Control of chronic excessive alcohol drinking by genetic manipulation of the Edinger-Westphal nucleus urocortin-1 neuropeptide system.

Midbrain neurons of the centrally projecting Edinger-Westphal nucleus (EWcp) are activated by alcohol, and enriched with stress-responsive neuropeptide modulators (including the paralog of corticotropin-releasing factor, urocortin-1). Evidence suggests that EWcp neurons promote behavioral processes for alcohol-seeking and consumption, but a definitive role for these cells remains elusive. Here we combined targeted viral manipulations and gene array profiling of EWcp neurons with mass behavioral phenotyping in C57BL/6 J mice to directly define the links between EWcp-specific urocortin-1 expression and voluntary binge alcohol intake, demonstrating a specific importance for EWcp urocortin-1 activity in escalation of alcohol intake.

Sweetened ethanol drinking during social isolation: enhanced intake, resistance to genetic heterogeneity and the emergence of a distinctive drinking pattern in adolescent mice.

With its ease of availability during adolescence, sweetened ethanol ('alcopops') is consumed within many contexts. We asked here whether genetically based differences in social motivation are associated with how the adolescent social environment impacts voluntary ethanol intake. Mice with previously described differences in sociability (BALB/cJ, C57BL/6J, FVB/NJ and MSM/MsJ strains) were weaned into isolation or same-sex pairs (postnatal day, PD, 21), and then given continuous access to two fluids on PDs 34-45: one containing water and the other containing an ascending series of saccharin-sweetened ethanol (3-6-10%). Prior to the introduction of ethanol (PDs 30-33), increased water and food intake was detected in some of the isolation-reared groups, and controls indicated that isolated mice also consumed more 'saccharin-only' solution. Voluntary drinking of 'ethanol-only' was also higher in a subset of the isolated groups on PDs 46-49. However, sweetened ethanol intake was increased in all isolated strain × sex combinations irrespective of genotype. Surprisingly, blood ethanol concentration (BEC) was not different between these isolate and socially housed groups 4 h into the dark phase. Using lickometer-based measures of intake in FVB mice, we identified that a predominance of increased drinking during isolation transpired outside of the typical circadian consumption peak, occurring ≈8.5 h into the dark phase, with an associated difference in BEC. These findings collectively indicate that isolate housing leads to increased consumption of rewarding substances in adolescent mice independent of their genotype, and that for ethanol this may be because of when individuals drink during the circadian cycle.

Prairie Voles as a Model to Screen Medications for the Treatment of Alcoholism and Addictions.

Most preclinical studies of medications to treat addictions are performed in mice and rats. These two rodent species belong to one phylogenetic subfamily, which narrows the likelihood of identifying potential mechanisms regulating addictions in other species, ie, humans. Expanding the genetic diversity of organisms modeling alcohol and drug abuse enhances our ability to screen for medications to treat addiction. Recently, research laboratories adapted the prairie vole model to study mechanisms of alcohol and drugs of abuse. This development not only expanded the diversity of genotypes used to screen medications, but also enhanced capabilities of such screens. Prairie voles belong to 3-5% of mammalian species exhibiting social monogamy. This unusual trait is reflected in their ability to form lasting long-term affiliations between adult individuals. The prairie vole animal model has high predictive validity for mechanisms regulating human social behaviors. In addition, these animals exhibit high alcohol intake and preference. In laboratory settings, prairie voles are used to model social influences on drug reward and alcohol consumption as well as effects of addictive substances on social bonding. As a result, this species can be adapted to screen medications whose effectiveness could be (a) resistant to social influences promoting excessive drug taking, (b) dependent on the presence of social support, and (c) medications affecting harmful social consequences of alcohol and drug abuse. This report reviews the literature on studies of alcohol and psychostimulants in prairie voles and discusses capabilities of this animal model as a screen for novel medications to treat alcoholism and addictions.

Effects of isoflurane and ethanol administration on c-Fos immunoreactivity in mice.

Noninvasive functional imaging holds great promise for the future of translational research, due to the ability to directly compare between preclinical and clinical models of psychiatric disorders. Despite this potential, concerns have been raised regarding the necessity to anesthetize rodent and monkey subjects during these procedures, because anesthetics may alter neuronal activity. For example, in studies on drugs of abuse and alcohol, it is not clear to what extent anesthesia can interfere with drug-induced neural activity. Therefore, the current study investigated whole-brain c-Fos activation following isoflurane anesthesia as well as ethanol-induced activation of c-Fos in anesthetized mice. In the first experiment, we examined effects of one or three sessions of gaseous isoflurane on c-Fos activation across the brain in male C57BL/6J mice. Isoflurane administration led to c-Fos activation in several areas, including the piriform cortex and lateral septum. Lower or similar levels of activation in these areas were detected after three sessions of isoflurane, suggesting that multiple exposures may eliminate some of the enhanced neuronal activation caused by acute isoflurane. In the second experiment, we investigated the ability of ethanol injection (1.5 or 2.5g/kgi.p.) to induce c-Fos activation under anesthesia. Following three sessions of isoflurane, 1.5g/kg of ethanol induced c-Fos in the central nucleus of amygdala and the centrally-projecting Edinger-Westphal nucleus (EWcp). This induction was lower after 2.5g/kg of ethanol. These results demonstrate that ethanol-induced neural activation can be detected in the presence of isoflurane anesthesia. They also suggest, that while habituation to isoflurane helps reduce neuronal activation, interaction between effects of anesthesia and alcohol can occur. Studies using fMRI imaging could benefit from using habituated animals and dose-response analyses.

Dissection of corticotropin-releasing factor system involvement in locomotor sensitivity to methamphetamine.

Sensitivity to the euphoric and locomotor-activating effects of drugs of abuse may contribute to risk for excessive use and addiction. Repeated administration of psychostimulants such as methamphetamine (MA) can result in neuroadaptive consequences that manifest behaviorally as a progressive escalation of locomotor activation, termed psychomotor sensitization. The present studies addressed the involvement of specific components of the corticotropin-releasing factor (CRF) system in locomotor activation and psychomotor sensitization induced by MA (1, 2 mg/kg) by utilizing pharmacological approaches, as well as a series of genetic knockout (KO) mice, each deficient for a single component of the CRF system: CRF-R1, CRF-R2, CRF, or the CRF-related peptide Urocortin 1 (Ucn1). CRF-R1 KO mice did not differ from wild-type mice in sensitization to MA, and pharmacological blockade of CRF-R1 with CP-154,526 (15, 30 mg/kg) in DBA/2J mice did not selectively attenuate either the acquisition or expression of MA-induced sensitization. Deletion of either of the endogenous ligands of CRF-R1 (CRF, Ucn1) either enhanced or had no effect on MA-induced sensitization, providing further evidence against a role for CRF-R1 signaling. Interestingly, deletion of CRF-R2 attenuated MA-induced locomotor activation, elucidating a novel contribution of the CRF system to MA sensitivity, and suggesting the participation of the endogenous urocortin peptides Ucn2 and Ucn3. Immunohistochemistry for Fos was used to visualize neural activation underlying CRF-R2-dependent sensitivity to MA, identifying the basolateral and central nuclei of the amygdala as neural substrates involved in this response. Our results support further examination of CRF-R2 involvement in neural processes associated with MA addiction.

Differential sensitivity of the perioculomotor urocortin-containing neurons to ethanol, psychostimulants and stress in mice and rats.

The perioculomotor urocortin-containing population of neurons (pIIIu: otherwise known as the non-preganglionic Edinger-Westphal nucleus) is sensitive to alcohol and is involved in the regulation of alcohol intake. A recent study indicated that this brain region is also sensitive to psychostimulants. Since pIIIu has been shown to respond to stress, we investigated how psychostimulant-induced pIIIu activation compares to stress- and ethanol-induced activation, and whether it is independent from a generalized stress response. Several experiments were performed to test how the pIIIu responds to psychostimulants by quantifying the number of Fos immunoreactive nuclei after acute i.p. injections of saline, 10-30 mg/kg cocaine, 5 mg/kg methamphetamine, 5 mg/kg amphetamine, 2.5 g/kg ethanol, 2 h of restraint stress, 10 min of swim stress, or six applications of mild foot shock in male C57BL/6 J mice. We also compared Fos immunoreactivity in pIIIu after acute (20 mg/kg cocaine) and repeated cocaine exposure (7 days of 20 mg/kg cocaine) injections in male C57BL/6 J mice in order to investigate the potential habituation of this response. Finally, we quantified the number of Fos immunoreactive nuclei in pIIIu after administration of saline, 2.5 g/kg ethanol, 20 mg/kg cocaine, or 2 h of restraint stress in male Sprague-Dawley rats. We found that exposure to psychostimulants and ethanol induced significantly higher Fos levels in pIIIu compared to stress in mice. Furthermore, repeated cocaine injections did not decrease Fos immunoreactivity as would be expected if this response were due to stress. In rats, exposure to ethanol, psychostimulant and restraint stress all induced pIIIu Fos immunoreactivity compared to saline-injected controls. In both mice and rats, ethanol- and cocaine-induced Fos immunoreactivity occurred exclusively in urocortin 1-positive, but not in tyrosine hydroxylase-positive, cells. These results provide evidence that the pIIIu Fos-response to psychostimulants is independent of a generalized stress in mice, but not rats. They additionally show that the pIIIu response to stress differs significantly between species.

Urocortin 1 microinjection into the mouse lateral septum regulates the acquisition and expression of alcohol consumption.

Previous studies using genetic and lesion approaches have shown that the neuropeptide urocortin 1 (Ucn1) is involved in regulating alcohol consumption. Ucn1 is a corticotropin releasing factor (CRF) -like peptide that binds CRF1 and CRF2 receptors. Perioculomotor urocortin-containing neurons (pIIIu), also known as the non-preganglionic Edinger-Westphal nucleus, are the major source of Ucn1 in the brain and are known to innervate the lateral septum. Thus, the present study tested whether Ucn1 could regulate alcohol consumption through the lateral septum. In a series of experiments Ucn1 or CRF was bilaterally injected at various doses into the lateral septum of male C57BL/6J mice. Consumption of 20% volume/volume ethanol or water was tested immediately after the injections using a modification of a 2-h limited access sweetener-free "drinking-in-the-dark" procedure. Ucn1 significantly suppressed ethanol consumption when administered prior to the third ethanol drinking session (the expression phase of ethanol drinking) at doses as low as 6 pmol. Ethanol intake was differentially sensitive to Ucn1, as equivalent doses of this peptide did not suppress water consumption. In contrast, CRF suppressed both ethanol and water intake at 40 and 60 pmol, but not at lower doses. Repeated administration of Ucn1 during the acquisition of alcohol consumption showed that 40 pmol (but not 2 or 0.1 pmol) significantly attenuated ethanol intake. Repeated administration of Ucn1 also resulted in a decrease of ethanol intake in sham-injected animals, a finding suggesting that the suppressive effect of Ucn1 on ethanol intake can be conditioned. Taken together, these studies confirm the importance of lateral septum innervation by Ucn1 in the regulation of alcohol consumption.

Urocortin 1 in the dorsal raphe regulates food and fluid consumption, but not ethanol preference in C57BL/6J mice.

The midbrain-localized Edinger-Westphal nucleus is a major site of production of urocortin 1. Urocortin 1 is a neuropeptide related to corticotropin-releasing factor that has high affinity for corticotropin-releasing factor type-1 and corticotropin-releasing factor type-2 receptors. In several mouse models, the amount of urocortin 1 neurons within the Edinger-Westphal nucleus is positively associated with ethanol preference. Central administration of urocortin 1 exerts potent anorectic actions, and implicates endogenous urocortin 1 in the regulation of food intake. It is possible that brain areas such as the dorsal raphe, which receives urocortin 1 from the Edinger-Westphal nucleus and highly expresses corticotropin-releasing factor type-2 receptors, mediate the actions of urocortin 1 on feeding and ethanol preference. In this study the amount of food, water and ethanol consumed over the dark cycle by ethanol-preferring C57BL/6J mice was measured after injection of artificial cerebrospinal fluid vehicle, urocortin 1, corticotropin-releasing factor and the corticotropin-releasing factor type-2 receptor-selective antagonist antisauvagine-30 onto the dorsal raphe. Compared with vehicle, corticotropin-releasing factor and antisauvagine-30, urocortin 1 induced a significant reduction in the amount of food consumed overnight. Also, compared with antisauvagine-30 treatment, urocortin 1 significantly reduced the amount of weight gained during this time. Urocortin 1 also significantly reduced the total amount of fluid consumed, but did not alter ethanol preference, which was high during all treatments. These results suggest that the dorsal raphe is a neuroanatomical substrate of urocortin 1-induced reductions in feeding, possibly through modulation of serotonergic activity from this nucleus. In addition, it is suggested that endogenous urocortin 1 in this area, such as from the Edinger-Westphal nucleus, does not regulate ethanol preference in C57BL/6J mice.

Urocortin 1-containing neurons in the human Edinger-Westphal nucleus.

The topographical location of neurons containing urocortin 1, a peptide related to corticotropin-releasing factor was investigated in human postmortem brain by immunohistochemistry, and compared with the location of neurons containing choline acetyltransferase, a marker for cholinergic cells. A three-dimensional computer reconstruction of the urocortin 1 and choline acetyltransferase-positive population of neurons within the oculomotor area was made. It was shown that the urocortin 1-positive neurons are located within the area identified as the Edinger-Westphal nucleus according to the human brain stem atlas, and that the neurons identified as Edinger-Westphal nucleus in the atlas are not choline acetyltransferase-positive. This finding agrees with recent animal studies showing that urocortin 1-positive neurons are not identical with the parasympathetic cholinergic neurons projecting to the ciliary ganglion. They indicate that the neurons identified as Edinger-Westphal nucleus in the human brain stem atlas belong to the non-preganglionic Edinger-Westphal nucleus, whereas the location of preganglionic Edinger-Westphal nucleus remains unidentified.

Urocortin 1 expression in five pairs of rat lines selectively bred for differences in alcohol drinking.

RATIONALE: There is accumulating evidence that the neuropeptide urocortin 1 (Ucn1) is involved in alcohol consumption. Thus far, however, most studies have been performed in mice. OBJECTIVES: The purpose of the present study was to characterize Ucn1 expression in rats selectively bred for either high or low alcohol intake. METHODS: Brains from naive male rats of five pairs of independently selected lines (iP/iNP, AA/ANA, HARF/LARF, HAD1/LAD1, and HAD2/LAD2) were analyzed by immunohistochemistry. RESULTS: Significant differences were found between iP/iNP, HARF/LARF, and HAD2/LAD2 in number of Ucn1-containing cells in the Edinger-Westphal (EW) nucleus (the main source of Ucn1 in the brain), whereas no significant differences were found between HAD1/LAD1 and AA/ANA. Similarly, significant differences in the optical density of Ucn1 immunoreactivity in EW were found between iP/iNP, HARF/LARF, and HAD2/LAD2, whereas no differences on this measure were found between HAD1/LAD1 and AA/ANA. In the lateral septum (LS, the main projection area of Ucn1-containing neurons in the rat), significant differences were found only between AA/ANA and HAD2/LAD2; however, a meta-analysis indicated that across all five lines, preferring animals had a significantly greater number of Ucn1-positive fibers than nonpreferring animals. CONCLUSIONS: These results provide evidence that, in rats, Ucn1 may be involved in regulation of alcohol intake, and that this regulation may occur through the Ucn1 projections to LS.

Ethanol versus lipopolysaccharide-induced hypothermia: involvement of urocortin.

The urocortin1 (Ucn1) neurons of the mid-brain-localized Edinger-Westphal nucleus (EW) are robustly responsive to ethanol (EtOH) administration, and send projections to the dorsal raphe nucleus (DRN), which contains corticotropin-releasing factor type 2 receptors (CRF2) that are responsive to Ucn1. In addition, the DRN has been shown to be involved in regulation of body temperature, a function greatly affected by EtOH administration. The goal of the present study was to identify the role that the urocortinergic projections from the EW to the DRN have in mediating EtOH-induced and lipopolysaccharide (LPS)-induced hypothermia. Male C57BL6/J mice were used. Groups of mice underwent cannulation of the DRN, and then received i.p. injections of EtOH (2g/kg) or LPS (600 microg/kg or 400 microg/kg), followed by intra-DRN injections of artificial cerebrospinal fluid (aCSF) or anti-sauvagine (aSVG) (55 pmol), a CRF2 antagonist. Separate groups of mice received single intra-DRN injections of Ucn1 (20 pmol), CRF (20 pmol) or aCSF. For all experiments, core temperatures were monitored rectally every 30 min for several hours post-injection. Both EtOH and LPS induced hypothermia, and aSVG significantly attenuated this effect after EtOH; however, there was no significant attenuation of hypothermia after either dose of LPS. Ucn1 injection also caused hypothermia, while CRF injection did not. These data demonstrate that EtOH-induced hypothermia, but not LPS-induced hypothermia, may involve Ucn1 from EW acting at CRF2 receptors in the DRN.

Urocortin 1 distribution in mouse brain is strain-dependent.

Urocortin 1 has been implicated in a number of specific behaviors, which include energy balance, stress reactivity and ethanol consumption. To elucidate genetically influenced differences in the mouse urocortin 1 system, we performed immunohistochemical characterization of urocortin 1 distribution in C57BL/6J and DBA/2J mouse brain. Urocortin 1 analysis reveals strain-dependent differences in distribution of urocortin 1 immunoreactive neurons and neuronal fibers. In both strains, the highest number of urocortin 1-positive neurons was observed in the Edinger-Westphal nucleus and lateral superior olive. Urocortin 1-positive neurons were detected in the dorsal nucleus of the lateral lemniscus of DBA/2J mice, but were absent in the C57BL/6J strain. Differences in urocortin 1 fibers were detected in many areas throughout the brain, and were most apparent in the septal areas, thalamic areas, several midbrain regions, and medulla. Strain-dependent distribution of urocortin 1-containing cells and fibers suggests that differences in this neuropeptide system may underlie differences in behavior and physiological responses between these strains. Further, we found that in both mouse strains, urocortin 1 in the Edinger-Westphal nucleus and choline acetyltransferase are not coexpressed. We show that the urocortin 1-positive neurons of this brain area form a separate population of cells that we propose to be called the non-preganglionic Edinger-Westphal nucleus.

Strain differences in urocortin expression in the Edinger-Westphal nucleus and its relation to alcohol-induced hypothermia.

The Edinger-Westphal nucleus is the primary source of urocortin in rodent brain. Mapping of inducible transcription factors has shown that the Edinger-Westphal nucleus is preferentially sensitive to ethanol self-administration. In the present study we have immunohistochemically compared expression of urocortin and c-Fos in naive and ethanol-treated C57BL/6J and DBA/2J mouse inbred strains. We found that C57BL/6J mice possess significantly higher numbers of urocortin-expressing cells in the Edinger-Westphal compared to DBA/2J mice. Subsequent histological analysis confirmed a lower number of large neurons in the DBA/2J Edinger-Westphal nucleus. Surprisingly, despite the differences in structure, no strain differences were observed in the number of c-Fos-containing cells after acute (0.6-4.8 g/kg, i.p.) and repeated (2.4 g/kg, 14 days, one injection/day) administration of ethanol. Double-label immunohistochemistry showed that ethanol-induced c-Fos expression is present in different sets of Edinger-Westphal cells between the strains. Specifically, expression of c-Fos in C57BL/6J mice is preferentially induced in urocortin cells, while c-Fos in DBA/2J mice occurs in a mixed population of cells. Behavioral analysis of the B6D2 F2 intercross, a heterogeneous mouse strain, showed that the number of urocortin cells is positively correlated with basal temperatures and ethanol-induced hypothermia. Involvement of the Edinger-Westphal in alcohol-induced hypothermia is further confirmed by analysis of urocortin cells in the HOT/COLD selected lines. These results provide evidence that C57BL/6J and DBA/2J mice have structural differences in the Edinger-Westphal that can result in activation of different populations of neurons upon alcohol intoxication contributing to differential thermoregulation between these inbred strains.

Expression of the c-fos gene in the mouse brain during the acquisition of defensive behavior habits.

Experimental data reported here provide evidence that marked defensive motivation in conditions of an unavoidable electrocutaneous stimulus is accompanied in mice by increases in the expression to the c-fos gene in the hippocampus and cerebral cortex. As reinforcement and learning progress and the animals become able to achieve a useful result--avoiding the electrocutaneous shock--expression of the c-fos gene in these brain structures decreases. The level of expression of the c-fos gene was found to be different in animals in which defensive behavior had different efficacies. Expression was higher in mice making large numbers of errors than in mice with few errors during acquisition of the defensive habit.

Expression of c-Fos in Alko alcohol rats responding for ethanol in an operant paradigm.

BACKGROUND: Identification of the brain regions involved in ethanol administration is important for understanding the neurobiology of ethanol addiction. Animal studies with different brain mapping techniques found that voluntary ethanol self-administration leads to changes in activity of specific brain regions in patterns that only partially overlap with patterns of brain regions affected by involuntary (i.e., experimenter-administered) ethanol administration. As an extension of studies mapping changes in neural activity after voluntary ethanol drinking, this study analyzed expression of the inducible transcription factor c-Fos after ethanol consumption in an operant procedure. METHODS: AA (Alko alcohol) rats were trained to operantly respond for water, 0.2% saccharin, 0.2% saccharin/10% (w/v) ethanol, or 10% ethanol in a 30-min limited-access procedure. Animals were allowed to self-administer solutions for at least 40 ethanol response sessions and were killed 1.5 hr after beginning of the last session. Forty-seven brain regions were immunohistochemically analyzed for c-Fos expression. RESULTS: In this paradigm, ethanol dose-dependently increased c-Fos expression in the Edinger-Westphal nucleus (EW) and decreased expression in the dorsal tenia tecta compared with no-ethanol controls. No effects of saccharin on c-Fos expression were found. CONCLUSIONS: Our results extend previous findings of preferential sensitivity of EW to alcohol in voluntary self-administration procedures to operant responding for ethanol and warrant further investigation of ethanol's effects on the EW. The finding that ethanol attenuated c-Fos expression in the tenia tecta is novel. Taken together, these findings confirm that voluntary ethanol self-administration leads to changes in activity of a limited number of brain regions with previously unexamined roles in ethanol sensitivity and addiction.

Interactive effects of nicotine and alcohol co-administration on expression of inducible transcription factors in mouse brain.

Nicotine and alcohol are abused substances that are often used concurrently. Despite their combined usage, little is known about how they interact to produce changes in behavior and neural activity. Two experiments were conducted to identify interactions on both behavior and neural targets resulting from the co-administration of nicotine and alcohol. In Experiment 1, male C57BL/6J mice were administered saline, alcohol (2.4 g/kg, i.p.), nicotine (0.5 mg/kg, i.p.) or an alcohol/nicotine mixture and returned to their home cage. In Experiment 2, a higher dose of nicotine (1.0 mg/kg, i.p.) was included and animals were exposed to a novel environment. Several behavioral measures were analysed during novelty exposure. Immunohistochemical detection of inducible transcription factors (c-Fos and Egr1) was used in both experiments to identify changes in neural activation. Behavioral results suggested that the drugs were interacting in the production of behaviors. In particular, alcohol produced locomotor stimulation while it suppressed counts of rearing and leaning. When co-administered, nicotine appeared to counteract the alcohol-enhanced locomotor activity. Several brain regions were observed to have altered transcription factor expression in response to the different drug treatments, including amygdalar, hippocampal and cortical subregions. In a subset of these brain areas, nicotine and alcohol counteracted one another in the expression of transcription factors. These results identify several interactive target sites within the hippocampus, extended amygdala and cortical regions. The interactions appear to be a result of antagonizing actions of nicotine and alcohol. Finally, the results suggest that the combined use of nicotine and alcohol may offset the effects of the drug administered independently.

ITF expression in mouse brain during acquisition of alcohol self-administration.

Expression of inducible transcription factors (ITFs) c-Fos and FosB was investigated during acquisition of alcohol drinking in C57BL/6J mice. A slight but statistically significant increase in c-Fos expression was found in the Edinger-Westphal nucleus (EW) of animals consuming 2% ethanol/10% sucrose for the first time. Stronger expression of c-Fos in EW was found in animals repeatedly consuming ethanol-containing solutions. These findings underscore the potential importance of EW in alcohol-related behaviors.

Cocaine- and alcohol-mediated expression of inducible transcription factors is blocked by pentobarbital anesthesia.

Identifying the neurocircuitry involved in behavioral responses to drugs of abuse is an important step towards understanding the mechanisms of drug addiction. The present study sought to distinguish brain regions involved in pharmacological effects of cocaine and ethanol from secondary effects by administering these drugs in the presence or absence of pentobarbital anesthesia. Changes in neuronal activity were assessed by immunohistochemical analysis of expression of an inducible transcription factor (ITF), c-Fos, in the brain of rats habituated to repeated pentobarbital anesthesia or saline administration. Cocaine administration (15 mg/kg, i.v.) in non-anesthetized animals produced a strong induction of c-Fos in the striatum and large number of other brain areas. Ethanol administration (2 g/kg, i.p.) induced c-Fos in a smaller number of characteristic brain areas, including the central nucleus of amygdala and paraventricular nucleus of hypothalamus. However, neither of these drugs was able to induce c-Fos in pentobarbital-anesthetized rats (50 mg/kg, i.v.). The suppressive effects of pentobarbital were not specific to c-Fos, such that pentobarbital also suppressed expression of ITFs FosB and Egr1 in the striatum of cocaine-treated rats. On the other hand, pentobarbital by itself strongly induced c-Fos expression in the lateral habenula of saline-, cocaine-, and ethanol-injected rats. It is not clear whether the suppressive effects of anesthesia on ITF expression in other areas are mediated by activation of lateral habenula, or are independent of this event. Our data suggest that in the absence of conscious awareness of drug-associated cues, cocaine and alcohol activate only a fraction of the neural elements engaged in the unanesthetized state.

ITF mapping after drugs of abuse: pharmacological versus perceptional effects.

Analysis of inducible transcription factors (ITFs) expression is often applied to map drug-induced changes of neuronal activity in brain. Administration of cocaine and alcohol induces ITFs in a large number of brain structures. However, induction of ITFs in a brain region does not necessarily indicate a pharmacological effect of the drug in this brain region. Many of the brain regions could be activated by secondary effects. Perception of stimulus properties of the drug or locomotor effects of the drug are possible secondary effects. Anesthesia can block induction of ITFs by cocaine and alcohol suggesting that ITF expression in a majority of brain regions is more sensitive to secondary effects than to pharmacological effects of these drugs. In agreement with this hypothesis is our finding that the majority of brain regions responding with ITF expression to alcohol administration do not respond to voluntary alcohol self-administration in animals. Only a few brain regions show similar ITF induction after both administration and self-administration of this drug. Presumably these brain regions could be responding to pharmacological effects of alcohol. Given the low resolution of invasive techniques, ITF mapping experiments will continually contribute to our understanding of mechanisms of drug addiction and alcoholism.

Alcohol drinking produces brain region-selective changes in expression of inducible transcription factors.

Mapping the effects of alcohol consumption on neural activity could provide valuable information on mechanisms of alcohol's effects on behavior. The present study sought to identify effects of alcohol consumption on expression of inducible transcription factors (ITFs) in mouse brain. C57BL/6J mice were trained to consume 10% ethanol/10% sucrose solution during a 30-min limited access period. Control animals were given access to 10% sucrose solution or water. Following the final day of the procedure, animals were sacrificed and immunohistochemical analyses were performed for three ITFs (c-Fos, FosB, and Zif268). Alcohol-consuming animals had increased ITF expression in several brain areas. Specifically, c-Fos was significantly induced in the nucleus accumbens core (AcbC), the medial posteroventral portion of the central nucleus of the amygdala (CeMPV), and the Edinger-Westphal nucleus (EW). Expression of c-Fos was significantly lower in the dentate gyrus of alcohol-consuming animals vs. sucrose-consuming animals. However, it was not significantly different from the water controls. Induction of c-Fos in AcbC, CeMPV and EW was significantly related to blood alcohol concentrations (BAC). Furthermore, FosB expression in the CeMPV and the EW was also significantly higher in the alcohol-consuming animals vs. water controls. FosB expression in the EW was significantly related to BAC. The significance of these results is two-fold. First, our experiments demonstrate that ITF mapping is an effective strategy in identifying alcohol-induced changes following voluntary consumption. Second, they suggest a relationship between ITF expression in AcbC, CeMPV and EW and the level of alcohol intoxication.