that have been implicated in the regulation of anxiety-like behavior, including corticotropin releasing factor (CRF) and NMDA-type glutamate receptors. Chronic ethanol exposure causes functional adaptations in both CRF and NMDA receptors that are likely to regulate anxiety-like behaviors expressed during withdrawal. However, the molecular mechanisms governing these adaptations remain unexplored. We therefore evaluated these neurotransmitter systems in Sprague–Dawley rats during chronic ingestion of an ethanol-containing liquid diet. Quantitative real-time reverse transcription–PCR demonstrated that preproCRF mRNA was significantly upregulated by chronic ethanol exposure, whereas mRNA expression of CRF binding protein did not change. There were also no significant changes observed in any of the NMDA subunit mRNAs, although there was a trend toward greater NR2A mRNA expression during chronic ethanol exposure. Using Western blotting analysis we measured NMDA receptor subunit protein expression. Chronic ethanol exposure did not affect protein levels of the NR1 and NR2B subunits. Like the mRNA measures, chronic ethanol exposure did influence NR2A protein levels but the effects were modest. Our results demonstrate that NMDA receptor subunit mRNA and protein expressions are not strongly influenced by exposure to chronic ethanol. This suggests that the functional NMDA receptor adaptations identified in previous studies [Roberto, M., Schweitzer, P., Madamba, S. G., Stouffer, D. G., Parsons, L. H., & Siggins, G. R. (2004). Acute and chronic ethanol exposure alter glutamatergic transmission in rat central amygdala: an in vitro and in vivo analysis. J Neurosci 24, 1594–1603] are likely to be mediated by post-translational events. In contrast, enhanced levels of CRF during/after chronic ethanol exposure are likely to be mediated by increased levels of prepro CRF mRNA. Together, our findings suggest that adaptations to chronic ethanol exposure by proanxiety factors expressed in the central nucleus appear to be mediated by distinct cellular and molecular mechanisms.

The neurobiological mechanisms governing alcohol-induced alterations in anxiety-like behaviors are not fully understood. Given that the amygdala is a major emotional center in the brain and regulates the expression of both learned fear and anxiety, neurotransmitter systems within the basolateral amygdala represent likely mechanisms governing the anxiety-related effects of acute ethanol exposure. It is well established that, within the glutamatergic system, N-methyl-d-aspartate (NMDA)-type receptors are particularly sensitive to intoxicating concentrations of ethanol. However, recent evidence suggests that kainate-type glutamate receptors are sensitive to ethanol as well. Therefore, we examined the effect of acute ethanol on kainate receptor (KA-R)-mediated synaptic transmission in the basolateral amygdala (BLA) of Sprague–Dawley rats. Acute ethanol decreased KA-R-mediated excitatory postsynaptic currents (EPSCs) in the BLA in a concentration-dependent manner. Ethanol also inhibited currents evoked by focal application of the kainate receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), and ethanol inhibition of kainate EPSCs was not associated with a change in paired-pulse ratio, suggesting a postsynaptic mechanism of ethanol action. The neurophysiological consequences of this acute sensitivity were tested by measuring ethanol's effects on KA-R-dependent modulation of synaptic plasticity. Acute ethanol, like the GluR5-specific antagonist (R,S)-3-(2-carboxybenzyl)willardiine (UBP 296), robustly diminished ATPA-induced increases in synaptic efficacy. Lastly, to better understand the relationship between KA-R activity and anxiety-like behavior, we bilaterally microinjected ATPA directly into the BLA. We observed an increase in measures of anxiety-like behavior, assessed in the light/dark box, with no change in locomotor activity. This evidence suggests that kainate receptors in the BLA are inhibited by pharmacologically relevant concentrations of ethanol and may contribute to some of the acute anxiolytic effects of this drug.

Withdrawal anxiety after chronic alcohol is likely to contribute to drug seeking and relapse in alcoholics. The brain regions regulating fear/anxiety behaviors, especially neurotransmitter systems with acute ethanol sensitivity, are potential targets for chronic ethanol-induced adaptations. We have therefore examined N-methyl-D-aspartate ( NMDA) receptors after chronic ethanol ingestion in rat lateral/basolateral amygdala. Whole cell patch-clamp measurements indicate that chronic ethanol ingestion significantly increased NMDA receptor current density. This enhanced NMDA receptor function was also associated with an increase in ifenprodil inhibition and a decrease in apparent calcium-dependent current inactivation. These findings suggest that NR2B-containing receptors may be specifically enhanced and suggest that processes dependent upon calcium influx through amygdala NMDA receptors may potentially be enhanced by chronic ethanol ingestion. We measured subunit mRNA expression to investigate possible molecular mechanisms that control functional receptor adaptations to chronic ethanol. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that NR1 subunit mRNA expression, but not NR2 or NR3 expression, was enhanced in samples from chronic ethanol-exposed animals. Single-cell RT-PCR was then used to confirm that NR2 mRNA expression was unaltered by chronic ethanol. Most GAD(-), presumed projection neurons expressed both NR2A and NR2B mRNAs, and this profile did not change during chronic ethanol exposure. Our results suggest that both transcriptional and nontranscriptional adaptations to chronic ethanol ultimately contribute to alterations in NMDA receptor function. Because amygdala NMDA receptors play a significant role in many learned fear behaviors, chronic ethanol-induced adaptations in these receptors may influence the expression of withdrawal anxiety.

Annals of the New York Academy of SciencesVolume 985, Issue 1 p. 479-480 Effects of Chronic Alcohol Ingestion on Rat Lateral/Basolateral Amygdala Ligand-Gated Chloride Channels S. K. BOTTING, Corresponding Author S. K. BOTTING Department of Medical Pharmacology and Toxicology, Texas A & M System Health Science Center, College Station, Texas, USA Department of Pathology, University of Arizona Health Sciences Center, Tucson, Arizona, USAAddress for correspondence: S.K. Botting, Department of Medical Pharmacology and Toxicology, Texas A & M System Health Science Center, College Station, TX 77843.Search for more papers by this authorG. D. FRYE, G. D. FRYE Department of Medical Pharmacology and Toxicology, Texas A & M System Health Science Center, College Station, Texas, USASearch for more papers by this authorM. D. PULIDO, M. D. PULIDO Department of Medical Pharmacology and Toxicology, Texas A & M System Health Science Center, College Station, Texas, USASearch for more papers by this authorB. A. McCOOL, B. A. McCOOL Department of Medical Pharmacology and Toxicology, Texas A & M System Health Science Center, College Station, Texas, USA Department of Physiology & Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USASearch for more papers by this author S. K. BOTTING, Corresponding Author S. K. BOTTING Department of Medical Pharmacology and Toxicology, Texas A & M System Health Science Center, College Station, Texas, USA Department of Pathology, University of Arizona Health Sciences Center, Tucson, Arizona, USAAddress for correspondence: S.K. Botting, Department of Medical Pharmacology and Toxicology, Texas A & M System Health Science Center, College Station, TX 77843.Search for more papers by this authorG. D. FRYE, G. D. FRYE Department of Medical Pharmacology and Toxicology, Texas A & M System Health Science Center, College Station, Texas, USASearch for more papers by this authorM. D. PULIDO, M. D. PULIDO Department of Medical Pharmacology and Toxicology, Texas A & M System Health Science Center, College Station, Texas, USASearch for more papers by this authorB. A. McCOOL, B. A. McCOOL Department of Medical Pharmacology and Toxicology, Texas A & M System Health Science Center, College Station, Texas, USA Department of Physiology & Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USASearch for more papers by this author First published: 24 January 2006 https://doi.org/10.1111/j.1749-6632.2003.tb07104.xCitations: 2 Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume985, Issue1THE AMYGDALA IN BRAIN FUNCTION: Basic and Clinical ApproachesApril 2003Pages 479-480 RelatedInformation

It is well known that the anxiolytic potential of ethanol is maintained during chronic exposure. We have confirmed this using a light-dark box paradigm following chronic ethanol ingestion via a liquid diet. However, cessation from chronic ethanol exposure is known to cause severe withdrawal anxiety. These opposing effects on anxiety likely result from neuro-adaptations of neurotransmitter systems within the brain regions regulating anxiety. Recent work highlights the importance of amygdala ligand-gated chloride channels in the expression of anxiety. We have therefore examined the effects of chronic ethanol exposure on GABA(A) and strychnine-sensitive glycine receptors expressed by acutely isolated adult rat lateral/basolateral amygdala neurons. Chronic ethanol exposure increased the functional expression of GABA(A) receptors in acutely isolated basolateral amygdala neurons without altering strychnine-sensitive glycine receptors. Neither the acute ethanol nor benzodiazepine sensitivity of either receptor system was affected. We explored the likelihood that subunit composition might influence each receptor's response to chronic ethanol. Importantly, when expressed in a mammalian heterologous system, GABA(A) receptors composed of unique alpha subunits were differentially sensitive to acute ethanol. Likewise, the presence of the beta subunit appeared to influence the acute ethanol sensitivity of glycine receptors containing the alpha(2) subunit. Our results suggest that the facilitation of GABA(A) receptors during chronic ethanol exposure may help explain the maintenance of ethanol's anti-anxiety effects during chronic ethanol exposure. Furthermore, the subunit composition of GABA(A) and strychnine-sensitive glycine receptors may ultimately influence the response of each system to chronic ethanol exposure.

A definitive association between the aberrant expression of cytokines and adhesion molecules in renal failure has been established. In this regard a relationship between cytokine and adhesion molecule expression is suggested but has not been shown in models of proximal tubular cell injury. To investigate the impact of acute injury on the relationship between transforming growth factor-beta 1 (TGF-beta1) and intercellular adhesion molecule-1 (ICAM-1) expression, two immortalized mouse proximal tubular epithelial cell lines were exposed to cadmium chloride or mercuric chloride (0-50 microM) for 0-8 h. ELISA and Western blot measured expression of secreted and intercellular TGF-beta1, respectively. Direct cellular ELISA or Western blot was used to assess ICAM-1 expression. Challenge with cadmium caused a greater loss of cell viability than did mercury. Interestingly, cadmium significantly decreased the amount of TGF-beta1 in the conditioned media. Although a similar trend was seen in mercury-challenged cells, no significant differences were observed. The decrease in TGF-beta1 in the culture media was not due to decreased expression of this cytokine, as intercellular levels were not affected by metal-induced injury. Significant increases in ICAM-1 protein expression were observed following cadmium and mercury challenge. The increase in ICAM-1 appears to be due to increased mRNA, as Northern blot analysis demonstrated increased message expression following a 4-h cadmium or mercury challenge. Supplementation of the culture media with exogenous TGF-beta1 decreased basal ICAM-1 expression and attenuated the cadmium-induced increase. These data suggest that metal-induced injury is associated with increased ICAM-1 expression. The mechanism of this induction may involve the decreased TGF-beta1 in the conditioned media following metal challenge. Taken together, these studies suggest a link between cytokine and adhesion molecule expression in renal injury.

The medial septum and nucleus of the diagonal band (MS/nDB) contain cholinergic and GABAergic neuronal populations that have been identified based on immunohistochemical staining and/or electrophysiological properties. We explored the molecular diversity of MS/nDB neurons using single-cell reverse transcription-polymerase chain reaction (scRT-PCR) to assess gene expression profiles during aging in individual neurons acutely isolated from young (2–4 months) and aged (26–27 months) F344 rats. Neuronal gene expression profiles were characterized by detection of mRNAs for choline acetyltransferase (ChAT, cholinergic) and glutamate decarboxylase (GAD67, GABAergic), as well as mRNAs for calcium binding proteins (CaBPs) calbindin-D28k, calretinin and parvalbumin. Four major neuronal populations were identified: ChAT-positive (ChAT+) cells, GAD-positive (GAD+) cells, ChAT+/GAD+ cells and ChAT negative/GAD negative (ChAT−/GAD−) cells. With age, the percentage of cells expressing ChAT mRNA decreased from 53% in young to 40%, and the expression of GAD67 mRNA was reduced from 56 to 35% of the cells tested. The percentage of cells with detectable levels of both ChAT and GAD67 mRNA was reduced from 24% in young to 9% in aged. Concomitantly, the percentage of ChAT−/GAD− cells increased from 15 to 34% with age. Of the CaBPs, calretinin expression was observed most frequently in this study, and its detection decreased from 33 to 22% of the cells with age. Observations concerning the CaBPs were confirmed using in situ hybridization. These results suggest a shift in the mRNA expression profiles of MS/nDB neuronal populations during aging and exemplify the molecular diversity of cholinergic and GABAergic cells.

Fetal alcohol syndrome includes brain damage from aberrant synaptogenesis, altered cell–cell signaling and blunted plasticity in surviving neurons. Distortion of neurotrophic GABA signals by ethanol-mediated allosteric modulation of GABAA receptor (GABAAR) activity during brain maturation may play a role. In this regard, early postnatal binge-like ethanol treatment on postnatal days (PDs) 4–9 acutely inhibits whole cell GABAAR Cl− current and subsequently blunts GABAAR function in medial septum/diagonal band (MS/DB) neurons and cerebellar Purkinje cells [Dev. Brain Res. 130 (2001) 25–40; Brain Res. 810 (1998) 100–113; Brain Res. 832 (1999) 124–135]. In light of these functional changes, we hypothesized that ethanol treatment also would decrease levels of proteins important for assembly of GABAergic synapses in maturing brain. To test this relationship, binge-like ethanol intubation was administered to rat pups on PDs 4–9 producing peak blood ethanol concentrations in the range of 302.5±6.3 mg/dl. GABAergic synaptic proteins were measured in brain tissue on PDs 13–14 when GABAAR currents in individual MS/DB neurons are reduced, but those of cerebellar Purkinje neurons are not yet altered [Dev. Brain Res. 130 (2001) 25–40; Brain Res. 810 (1998) 100–113; Brain Res. 832 (1999) 124–135]. Surprisingly, ethanol did not decrease protein levels of GABAAR α1/β2 subunits, GAD67 or gephyrin in MS/DB at this time when whole cell recordings indicate GABAAR function is impaired in acutely dissociated individual neurons. However, in cerebellum where ethanol treated Purkinje cell GABAAR function remains normal on PDs 13–14 [Brain Res. 832 (1999) 124–135], reduced levels of several GABAergic synaptic proteins including: GAD67, GABAAR α1 subunit, ClC-2 a voltage-gated Cl− channel, synaptotagmin a synaptic vesicle protein, and N-cadherin, a synapse associated cell adhesion molecule, were found. These results indicate that binge-like ethanol exposure differentially decreases GABAergic synaptic proteins in some brain areas in a pattern that does not parallel reductions in GABAAR function of individual neurons that survive this ethanol insult.

1. The anticonvulsant properties of 2-chloroadenosine (CADO) in the basolateral amygdala rely on the activation of adenosine-specific heptahelical receptors. We have utilized whole-cell voltage-clamp electrophysiology to examine the modulatory effects of CADO and other adenosine receptor agonists on voltage-gated calcium channels in dissociated basolateral amygdala neurons. 2. CADO, adenosine, and the A1 subtype-selective agonists N6-(L-2-Phenylisopropyl)adenosine (R-PIA) and 2-chloro-N6-cyclopentyladenosine (CCPA) reversibly modulated whole cell Ba2+ currents in a concentration-dependent fashion. CADO inhibition of barium currents was also sensitive to the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). 3. The A2A-selective agonist 4-[2-[[6-Amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS21680) was without effect. 4. CADO inhibition was predominantly voltage-dependent and sensitive to the sulphydryl-modifying reagent N:-ethylmaleimide, implicating a membrane-delimited, G(i/o)-coupled signal transduction pathway in the channel regulation. 5. Using Ca2+ channel subtype-selective antagonists, CADO inhibition appeared to target multiple channel subtypes, with the inhibition of omega-conotoxin GVIA-sensitive calcium channels being more prominent. 6. Our results indicate that the anti-convulsant effects CADO in the basolateral amygdala may be mediated, in part, by the A1 receptor-dependent inhibition of voltage gated calcium channels.

In neonatal rats, strychnine‐sensitive glycine receptors are widely expressed in the spinal cord, brainstem and forebrain. During development, these ‘neonatal’ receptors are replaced by an adult isoform, the expression of which becomes restricted primarily to brain stem and spinal cord. Unlike most forebrain regions, functional strychnine‐sensitive glycine receptors appear to persist within adult rat amygdala. However, the subunit composition of glycine receptors expressed by amygdala neurons and its relationship to the adult isoform in brain stem/spinal cord has not been defined precisely. In this report, we have utilized RT‐PCR and single‐cell RT‐PCR to demonstrate that the ‘neonatal’α2‐subunit mRNA persists in adult rat amygdala neurons and is the predominant α‐subunit. We further demonstrate that native amygdala glycine receptors are relatively insensitive to the receptor antagonist picrotoxin, suggesting that α2‐ and β‐subunits may be present together in the same multisubunit complex. We further demonstrate that α2‐ and β‐subunits cloned from adult rat amygdala can form functional channels when expressed in a heterologous system. Together, these studies highlight both the unique characteristics of strychnine‐sensitive glycine receptors in the adult rat amygdala as well as the possibility that α2/β channels may represent the adult forebrain isoform of the strychnine‐sensitive glycine receptor.

Large concentrations of the beta-amino acid, taurine, can be found in many forebrain areas such as the basolateral amygdala, a portion of the limbic forebrain intimately associated with the regulation of fear/anxiety-like behaviors. In addition to its cytoprotective and osmoregulatory roles, taurine may also serve as an agonist at GABA(A)- and strychnine-sensitive glycine receptors. In this latter context, the present study demonstrates that application of taurine to acutely isolated neurons from the basolateral amygdala of adult rats causes significant alterations in resting membrane current, as measured by whole-cell patch clamp electrophysiology. Using standard pharmacological approaches, we find that currents gated by concentrations of taurine =3 mM are predominantly mediated by strychnine-sensitive receptors. Furthermore, these strychnine-sensitive receptors are shown to be pharmacologically and biophysically similar to 'classic' strychnine-sensitive, chloride-conducting glycine receptors expressed in brainstem and spinal cord. While amygdala glycine receptors can be distinguished from GABA(A) receptors expressed by the same neurons, these two chloride channels are functionally expressed at comparable levels. Given that a number of clinically relevant compounds are associated with the regulation of GABA(A) receptors in this brain region, the presence of both strychnine-sensitive glycine receptors and their agonist, taurine, in the basolateral amygdala may suggest an important role for these receptors in the limbic forebrain of adult rats.

We have shown previously that metabotropic glutamate receptors with group I-like pharmacology couple to N-type and P/Q-type calcium channels in acutely isolated cortical neurons using G proteins most likely belonging to the Gi/Go subclass. To better understand the potential mechanisms forming the basis for group I mGluR modulation of voltage-gated calcium channels in the CNS, we have examined the ability of specific mGluRs to couple to neuronal N-type (alpha1B-1/alpha2delta/beta1b) and P/Q-type (alpha1A-2/alpha2delta/beta1b) voltage-gated calcium channels in an HEK 293 heterologous expression system. Using the whole cell patch-clamp technique where intracellular calcium is buffered to low levels, we have shown that group I receptors inhibit both N-type and P/Q-type calcium channels in a voltage-dependent fashion. Similar to our observations in cortical neurons, this voltage-dependent inhibition is mediated almost entirely by N-ethylmaleimide (NEM)-sensitive heterotrimeric G proteins, strongly suggesting that these receptors can use Gi/Go-like G proteins to couple to N-type and P/Q-type calcium channels. However, inconsistent with the apparent NEM sensitivity of group I modulation of calcium channels, modulation of N-type channels in group I mGluR-expressing cells was only partially sensitive to pertussis toxin (PTX), indicating the potential involvement of both PTX-sensitive and -resistant G proteins. The PTX-resistant modulation was voltage dependent and entirely resistant to NEM and cholera toxin. A time course of treatment with PTX revealed that this toxin caused group I receptors to slowly shift from using a primarily NEM-sensitive G protein to using a NEM-resistant form. The PTX-induced switch from NEM-sensitive to -resistant modulation was also dependent on protein synthesis, indicating some reliance on active cellular processes. In addition to these voltage-dependent pathways, perforated patch recordings on group I mGluR-expressing cells indicate that another slowly developing, calcium-dependent form of modulation for N-type channels may be seen when intracellular calcium is not highly buffered. We conclude that group I mGluRs can modulate neuronal Ca2+ channels using a variety of signal transduction pathways and propose that the relative contributions of different pathways may exemplify the diversity of responses mediated by these receptors in the CNS.