Alcohol binge drinking during adolescence or dependence during adulthood reduces prefrontal myelin in male rats.

Teen binge drinking is associated with low frontal white matter integrity and increased risk of alcoholism in adulthood. This neuropathology may result from alcohol exposure or reflect a pre-existing condition in people prone to addiction. Here we used rodent models with documented clinical relevance to adolescent binge drinking and alcoholism in humans to test whether alcohol damages myelinated axons of the prefrontal cortex. In Experiment 1, outbred male Wistar rats self-administered sweetened alcohol or sweetened water intermittently for 2 weeks during early adolescence. In adulthood, drinking behavior was tested under nondependent conditions or after dependence induced by 1 month of alcohol vapor intoxication/withdrawal cycles, and prefrontal myelin was examined 1 month into abstinence. Adolescent binge drinking or adult dependence induction reduced the size of the anterior branches of the corpus callosum, i.e., forceps minor (CCFM), and this neuropathology correlated with higher relapse-like drinking in adulthood. Degraded myelin basic protein in the gray matter medial to the CCFM of binge rats indicated myelin was damaged on axons in the mPFC. In follow-up studies we found that binge drinking reduced myelin density in the mPFC in adolescent rats (Experiment 2) and heavier drinking predicted worse performance on the T-maze working memory task in adulthood (Experiment 3). These findings establish a causal role of voluntary alcohol on myelin and give insight into specific prefrontal axons that are both sensitive to alcohol and could contribute to the behavioral and cognitive impairments associated with early onset drinking and alcoholism.

Is the alcohol deprivation effect genetically mediated? Studies with HXB/BXH recombinant inbred rat strains.

BACKGROUND: Two features of alcohol addiction that have been widely studied in animal models are relapse drinking following periods of alcohol abstinence and the escalation of alcohol consumption after chronic continuous or intermittent alcohol exposure. The genetic contribution to these phenotypes has not been systematically investigated. METHODS: HXB/BXH recombinant inbred (RI) rat strains were given access to alcohol sequentially as follows: alcohol (10%) as the only fluid for 1 week; alcohol (10%) and water in a 2-bottle choice paradigm for 7 weeks ("pre-alcohol deprivation effect [ADE] alcohol consumption"); 2 weeks of access to water only (alcohol deprivation); and 2 weeks of reaccess to 10% alcohol and water ("post-ADE alcohol consumption"). The periods of deprivation and reaccess to alcohol were repeated 3 times. The ADE was defined as the amount of alcohol consumed in the first 24 hours after deprivation minus the average daily amount of alcohol consumed in the week prior to deprivation. Heritability of the phenotypes was determined by analysis of variance, and quantitative trait loci (QTLs) were identified. RESULTS: All strains showed increased alcohol consumption, compared to the predeprivation period, in the first 24 hours after each deprivation (ADE). Broad-sense heritability of the ADEs was low (ADE1, 9.1%; ADE2, 26.2%; ADE3, 16.3%). Alcohol consumption levels were relatively stable over weeks 2 to 7. Post-ADE alcohol consumption levels consistently increased in some strains and were decreased or unchanged in others. Heritability of pre- and post-ADE alcohol consumption was high and increased over time (week 2, 38.5%; week 7, 51.1%; week 11, 56.8%; week 15, 63.3%). QTLs for pre- and post-ADE alcohol consumption were similar, but the strength of the QTL association with the phenotype decreased over time. CONCLUSIONS: In the HXB/BXH RI rat strains, genotypic variance does not account for a large proportion of phenotypic variance in the ADE phenotype (low heritability), suggesting a role of environmental factors. In contrast, a large proportion of the variance across the RI strains in pre- and post-ADE alcohol consumption is due to genetically determined variance (high heritability).

Alcohol drinking during early adolescence activates microglial cells and increases frontolimbic Interleukin-1 beta and Toll-like receptor 4 gene expression, with heightened sensitivity in male rats compared to females.

Adolescent drinking is risky because neural circuits in the frontal lobes are undergoing maturational processes important for cognitive function and behavioral control in adulthood. Previous studies have shown that myelinated axons in the medial prefrontal cortex (mPFC) are particularly sensitive to alcohol drinking, especially in males. Pro-inflammatory mediators like toll-like receptor 4 (TLR4) and interleukin-1 beta (IL1b) have been implicated in alcohol induced-inflammation and demyelination; thus, herein we test the hypothesis that voluntary alcohol drinking early in adolescence elicits a pro-inflammatory state that is more pronounced in the brain of males compared to females. Adolescent male and female Wistar rats self-administered sweetened alcohol or sweetened water from postnatal days 28-42 and separate sets of brains were processed for 1) immunolabeling for ionized calcium-binding adapter molecule 1 to analyze microglial cell morphology, or 2) qPCR analysis of gene expression of pro-inflammatory mediators. Binge drinking alcohol activated microglia in the mPFC and hippocampus of both males and females, suggesting that voluntary alcohol exposure initiates an inflammatory response. Il1b mRNA was upregulated in the mPFC of both sexes. Conversely, Tlr4 mRNA levels were elevated after drinking only in males, which could explain more robust effects of alcohol on myelin in this region in developing males compared to females. Il1b mRNA changes were not observed in the hippocampus, but alcohol elevated Tlr4 mRNA in both sexes, highlighting regional specificity in inflammatory responses to alcohol. Overall, these findings give insight into potential mechanisms by which low-to-moderate voluntary alcohol intake impacts the developing brain. This article is part of the special Issue on 'Vulnerabilities to Substance Abuse'.

Shortening time for access to alcohol drives up front-loading behavior, bringing consumption in male rats to the level of females.

BACKGROUND: Incentives to promote drinking ("happy hour") can encourage faster rates of alcohol consumption, especially in women. Sex differences in drinking dynamics may underlie differential health vulnerabilities relating to alcohol in women versus men. Herein, we used operant procedures to model the happy hour effect and gain insight into the alcohol drinking dynamics of male and female rats. METHODS: Adult male and female Wistar rats underwent operant training to promote voluntary drinking of 10% (w/v) alcohol (8 rats/sex). We tested how drinking patterns changed after manipulating the effort required for alcohol (fixed ratio, FR), as well as the length of time in which rats had access to alcohol (self-administration session length). Rats were tested twice within the 12 h of the dark cycle, first at 2 h (early phase of the dark cycle, "early sessions") and then again at 10 h into the dark cycle (late phase of the dark cycle, "late sessions") with an 8-h break between the two sessions in the home cage. RESULTS: Adult females consumed significantly more alcohol (g/kg) than males in the 30-min sessions with the FR1 schedule of reinforcement when tested late in the dark cycle. Front-loading of alcohol was the primary factor driving higher consumption in females. Changing the schedule of reinforcement from FR1 to FR3 reduced total consumption. Notably, this manipulation had minimal effect on front-loading behavior in females, whereas front-loading behavior was significantly reduced in males when more effort was required to access alcohol. Compressing drinking access to 15 min to model a happy hour drove up front-loading behavior, generating alcohol drinking patterns in males that were similar to patterns in females (faster drinking and higher intake). CONCLUSIONS: This strategy could be useful for exploring sex differences in the neural mechanisms underlying alcohol drinking and related health vulnerabilities. Our findings also highlight the importance of the time of testing for detecting sex differences in drinking behavior.

Protracted withdrawal from alcohol and drugs of abuse impairs long-term potentiation of intrinsic excitability in the juxtacapsular bed nucleus of the stria terminalis.

The juxtacapsular bed nucleus of the stria terminalis (jcBNST) is activated in response to basolateral amygdala (BLA) inputs through the stria terminalis and projects back to the anterior BLA and to the central nucleus of the amygdala. Here we show a form of long-term potentiation of the intrinsic excitability (LTP-IE) of jcBNST neurons in response to high-frequency stimulation of the stria terminalis. This LTP-IE, which was characterized by a decrease in the firing threshold and increased temporal fidelity of firing, was impaired during protracted withdrawal from self-administration of alcohol, cocaine, and heroin. Such impairment was graded and was more pronounced in rats that self-administered amounts of the drugs sufficient to maintain dependence. Dysregulation of the corticotropin-releasing factor (CRF) system has been implicated in manifestation of protracted withdrawal from dependent drug use. Administration of the selective corticotropin-releasing factor receptor 1 (CRF(1)) antagonist R121919 [2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7-dipropylamino-pyrazolo[1,5-a]pyrimidine)], but not of the CRF(2) antagonist astressin(2)-B, normalized jcBNST LTP-IE in animals with a history of alcohol dependence; repeated, but not acute, administration of CRF itself produced a decreased jcBNST LTP-IE. Thus, changes in the intrinsic properties of jcBNST neurons mediated by chronic activation of the CRF system may contribute to the persistent emotional dysregulation associated with protracted withdrawal.

Genetical genomic determinants of alcohol consumption in rats and humans.

BACKGROUND: We have used a genetical genomic approach, in conjunction with phenotypic analysis of alcohol consumption, to identify candidate genes that predispose to varying levels of alcohol intake by HXB/BXH recombinant inbred rat strains. In addition, in two populations of humans, we assessed genetic polymorphisms associated with alcohol consumption using a custom genotyping array for 1,350 single nucleotide polymorphisms (SNPs). Our goal was to ascertain whether our approach, which relies on statistical and informatics techniques, and non-human animal models of alcohol drinking behavior, could inform interpretation of genetic association studies with human populations. RESULTS: In the HXB/BXH recombinant inbred (RI) rats, correlation analysis of brain gene expression levels with alcohol consumption in a two-bottle choice paradigm, and filtering based on behavioral and gene expression quantitative trait locus (QTL) analyses, generated a list of candidate genes. A literature-based, functional analysis of the interactions of the products of these candidate genes defined pathways linked to presynaptic GABA release, activation of dopamine neurons, and postsynaptic GABA receptor trafficking, in brain regions including the hypothalamus, ventral tegmentum and amygdala. The analysis also implicated energy metabolism and caloric intake control as potential influences on alcohol consumption by the recombinant inbred rats. In the human populations, polymorphisms in genes associated with GABA synthesis and GABA receptors, as well as genes related to dopaminergic transmission, were associated with alcohol consumption. CONCLUSION: Our results emphasize the importance of the signaling pathways identified using the non-human animal models, rather than single gene products, in identifying factors responsible for complex traits such as alcohol consumption. The results suggest cross-species similarities in pathways that influence predisposition to consume alcohol by rats and humans. The importance of a well-defined phenotype is also illustrated. Our results also suggest that different genetic factors predispose alcohol dependence versus the phenotype of alcohol consumption.

Sex Differences in the Neurobiology of Alcohol Use Disorder.

Sex differences may play a critical role in modulating how chronic or heavy alcohol use impacts the brain to cause the development of alcohol use disorder (AUD). AUD is a multifaceted and complex disorder driven by changes in key neurobiological structures that regulate executive function, memory, and stress. A three-stage framework of addiction (binge/intoxication; withdrawal/negative affect; preoccupation/anticipation) has been useful for conceptualizing the complexities of AUD and other addictions. Initially, alcohol drinking causes short-term effects that involve signaling mediated by several neurotransmitter systems such as dopamine, corticotropin releasing factor, and glutamate. With continued intoxication, alcohol leads to dysfunctional behaviors that are thought to be due in part to alterations of these and other neurotransmitter systems, along with alterations in neural pathways connecting prefrontal and limbic structures. Using the three-stage framework, this review highlights examples of research examining sex differences in drinking and differential modulation of neural systems contributing to the development of AUD. New insights addressing the role of sex differences in AUD are advancing the field forward by uncovering the complex interactions that mediate vulnerability.

Permanent impairment of birth and survival of cortical and hippocampal proliferating cells following excessive drinking during alcohol dependence.

Experimenter-delivered alcohol decreases adult hippocampal neurogenesis and hippocampal-dependent learning and memory. The present study used clinically relevant rodent models of nondependent limited access alcohol self-administration and excessive drinking during alcohol dependence (alcohol self-administration followed by intermittent exposure to alcohol vapors over several weeks) to compare alcohol-induced effects on cortical gliogenesis and hippocampal neurogenesis. Alcohol dependence, but not nondependent drinking, reduced proliferation and survival in the medial prefrontal cortex (mPFC). Apoptosis was reduced in both alcohol groups within the mPFC, which may reflect an initiation of a reparative environment following alcohol exposure as decreased proliferation was abolished after prolonged dependence. Reduced proliferation, differentiation, and neurogenesis were observed in the hippocampus of both alcohol groups, and prolonged dependence worsened the effects. Increased hippocampal apoptosis and neuronal degeneration following alcohol exposure suggest a loss in neuronal turnover and indicate that the hippocampal neurogenic niche is highly vulnerable to alcohol.

Operant behavior and alcohol levels in blood and brain of alcohol-dependent rats.

BACKGROUND: The purpose of the present investigation was to more clearly define blood-alcohol parameters associated with alcohol dependence produced by alcohol vapor inhalation and alcohol-containing liquid diet. METHODS: Alcohol levels in blood and brain were compared during and after 4 hours of acute alcohol vapor exposure; also, brain-alcohol levels were assessed in alcohol-exposed (14-day alcohol vapor) and alcohol-naïve rats during and after 4 hours of acute alcohol vapor exposure. A separate group of rats were implanted with i.v. catheters, made dependent on alcohol via vapor inhalation, and tested for operant alcohol responding; blood-alcohol levels (BALs) were measured throughout operant alcohol drinking sessions during alcohol withdrawal. A final group of rats consumed an alcohol-liquid diet until they were dependent, and those rats were then tested for operant behavior at various withdrawal time points; BALs were measured at different withdrawal time points and after operant sessions. RESULTS: Blood- and brain-alcohol levels responded similarly to vapor, but brain-alcohol levels peaked at a higher point and more slowly returned to zero in alcohol-naïve rats relative to alcohol-exposed rats. Alcohol vapor exposure also produced an upward shift in subsequent operant alcohol responding and resultant BALs. Rats consumed large quantities of alcohol-liquid diet, most of it during the dark cycle, sufficient to produce high blood-alcohol levels and elevated operant alcohol responding when tested during withdrawal from liquid diet. CONCLUSIONS: These results emphasize that the key determinants of excessive alcohol drinking behavior are the BAL range and pattern of chronic high-dose alcohol exposure.

Corticotropin-releasing factor-1 receptor antagonists decrease heroin self-administration in long- but not short-access rats.

Dysregulation of the stress-related corticotropin-releasing factor (CRF) system has been implicated in the development of drug dependence. The present study examined the effects of administering CRF type 1 (CRF(1)) receptor antagonists on heroin self-administration in animals allowed short (1 hour) or long (8-12 hours) access to intravenous heroin self-administration sessions. The nonpeptide CRF(1) antagonists MJL-1-109-2 (1 hour versus 8 hours access) or R121919 (1 hour versus 12 hours access) were systemically injected in both short- and long-access rats. MJL-1-109-2 (10 mg/kg) and R121919 (10 and 20 mg/kg) reduced heroin self-administration in long-access animals without altering heroin intake in short-access animals. Both MJL-1-109-2 and R121919 decreased first-hour intravenous heroin self-administration selectively in long-access rats, with R121919 decreasing cumulative heroin intake across the 12-hour session. The results demonstrate that blockade of the CRF-CRF(1) receptor system attenuates the increased heroin intake of rats with extended access to the drug.

Sex Differences in the Effect of Alcohol Drinking on Myelinated Axons in the Anterior Cingulate Cortex of Adolescent Rats.

Cognitive deficits associated with teenage drinking may be due to disrupted myelination of prefrontal circuits. To better understand how alcohol affects myelination, male and female Wistar rats (n = 7-9/sex/treatment) underwent two weeks of intermittent operant self-administration of sweetened alcohol or sweetened water early in adolescence (postnatal days 28-42) and we tested for macro- and microstructural changes to myelin. We previously reported data from the males of this study showing that alcohol drinking reduced myelinated fiber density in layers II-V of the anterior cingulate division of the medial prefrontal cortex (Cg1); herein, we show that myelinated fiber density was not significantly altered by alcohol in females. Alcohol drinking patterns were similar in both sexes, but males were in a pre-pubertal state for a larger proportion of the alcohol exposure period, which may have contributed to the differential effects on myelinated fiber density. To gain more insight into how alcohol impacts myelinated axons, brain sections from a subset of these animals (n = 6/sex/treatment) were used for microstructural analyses of the nodes of Ranvier. Confocal analysis of nodal domains, flanked by immunofluorescent-labeled contactin-associated protein (Caspr) clusters, indicated that alcohol drinking reduced nodal length-to-width ratios in layers II/III of the Cg1 in both sexes. Despite sex differences in the underlying cause (larger diameter axons after alcohol in males vs. shorter nodal lengths after alcohol in females), reduced nodal ratios could have important implications for the speed and integrity of neural transmission along these axons in both males and females. Alcohol-induced changes to myelinated axonal populations in the Cg1 may contribute to long-lasting changes in prefrontal function associated with early onset drinking.

Methamphetamine self-administration and voluntary exercise have opposing effects on medial prefrontal cortex gliogenesis.

Psychostimulant abuse produces deficits in prefrontal cortex (PFC) function, whereas physical activity improves PFC-dependent cognition and memory. The present study explored the vulnerability of medial PFC (mPFC) precursor proliferation and survival to methamphetamine self-administration and voluntary exercise, factors that may have opposing effects on mPFC plasticity to facilitate functional consequences. Intermittent 1 h access to methamphetamine (I-ShA) increased, but daily 1 and 6 h access decreased, proliferation and survival, with dose-dependent effects on mature cell phenotypes. All groups showed increased cell death. Voluntary exercise enhanced proliferation and survival but, in contrast to methamphetamine exposure, did not alter cell death or mature phenotypes. Furthermore, enhanced cell survival by I-ShA and voluntary exercise had profound effects on gliogenesis with differential regulation of oligodendrocytes versus astrocytes. In addition, new cells in the adult mPFC stain for the neuronal marker neuronal nuclear protein, although enhanced cell survival by I-ShA and voluntary exercise did not result in increased neurogenesis. Our findings demonstrate that mPFC gliogenesis is vulnerable to psychostimulant abuse and physical activity with distinct underlying mechanisms. The susceptibility of mPFC gliogenesis to even modest doses of methamphetamine could account for the pronounced pathology linked to psychostimulant abuse.

Alcohol self-administration acutely stimulates the hypothalamic-pituitary-adrenal axis, but alcohol dependence leads to a dampened neuroendocrine state.

Clinical studies link disruption of the neuroendocrine stress system with alcoholism, but remaining unknown is whether functional differences in the hypothalamic-pituitary-adrenal (HPA) axis precede alcohol abuse and dependence or result from chronic exposure to this drug. Using an operant self-administration animal model of alcohol dependence and serial blood sampling, we show that longterm exposure to alcohol causes significant impairment of HPA function in adult male Wistar rats. Acute alcohol (voluntary self-administration or experimenter-administered) stimulated the release of corticosterone and its upstream regulator, adrenocorticotropic hormone, but chronic exposure sufficient to produce dependence led to a dampened neuroendocrine state. HPA responses to alcohol were most robust in 'low-responding' non-dependent animals (averaging < 0.2 mg/kg/session), intermediate in nondependent animals (averaging approximately 0.4 mg/kg/session), and most blunted in dependent animals (averaging approximately 1.0 mg/kg/session) following several weeks of daily 30-min self-administration sessions, suggesting that neuroendocrine tolerance can be initiated prior to dependence and relates to the amount of alcohol consumed. Decreased expression of corticotropin-releasing factor (CRF) mRNA expression in the paraventricular nucleus of the hypothalamus and reduced sensitivity of the pituitary to CRF may contribute to, but do not completely explain, neuroendocrine tolerance. The present results, combined with previous studies, suggest that multiple adaptations to stress regulatory systems may be brought about by excessive drinking, including a compromised hormonal response and a sensitized brain stress response that together contribute to dependence.

Effects of CRF1-receptor and opioid-receptor antagonists on dependence-induced increases in alcohol drinking by alcohol-preferring (P) rats.

BACKGROUND: Selective breeding of rats over generations and induction of alcohol dependence via chronic vapor inhalation both enhance alcohol consumption in animal models. The purpose of this study was to determine whether dependence-induced increases in alcohol consumption by P rats is sensitive to naltrexone, a general opioid receptor antagonist (but with highest affinity at the mu-opioid receptor at low doses), and the recently characterized small molecule CRF(1)-receptor antagonist MPZP (N,N-bis(2-methoxyethyl)-3-(4-methoxy-2-methylphenyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidin-7-amine). METHODS: P rats (n = 20) were trained to respond for alcohol and water in a 2-lever operant situation during daily 30-minute sessions. P rats were then matched for alcohol intake and exposed to chronic intermittent alcohol vapor (n = 10) or ambient air (n = 10) for approximately 10 weeks. All rats were then administered MPZP and naltrexone in 2 separate and consecutive Latin-square designs. RESULTS: MPZP attenuated dependence-induced increases in alcohol intake by P rats while having no effect on alcohol consumption by nondependent controls. Conversely, operant alcohol responding was reduced similarly in dependent and nondependent P rats by naltrexone. CONCLUSIONS: These results confirm a role for brain CRF(1)-receptor systems in dependence-induced changes in the reinforcing properties of alcohol, and CRF(1)-receptor blockade appears to suppress dependence-induced drinking at lower doses in P rats relative to other rat lines. Therefore, brain CRF(1)-receptor systems are important in the regulation of dependence-induced alcohol consumption, whereas brain opioid systems are important in the regulation of basal alcohol consumption by rats.

MPZP: a novel small molecule corticotropin-releasing factor type 1 receptor (CRF1) antagonist.

The extrahypothalamic stress peptide corticotropin-releasing factor (CRF) system is an important regulator of behavioral responses to stress. Dysregulation of CRF and the CRF type 1 receptor (CRF(1)) system is hypothesized to underlie many stress-related disorders. Modulation of the CRF(1) system by non-peptide antagonists currently is being explored as a therapeutic approach for anxiety disorders and alcohol dependence. Here, we describe a new, less hydrophilic (cLogP approximately 2.95), small molecule, non-peptide CRF(1) antagonist with high affinity (K(i)=4.9 nM) and specificity for CRF(1) receptors: N,N-bis(2-methoxyethyl)-3-(4-methoxy-2-methylphenyl)-2,5-dimethyl-pyrazolo[1,5-a] pyrimidin-7-amine (MPZP). The compound was systemically administered to adult male rats in two behavioral models dependent on the CRF(1) system: defensive burying (0, 5, 20 mg/kg, n=6-11 for each dose) and alcohol dependence (0, 5, 10, 20 mg/kg, n=8 for each self-administration group). Acute administration of MPZP reduced burying behavior in the defensive burying model of active anxiety-like behavior. MPZP also attenuated withdrawal-induced excessive drinking in the self-administration model of alcohol dependence without affecting nondependent alcohol drinking or water consumption. The present findings support the proposed significance of the CRF(1) system in anxiety and alcohol dependence and introduce a promising new compound for further development in the treatment of alcohol dependence and stress-related disorders.

Myelination of Axons Corresponds with Faster Transmission Speed in the Prefrontal Cortex of Developing Male Rats.

Myelination of prefrontal circuits during adolescence is thought to lead to enhanced cognitive processing and improved behavioral control. However, while standard neuroimaging techniques commonly used in human and animal studies can measure large white matter bundles and residual conduction speed, they cannot directly measure myelination of individual axons or how fast electrical signals travel along these axons. Here we focused on a specific population of prefrontal axons to directly measure conduction velocity and myelin microstructure in developing male rats. An in vitro electrophysiological approach enabled us to isolate monosynaptic projections from the anterior branches of the corpus callosum (corpus callosum-forceps minor, CCFM) to the anterior cingulate subregion of the medial prefrontal cortex (Cg1) and to measure the speed and direction of action potentials propagating along these axons. We found that a large number of axons projecting from the CCFM to neurons in Layer V of Cg1 are ensheathed with myelin between pre-adolescence [postnatal day (PD)15] and mid-adolescence (PD43). This robust increase in axonal myelination is accompanied by a near doubling of transmission speed. As there was no age difference in the diameter of these axons, myelin is likely the driving force behind faster transmission of electrical signals in older animals. These developmental changes in axonal microstructure and physiology may extend to other axonal populations as well, and could underlie some of the improvements in cognitive processing between childhood and adolescence.

Corticotropin-releasing factor in ventromedial prefrontal cortex mediates avoidance of a traumatic stress-paired context.

Post-traumatic stress disorder (PTSD) affects 7.7 million Americans. One diagnostic criterion for PTSD is avoidance of stimuli that are related to the traumatic stress. Using a predator odor stress conditioned place aversion (CPA) model, rats can be divided into groups based on stress reactivity, as measured by avoidance of the odor-paired context. Avoider rats, which show high stress reactivity, exhibit persistent avoidance of stress-paired context and escalated alcohol drinking. Here, we examined the potential role of corticotropin-releasing factor (CRF), a neuropeptide that promotes anxiety-like behavior in mediating avoidance and escalated alcohol drinking after stress. CRF is expressed in the medial prefrontal cortex (mPFC). The dorsal and ventral sub-regions of the mPFC (dmPFC and vmPFC) have opposing roles in stress reactivity and alcohol drinking. We hypothesized that vmPFC CRF-CRFR1 signaling contributes functionally to stress-induced avoidance and escalated alcohol self-administration. In Experiment 1, adult male Wistar rats were exposed to predator odor stress in a CPA paradigm, indexed for avoidance of odor-paired context, and brains processed for CRF-immunoreactive cell density in vmPFC and dmPFC. Post-stress, Avoiders exhibited higher CRF cell density in vmPFC, but not the dmPFC. In Experiment 2, rats were tested for avoidance of a context repeatedly paired with intra-vmPFC CRF infusions. In Experiment 3, rats were stressed and indexed, then tested for the effects of intra-vmPFC CRFR1 antagonism on avoidance and alcohol self-administration. Intra-vmPFC CRF infusion produced avoidance of a paired context, and intra-vmPFC CRFR1 antagonism reversed avoidance of a stress-paired context, but did not alter post-stress alcohol self-administration. These findings suggest that vmPFC CRF-CRFR1 signaling mediates avoidance of stimuli paired with traumatic stress.

Exposure to repetitive versus varied stress during prenatal development generates two distinct anxiogenic and neuroendocrine profiles in adulthood.

Early life experiences can shape brain function and behavior in adulthood. The present study sought to elucidate the effects of repetitive, predictable vs. varied, unpredictable prenatal stress on sexually dichotomous neuroendocrine and anxiety-related behavioral responses in adult offspring. Rat dams were exposed repeatedly during the last week of pregnancy to no stress, only restraint stress [prenatal stress (PS)-restraint], or a randomized sequence of varied stressors (PS-random), and several behavioral and endocrine measures were assessed in adult male and female offspring. Repeated exposure to the same stressor (restraint) generated the most robust changes, including increased anxiety-related behaviors (both passive, measured on the elevated plus maze, and active, measured using defensive burying tests), a delayed and prolonged hypothalamic-pituitary-adrenal (HPA) axis response to stress in female offspring. Conversely, PS-restraint males showed no changes in anxiety-like behavior and had elevated basal ACTH and a blunted HPA response to stress; consistent with attenuated HPA responsivity was an increase in glucocorticoid receptor immunoreactivity in the hippocampus, suggesting increased negative feedback on the HPA axis in these animals. Prenatal exposure to a varied, unpredictable pattern of stressors did not have as much effect on HPA function, with most neuroendocrine measures residing intermediate to PS-restraint and control animals within each sex. Gonadal steroids were altered independent of the type of prenatal stress, but changes were measurable only in males (lower testosterone). The present data exemplify the differential sensitivity of the developing nervous and endocrine systems to stress, depending on not only gender but also the nature of the stressful experience endured by the mother during pregnancy.

Diminished nicotine withdrawal in adolescent rats: implications for vulnerability to addiction.

RATIONALE: Enhanced reinforcing effects of nicotine during adolescence appear to contribute to the rapid development of dependence in this age group. However, the contribution of nicotine withdrawal to dependence in adolescents is unclear. OBJECTIVE: We compared motivational and somatic signs of nicotine withdrawal in adolescent and adult rats. MATERIALS AND METHODS: In experiment 1, motivational signs of nicotine withdrawal were compared using intracranial self-stimulation procedures after administration of mecamylamine (1.5 mg/kg, i.p.) in adolescent and adult rats made dependent on nicotine (9 mg/kg/day). Somatic signs of withdrawal were compared in two experiments using various doses of nicotine (adolescent doses: 0, 1.6, 3.2, 4.7 mg/kg/day; adult doses: 0, 1, 2.1, 3.2 mg/kg/day, expressed as nicotine base) to produce dependence and one dose of mecamylamine (1.5 mg/kg, i.p.) to precipitate withdrawal (experiment 2) and in a subsequent experiment, using various doses of mecamylamine (0, 0.75, 1.5, 3.0 mg/kg, i.p.) to precipitate withdrawal and a dose of nicotine (adolescent dose: 4.7 mg/kg/day; adult dose: 3.2 mg/kg/day) that produced equivalent nicotine blood levels in these age groups (experiment 3). RESULTS: Adolescents did not display the decreases in brain reward function observed in adults experiencing withdrawal, and displayed fewer somatic signs of nicotine withdrawal relative to adults regardless of the dosing procedure used. CONCLUSION: The negative effects of nicotine withdrawal are lower during adolescence relative to later periods of development. Both the enhanced rewarding effects and the diminished nicotine withdrawal likely contribute to the rapid development of nicotine use during adolescence.

Traumatic Stress Promotes Hyperalgesia via Corticotropin-Releasing Factor-1 Receptor (CRFR1) Signaling in Central Amygdala.

Hyperalgesia is an exaggerated response to noxious stimuli produced by peripheral or central plasticity. Stress modifies nociception, and humans with post-traumatic stress disorder (PTSD) exhibit co-morbid chronic pain and amygdala dysregulation. Predator odor stress produces hyperalgesia in rodents. Systemic blockade of corticotropin-releasing factor (CRF) type 1 receptors (CRFR1s) reduces stress-induced thermal hyperalgesia. We hypothesized that CRF-CRFR1 signaling in central amygdala (CeA) mediates stress-induced hyperalgesia in rats with high stress reactivity. Adult male Wistar rats were exposed to predator odor stress in a conditioned place avoidance paradigm and indexed for high (Avoiders) and low (Non-Avoiders) avoidance of predator odor-paired context, or were unstressed Controls. Rats were tested for the latency to withdraw hindpaws from thermal stimuli (Hargreaves test). We used pharmacological, molecular, and immunohistochemical techniques to assess the role of CRF-CRFR1 signaling in CeA in stress-induced hyperalgesia. Avoiders exhibited higher CRF peptide levels in CeA that did not appear to be locally synthesized. Intra-CeA CRF infusion mimicked stress-induced hyperalgesia. Avoiders exhibited thermal hyperalgesia that was reversed by systemic or intra-CeA injection of a CRFR1 antagonist. Finally, intra-CeA infusion of tetrodotoxin produced thermal hyperalgesia in unstressed rats and blocked the anti-hyperalgesic effect of systemic CRFR1 antagonist in stressed rats. These data suggest that rats with high stress reactivity exhibit hyperalgesia that is mediated by CRF-CRFR1 signaling in CeA.