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Background: Protein kinase C (PKC) is a family of isoenzymes that regulate a variety of functions in the central nervous system including neurotransmitter release, ion channel activity, and cell differentiation. Growing evidence suggests that specific isoforms of PKC influence a variety of behavioral, biochemical, and physiological effects of ethanol in mammals. The purpose of this study was to determine whether acute ethanol exposure alters phosphorylation of conventional PKC isoforms at a threonine 674 (p-cPKC) site in the hydrophobic domain of the kinase, which is required for its catalytic activity., Methods: Male rats were administered a dose range of ethanol (0, 0.5, 1, or 2 g/kg, intragastric) and brain tissue was removed 10 minutes later for evaluation of changes in p-cPKC expression using immunohistochemistry and Western blot methods., Results: Immunohistochemical data show that the highest dose of ethanol (2 g/kg) rapidly increases p-cPKC immunoreactivity specifically in the nucleus accumbens (core and shell), lateral septum, and hippocampus (CA3 and dentate gyrus). Western blot analysis further showed that ethanol (2 g/kg) increased p-cPKC expression in the P2 membrane fraction of tissue from the nucleus accumbens and hippocampus. Although p-cPKC was expressed in numerous other brain regions, including the caudate nucleus, amygdala, and cortex, no changes were observed in response to acute ethanol. Total PKCgamma immunoreactivity was surveyed throughout the brain and showed no change following acute ethanol injection., Conclusions: These results suggest that ethanol rapidly promotes phosphorylation of cPKC in limbic brain regions, which may underlie effects of acute ethanol on the nervous system and behavior.

Recent work has identified a role for metabotropic glutamate receptor subtype 5 (mGlu(5)) in the discriminative stimulus properties of investigator-administered ethanol. The purpose of this study was to determine if mGlu(5) receptors modulate the discriminative stimulus properties of self-administered ethanol. Results show that the mGlu(5) receptor antagonist 6-Methyl-2-(phenylethynyl)pyridine (MPEP; 10 mg/kg) inhibited the discriminative stimulus properties of consumed ethanol during a self-administration test session. Further, 10 mg/kg MPEP increased and 1 mg/kg MPEP decreased the amount of self-administered ethanol required to produce full substitution. These results indicate that mGlu(5) receptors are involved in the expression of the discriminative stimulus properties of self-administered ethanol.

Protein kinase C (PKC) regulates a variety of neural functions, including ion channel activity, neurotransmitter release, receptor desensitization and differentiation. We have shown previously that mice lacking the epsilon-isoform of PKC (PKCepsilon) self-administer 75% less ethanol and exhibit supersensitivity to acute ethanol and allosteric positive modulators of GABA(A) receptors when compared with wild-type controls. The purpose of the present study was to examine involvement of PKCepsilon in GABA(A) receptor regulation of voluntary ethanol drinking. To address this question, PKCepsilon null-mutant and wild-type control mice were allowed to drink ethanol (10% v/v) vs. water on a two-bottle continuous access protocol. The effects of diazepam (nonselective GABA(A) BZ positive modulator), zolpidem (GABA(A) alpha1 agonist), L-655,708 (BZ-sensitive GABA(A) alpha5 inverse agonist), and flumazenil (BZ antagonist) were then tested on ethanol drinking. Ethanol intake (grams/kg/day) by wild-type mice decreased significantly after diazepam or zolpidem but increased after L-655,708 administration. Flumazenil antagonized diazepam-induced reductions in ethanol drinking in wild-type mice. However, ethanol intake by PKCepsilon null mice was not altered by any of the GABAergic compounds even though effects were seen on water drinking in these mice. Increased acute sensitivity to ethanol and diazepam, which was previously reported, was confirmed in PKCepsilon null mice. Thus, results of the present study show that PKCepsilon null mice do not respond to doses of GABA(A) BZ receptor ligands that regulate ethanol drinking by wild-type control mice. This suggests that PKCepsilon may be required for GABA(A) receptor regulation of chronic ethanol drinking.

Background: This work was conducted in an effort to establish an oral intake model system in which the effects of ethanol insult that occur during early stages of embryogenesis can be easily examined and in which agents that may modulate ethanol's teratogenicity can be readily tested in vivo. The model system described utilizes the alcohol deprivation effect to obtain teratogenic levels of maternal ethanol intake on days 7 and 8 of pregnancy in C57Bl/6J mice. Ocular defects including microphthalmia and uveal coloboma, which have previously been shown to result from ethanol administered by gavage or via intraperitoneal injection on these days, served as the developmental end point for this study. The ocular defects are readily identifiable and their degree of severity is expected to correlate with concurrently developing defects of the central nervous system (CNS)., Methods: Female C57Bl/6J mice were maintained on an ethanol-containing (4.8% v/v) liquid diet for 14 days and then mated during a subsequent abstinence period. Mice were then reexposed to ethanol on days 7 and 8 of pregnancy only. Control as well as ethanol-exposed dams were killed on their 14th day of pregnancy. Fetuses were then weighed, measured for crown rump length, photographed, and analyzed for ocular abnormalities. Globe size, palpebral fissure length, and pupil size and shape were noted for both the right and left eyes of all fetuses and informative comparisons were made., Results: This exposure paradigm resulted in peak maternal blood alcohol concentrations that ranged from 170 to 220 mg/dL on gestational day (GD) 8. Compared with the GD 14 fetuses from the normal control group, the pair-fed, acquisition controls, as well as the ethanol-exposed fetuses, were developmentally delayed and had reduced weights. Confirming previous studies, comparison of similarly staged control and treated GD 8 embryos illustrated reductions in the size of the forebrain in the latter. Subsequent ocular malformations were noted in 33% of the right eyes and 25% of the left eyes of the 103 GD 14 ethanol-exposed fetuses examined. This incidence of defects is twice that observed in the control groups. Additionally, it was found that the palpebral fissure length is directly correlated with globe size., Conclusions: The high incidence of readily identifiable ocular malformations produced by oral ethanol intake in this model and their relevance to human fetal alcohol spectrum disorders (FASD) makes this an excellent system for utilization in experiments involving factors administered to the embryo that might alter ethanol's teratogenic effects. Additionally, the fact that early ethanol insult yields ocular and forebrain abnormalities that are developmentally associated allows efficient specimen selection for subsequent detailed analyses of CNS effects in this in vivo mammalian FASD model.

Rationale: Many of the biochemical, physiological, and behavioral effects of ethanol are known to be mediated by ionotropic glutamate receptors. Emerging evidence implicates metabotropic glutamate receptors (mGluRs) in the biobehavioral effects of ethanol and other drugs of abuse, but there is little information regarding the role of mGluRs in the reinforcing effects of ethanol., Materials and Methods: Male C57BL/6J mice were trained to lever-press on a concurrent fixed ratio 1 schedule of ethanol (10% v/v) vs water reinforcement during 16-h sessions. Effects of mGluR1, mGluR2/3, and mGluR5 antagonists were then tested on parameters of ethanol self-administration behavior., Results: The mGluR5 antagonist MPEP (1-10 mg/kg, i.p.) dose-dependently reduced ethanol-reinforced responding but had no effect on concurrent water-reinforced responding. Analysis of the temporal pattern of responding showed that MPEP reduced ethanol-reinforced responding during peak periods of behavior occurring during the early hours of the dark cycle. Further analysis showed that MPEP reduced the number of ethanol response bouts and bout-response rate. MPEP also produced a 13-fold delay in ethanol response onset (i.e., latency to the first response) with no corresponding effect on water response latency or locomotor activity. The mGluR1 antagonist CPCCOEt (1-10 mg/kg, i.p.) or the mGluR2/3 antagonist LY 341495 (1-30 mg/kg, i.p.) failed to alter ethanol- or water-reinforced responding., Conclusions: These data indicate that mGlu5 receptors selectively regulate the onset and maintenance of ethanol self-administration in a manner that is consistent with reduction in ethanol's reinforcement function.

Rats and mice have a tendency to interact more with a novel object than with a familiar object. This tendency has been used by behavioral pharmacologists and neuroscientists to study learning and memory. A popular protocol for such research is the object-recognition task. Animals are first placed in an apparatus and allowed to explore an object. After a prescribed interval, the animal is returned to the apparatus, which now contains the familiar object and a novel object. Object recognition is distinguished by more time spent interacting with the novel object. Although the exact processes that underlie this 'recognition memory' requires further elucidation, this method has been used to study mutant mice, aging deficits, early developmental influences, nootropic manipulations, teratological drug exposure and novelty seeking.

Using a locomotor conditioning preparation, we examined whether manipulating time between exposure to distinct environmental cues and nicotine administration affected conditioned responding. Rats that received nicotine (0.42 mg/kg base) immediately before placement in an environment for 30 min on eight separate occasions displayed hyperactivity relative to controls in a subsequent injection/drug-free test. This conditioned hyperactivity was weaker if nicotine was administered 15 min before environment exposure. Conditioning was not evidenced when nicotine was administered 15 min after placement or upon removal from the environment. In a follow-up experiment, rats received 45 min in the environment; nicotine was administered 15 min after placement. This group showed conditioning that was localized to the last two-thirds of a 45 min test indicating that a 15 min delay did not prevent conditioning given 30 min of environment/nicotine overlap. This apparent timing of conditioned responding was not due to increasing environment exposure to 45 min. Further, a state-dependent environmental familiarization account of locomotor hyperactivity during testing was eliminated by the finding that rats displayed temporally specific increases in activity on the test day despite the fact that the context was previously experienced without drug for 15 min on eight consecutive days.

The discriminative stimulus properties of ethanol are mediated in part by positive modulation of GABA(A) receptors. Recent evidence indicates that metabotropic glutamate receptor subtype 5 (mGluR5) activity can influence GABA(A) receptor function. Therefore, the purpose of this work was to examine the potential involvement of mGluR5 in the discriminative stimulus effects of ethanol. In rats trained to discriminate ethanol (1 g/kg, intragastric gavage (i.g.)) from water, 2-methyl-6-(phenylethyl)-pyridine (MPEP) (1-50 mg/kg, i.p.) a selective noncompetitive antagonist of the mGlu5 receptor did not produce ethanol-like stimulus properties. However, pretreatment with MPEP (30 mg/kg) reduced the stimulus properties of ethanol as indicated by significant reductions in ethanol-appropriate responding, specifically at 0.5 and 1 g/kg ethanol, and a failure of ethanol test doses (1 and 2 g/kg) to fully substitute for the ethanol training dose. To test whether mGluR5 antagonism altered the GABA(A) receptor component of the ethanol stimulus, the ability of MPEP to modulate pentobarbital and diazepam substitution for ethanol was assessed. Pentobarbital substitution (1-10 mg/kg, i.p.) for ethanol was not altered by MPEP pretreatment. However, MPEP pretreatment inhibited the ethanol-like stimulus properties of diazepam (5 mg/kg, i.p.). To examine a potential anatomical basis for these pharmacological findings, expression patterns of mGluR5- and benzodiazepine-sensitive GABA(A) alpha1-containing receptors were examined by dual-label fluorescent immunohistochemistry with visualization by confocal microscopy. Results indicated that mGluR5- and GABA(A) alpha1-containing receptors were both coexpressed in limbic brain regions and colocalized on the same cells in specific brain regions including the amygdala, hippocampus, globus pallidus, and ventral pallidum. Together, these findings suggest an interaction between mGluR5- and benzodiazepine-sensitive GABA(A) receptors in mediating ethanol discrimination.

A decrease in sensitivity to pleasurable stimuli, anhedonia, is a major symptom of depression in humans. Several animal models have been developed to simulate this symptom (e.g. drug withdrawal, learned helplessness) using reward-sensitive procedures such as intracranial self-stimulation and progressive ratio responding as a measure of reward function. Recently, we introduced the use of another procedure, novel-object place conditioning in rats, to measure reward function in an associative learning situation. Withdrawal from chronic nicotine blocked a place preference conditioned by access to novel objects. This blockade was not due to impairment of object interaction, general activity, novelty detection, environmental familiarization, or expression of learning. Consequently, nicotine withdrawal directly reduced the rewarding properties of novelty. It is proposed that the novel-object place conditioning procedure could be usefully extended to other experimental situations and to genetically altered mice, so as to better understand the processes underlying changes in reward function.

The ionotropic serotonin subtype-3 (5-HT3) receptor has emerged as a potential therapeutic target in the treatment of alcohol abuse and alcoholism because selective pharmacological antagonists reduce alcohol consumption in preclinical and clinical models. 5-HT binds to the extracellular N-terminus of the 5-HT(3A) receptor subunit but receptor activation is also enhanced by distinct allosteric sites, which indicates the presence of other receptor subunits. It is not known if specific molecular subunits of the 5-HT3 receptor modulate alcohol drinking. To address this issue, we characterized acute locomotor response to alcohol and alcohol consumption in a two-bottle home-cage procedure by congenic C57BL/6J mice with a targeted deletion of the 5-HT(3A) receptor subunit gene. 5-HT(3A)-null mice did not differ from wild-type littermate controls on measures of spontaneous locomotor activity, habituation to a novel environment, or locomotor response to ethanol (0, 0.5, 1, or 2 g/kg). Moreover, null mice did not differ from controls on measures of ethanol (2-10%) intake and preference during or after a two-bottle home-cage sucrose fading procedure. Systemic administration of the 5-HT3 antagonist LY-278,584 (0-10 mg/kg) decreased intake of both sweetened (2% sucrose+10% ethanol) and unsweetened (10% ethanol) ethanol in wild-type mice only. These findings indicate that reduction of alcohol drinking produced by 5-HT3 antagonism is dependent on the presence of 5-HT(3A)-containing receptors.

Rationale: A growing number of studies suggest that gamma-aminobutyric acid type-B (GABA(B)) receptor agonists reduce alcohol use and craving., Objectives: This study was designed to further clarify behavioral mechanism(s) by which GABA(B) agonists may decrease alcohol reinforcement., Methods: Male C57BL/6 J mice were trained to lever press on a concurrent schedule of ethanol (10% v/v) and water reinforcement during 16-h overnight sessions. Effects of the GABA(B) agonist baclofen (0-17 mg/kg, IP) or SKF 97541 (0-1 mg/kg, IP) were examined on parameters of self-administration. Subsequently, potential motor inhibition and interaction with ethanol-induced sedation by GABA(B) agonists was examined in ethanol naive and self-administering mice., Results: Baclofen (10 mg/kg) and SKF 97541 (0.3 mg/kg) reduced ethanol-reinforced responding. In a locomotor activity test, these doses of the GABA(B) agonists inhibited locomotion in the ethanol-experienced mice and in a group of ethanol-inexperienced mice; locomotor suppression was greater in the ethanol-inexperienced mice. These doses of the GABA(B) agonists also potentiated the sedative effects of ethanol (4 g/kg) and converted a nonsedative dose of ethanol (2 g/kg) into a fully sedative dose. GABA(B) agonist enhancement of the sedative effects of ethanol was less pronounced in ethanol self-administering mice, suggesting cross-tolerance at the low dose of ethanol., Conclusions: GABA(B) agonists decrease the reinforcing effects of ethanol at doses that inhibit locomotor activity and potentiate the sedative hypnotic effects of ethanol. These nonspecific effects of GABA(B) agonists were reduced in alcohol experienced mice, suggesting cross-tolerance to the inhibitory properties of GABA(B) positive modulation. These data question the safety of prescribing GABA(B) agonists to alcoholics since these drugs may potentiate ethanol's sedative/hypnotic effects during relapse.

Rationale: In Pavlovian conditioning research, nicotine is typically conceptualized as the unconditioned stimulus (US) that becomes associated with an exteroceptive conditioned stimulus (CS). This research has not explored the possibility that nicotine can also function as a CS., Objectives: The present research examined whether nicotine served as a CS for the presence (CS+) or absence (CS-) of sucrose and started defining its specificity., Methods and Results: Rats trained in the CS+ condition had nicotine (0.4 mg/kg, base) paired intermittently with brief access to sucrose. Intermixed were saline sessions without sucrose. Nicotine acquired the ability to evoke goal tracking. This conditioned response (CR) decreased across extinction sessions. The CR was sensitive to nicotine dose (ED(50)=0.113 mg/kg) and administration to testing interval; 0-min and 100-min delays produced no CR. The CS properties were specific to nicotine in that amphetamine and bupropion substitution was incomplete. Rats in the CS- condition received similar discrimination training except that sucrose was paired with saline. Nicotine also served as a CS-; the saline state CS+ acquired control of goal tracking. Mecamylamine, but not hexamethonium, blocked nicotine's ability to serve as a CS+ and CS-, indicating a role for central nicotinic acetylcholine receptors., Conclusions: Nicotine served as a signal for the presence or absence of sucrose. The extinction, CS-, and substitution results eliminated a psychomotor stimulant account. The conceptualization of nicotine as a CS suggests novel empirical research in which a drug acquires additional inhibitory and/or excitatory value based on other outcomes present during its effects.

In the present experiments, we investigated the effects of several commonly employed antiepileptic drugs on the performance of adult rats in a Morris water maze task. We found that phenytoin treatment produced the most deleterious performance impairments across all days of training, and that these performance deficits are not likely due to any general sensorimotor impairments. Carbamazepine had milder, but detectable negative effects, as carbamazepine-treated animals exhibited initial acquisition deficits, but rapidly achieved escape levels comparable to controls. In marked contrast, valproate and ethosuximide had no detectable effects on learning in the water maze. These results parallel previous findings in rats treated with these compounds and tested in an instrumental learning task, and are in general agreement with the human clinical literature. To the extent that one might wish to minimize learning deficits associated with maintenance on antiepileptic drugs, phenytoin is definitely not the treatment of choice, while valproate or ethosuximide are apparently much less disruptive.

The authors tested the decreased reward function hypothesis of nicotine withdrawal using a novel-object place conditioning task. A conditioned place preference was evident in controls and in rats that had experienced 4 nicotine withdrawal days, but not in rats that had experienced 1-3 withdrawal days. This implies that the rewarding properties of interacting with novel objects were not readily associated with the environment in which they were paired. Follow-up experiments eliminated other explanations based on withdrawal-induced failures to process object or environment information. Also, expression of conditioning was not affected, indicating that withdrawal likely altered acquisition. Further investigation into the neurochemical and behavioral changes that accompany nicotine withdrawal will lead to a better understanding of the withdrawal syndrome.

Background: Activation of GABA(A) receptors in the amygdala or nucleus accumbens produces discriminative stimulus effects that substitute fully for those of systemically administered ethanol. This study was conducted to determine if GABA(A) receptors in the amygdala and nucleus accumbens interactively modulate ethanol discrimination., Methods: Male Long-Evans rats were trained to discriminate between intraperitoneal injections of ethanol (1 g/kg) and saline on a 2-lever drug discrimination task. The rats were then surgically implanted with bilateral injection cannulae aimed at the nucleus accumbens and the amygdala., Results: Infusion of the GABA(A) agonist muscimol in the nucleus accumbens resulted in full substitution for systemically administered ethanol. Concurrent infusion of the GABA(A) antagonist bicuculline in the amygdala shifted the muscimol substitution curve in the nucleus accumbens 10-fold to the right., Conclusions: These results indicate that blockade of GABA(A) receptors in the amygdala significantly reduces the potency of the GABA(A) agonist in the nucleus accumbens. This suggests that the ethanol-like stimulus effects of GABA(A) receptor activation in the nucleus accumbens are modulated by GABA(A) receptor activity in the amygdala. These data support the hypothesis that the addictive stimulus properties of alcohol are mediated by GABAergic transmission in a neural circuit involving the amygdala and nucleus accumbens.

We have attempted to investigate the role of extraretinal influences in controlling visual system development by rearing cats with esotropia in combination with sagittal transection of the optic chiasm. This combination leave two intact A1 laminae, one innervated by the deviated eye and one innervated by the unoperated eye and thus minimizes the contributions of retinally mediated influences. Cell size measurements in the dorsal lateral geniculate nucleus revealed that the neurons in the A1 lamina innervated by the deviated eye were, on average, 10-15% smaller than their counterparts in the contralateral A1 lamina.