Elena Crawford

Corticotropin-releasing factor within the central nucleus of the amygdala mediates enhanced ethanol self-administration in withdrawn, ethanol-dependent rats.

Alcohol dependence is characterized by excessive consumption, loss of control over intake, and the presence of a withdrawal syndrome, including both motivational and physical symptoms. The motivational symptoms, including anxiety, have been hypothesized to be important factors eliciting excessive drinking during abstinence. Previous work has shown that ethanol-dependent rats also display enhanced anxiety-like behaviors and enhanced ethanol self-administration during withdrawal, likely resulting from dysregulation of brain corticotropin-releasing factor (CRF) stress systems. The present study was designed to explore the brain sites within the extended amygdala [central nucleus of the amygdala (CeA), lateral bed nucleus of the stria terminalis (BNST), and nucleus accumbens shell (NAcSh)] that mediate the increased ethanol self-administration observed during withdrawal. Ethanol-dependent animals showed an increase in ethanol self-administration after acute withdrawal relative to nondependent controls. The CRF antagonist d-Phe-CRF12–41 ([d-Phe12,Nle21,38,Cα MeLeu37]-rCRF(12–41)) was administered into the CeA, lateral BNST, or NAcSh of acute-withdrawn dependent and nondependent rats. Administered into the CeA, the antagonist reduced ethanol self-administration in dependent animals, with no effect in nondependent animals. Administration of d-Phe-CRF12–41 into the lateral BNST and NAcSh was without effect on ethanol self-administration in dependent and nondependent animals. At the same time point of withdrawal, there was a decrease in CRF immunoreactivity within the CeA, suggesting an increased extracellular release of CRF during withdrawal. There was no change in CRF immunoreactivity in the BNST or NAcSh. These results indicate that CRF, specifically within the CeA, plays a role in mediating excessive ethanol consumption in ethanol-dependent animals.

Recruitment of medial prefrontal cortex neurons during alcohol withdrawal predicts cognitive impairment and excessive alcohol drinking

Chronic intermittent access to alcohol leads to the escalation of alcohol intake, similar to binge drinking in humans. Converging lines of evidence suggest that impairment of medial prefrontal cortex (mPFC) cognitive function and overactivation of the central nucleus of the amygdala (CeA) are key factors that lead to excessive drinking in dependence. However, the role of the mPFC and CeA in the escalation of alcohol intake in rats with a history of binge drinking without dependence is currently unknown. To address this issue, we examined FBJ murine osteosarcoma viral oncogene homolog (Fos) expression in the mPFC, CeA, hippocampus, and nucleus accumbens and evaluated working memory and anxiety-like behavior in rats given continuous (24 h/d for 7 d/wk) or intermittent (3 d/wk) access to alcohol (20% vol/vol) using a two-bottle choice paradigm. The results showed that abstinence from alcohol in rats with a history of escalation of alcohol intake specifically recruited GABA and corticotropin-releasing factor (CRF) neurons in the mPFC and produced working memory impairments associated with excessive alcohol drinking during acute (24–72 h) but not protracted (16 –68 d) abstinence. Moreover, abstinence from alcohol was associated with a functional disconnection of the mPFC and CeA but not mPFC and nucleus accumbens. These results show that recruitment of a subset of GABA and CRF neurons in the mPFC during withdrawal and disconnection of the PFC–CeA pathway may be critical for impaired executive control over motivated behavior, suggesting that dysregulation of mPFC interneurons may be an early index of neuroadaptation in alcohol dependence.

Chronic ethanol exposure and protracted abstinence alter NMDA receptors in central amygdala

We recently reported that chronic ethanol treatment (CET) and early withdrawal (2–8 h) altered glutamatergic transmission at both pre- and postsynaptic sites in central nucleus of the amygdala (CeA). Acute ethanol (44 mM) inhibited the NMDA receptor (NMDAR)-mediated EPSCs (NMDA-EPSCs) more in CeA neurons from CET rats than from naïve rats and also decreased paired-pulse facilitation (PPF) of NMDA-EPSCs only in CET rats. To determine whether these CET effects persisted after prolonged withdrawal, we recorded intracellularly in rat CeA slices and measured mRNA and protein expression of CeA NMDAR subunits from CET rats and those withdrawn from ethanol for 1 or 2 weeks. At 1 week withdrawal, acute ethanol decreased evoked NMDA-EPSC amplitudes and NMDA currents induced by exogenous NMDA (∼20%) equally to that in naïve rats, indicating that CET effects on postsynaptic mechanisms reversed 1 week after CET cessation. However, acute ethanol still decreased PPF of NMDA-EPSCs, indicating that the acute ethanol-induced increase in glutamate release in CeA seen in CET rats was still present at this time. CET also significantly increased mRNA levels of NR1 and NR2B NMDAR subunits compared to control rats. At 1 week withdrawal, mRNA levels for NR1 and NR2B subunits were significantly decreased. These changes reversed at 2 weeks withdrawal. In Western blots, a significant increase in protein for all three subunits occurred in CeA from CET rats, but not after 1 and 2 weeks of withdrawal. These data indicate that CET induces reversible neuroadaptations in synaptic function, gene expression, and protein composition of NMDAR at CeA synapses.

Varied Access to Intravenous Methamphetamine Self-Administration Differentially Alters Adult Hippocampal Neurogenesis

BACKGROUND Chronic abuse of methamphetamine produces deficits in hippocampal function, perhaps by altering hippocampal neurogenesis and plasticity. We examined how intravenous methamphetamine self-administration modulates active division, proliferation of late progenitors, differentiation, maturation, survival, and mature phenotype of hippocampal subgranular zone (SGZ) progenitors. METHODS Adult male Wistar rats were given access to methamphetamine 1 hour twice weekly (intermittent short), 1 hour daily (short), or 6 hours daily (long). Rats received one intraperitoneal injection of bromodeoxyuridine (BrdU) to label progenitors in the synthesis (S) phase, and 28-day-old surviving BrdU-immunoreactive (IR) cells were quantified. Ki-67, doublecortin (DCX), and activated caspase-3 (AC-3) were used to visualize and quantify proliferating, differentiating, maturing, and apoptotic cells. Terminal corticosterone was measured to determine changes in adrenal steroids. RESULTS Intermittent access to methamphetamine increased Ki-67 and DCX-IR cells, but opposing effects on late progenitors and postmitotic neurons resulted in no overall change in neurogenesis. Daily access to methamphetamine decreased all studied aspects of neurogenesis and reduced hippocampal granule neurons and volume, changes that likely are mediated by decreased proliferative and neurogenic capacity of the SGZ. Furthermore, methamphetamine self-administration relative to the amount of methamphetamine intake produced a biphasic effect on hippocampal apoptosis and reduced corticosterone levels. CONCLUSIONS Intermittent (occasional access) and daily (limited and extended access) self-administration of methamphetamine impact different aspects of neurogenesis, the former producing initial pro-proliferative effects and the latter producing downregulating effects. These findings suggest that altered hippocampal integrity by even modest doses of methamphetamine could account for pronounced pathology linked to methamphetamine abuse.

Long-lasting reduction in hippocampal neurogenesis by alcohol consumption in adolescent nonhuman primates

Binge alcohol consumption in adolescents is increasing, and studies in animal models show that adolescence is a period of high vulnerability to brain insults. The purpose of the present study was to determine the deleterious effects of binge alcohol on hippocampal neurogenesis in adolescent nonhuman primates. Heavy binge alcohol consumption over 11 mo dramatically and persistently decreased hippocampal proliferation and neurogenesis. Combinatorial analysis revealed distinct, actively dividing hippocampal neural progenitor cell types in the subgranular zone of the dentate gyrus that were in transition from stem-like radial glia-like cells (type 1) to immature transiently amplifying neuroblasts (type 2a, type 2b, and type 3), suggesting the evolutionary conservation of milestones of neuronal development in macaque monkeys. Alcohol significantly decreased the number of actively dividing type 1, 2a, and 2b cell types without significantly altering the early neuronal type 3 cells, suggesting that alcohol interferes with the division and migration of hippocampal preneuronal progenitors. Furthermore, the lasting alcohol-induced reduction in hippocampal neurogenesis paralleled an increase in neural degeneration mediated by nonapoptotic pathways. Altogether, these results demonstrate that the hippocampal neurogenic niche during adolescence is highly vulnerable to alcohol and that alcohol decreases neuronal turnover in adolescent nonhuman primate hippocampus by altering the ongoing process of neuronal development. This lasting effect, observed 2 mo after alcohol discontinuation, may underlie the deficits in hippocampus-associated cognitive tasks that are observed in alcoholics.

Age-independent and age-related deficits in visuospatial learning, sleep-wake states, thermoregulation and motor activity in PDAPP mice.

Recent studies demonstrated that mice overexpressing the human mutant beta-amyloid precursor protein (hbetaAPP; PDAPP mice) show age-independent and age-related deficits in spatial learning. We used behavioral and electrophysiological techniques to determine in young and aged PDAPP mice whether deficits in spatial learning also involve alterations in sleep-wake states, thermoregulation and motor activity. Consistent with earlier studies, young PDAPP mice exhibited selective age-independent deficits using spatial, but not random and serial strategies in the circular maze. Aged PDAPP mice exhibited deficits using all search strategies. The core body temperature (Tb) in young and aged PDAPP mice was significantly lower than in age-matched non-transgenic (non-Tg) littermates. During the dark period, the motor activity (LMA) was significantly increased in young PDAPP mice, but not in aged PDAPP mice. During the light period, young PDAPP mice showed a reduction in the generation of rapid-eye-movement (REM) sleep. In contrast, aged PDAPP mice exhibited a reduction in the amount of time spent in W and an increase in SWS during the light period. Aged PDAPP mice also showed an increase in the amount of time spent in W and a reduction in REM sleep during the dark period. Our findings support previous reports indicating deficits in spatial learning in young and aged PDAPP mice. These data also suggest that PDAPP mice exhibit age-independent and age-related deficits in neural mechanisms regulating visuospatial learning, the total amount and the circadian distribution of sleep-wake states, thermoregulation and motor activity.

Stress experienced in utero reduces sexual dichotomies in neurogenesis, microenvironment, and cell death in the adult rat hippocampus

Hippocampal function and plasticity differ with gender, but the regulatory mechanisms underlying sex differences remain elusive and may be established early in life. The present study sought to elucidate sex differences in hippocampal plasticity under normal developmental conditions and in response to repetitive, predictable versus varied, unpredictable prenatal stress (PS). Adult male and diestrous female offspring of pregnant rats exposed to no stress (control), repetitive stress (PS‐restraint), or a randomized sequence of varied stressors (PS‐random) during the last week of pregnancy were examined for hippocampal proliferation, neurogenesis, cell death, and local microenvironment using endogenous markers. Regional volume was also estimated by stereology. Control animals had comparable proliferation and regional volume regardless of sex, but females had lower neurogenesis compared to males. Increased cell death and differential hippocampal precursor kinetics both appear to contribute to reduced neurogenesis in females. Reduced local interleukin‐1beta (IL‐1β) immunoreactivity (IR) in females argues for a mechanistic role for the anti‐apoptotic cytokine in driving sex differences in cell death. Prenatal stress significantly impacted the hippocampus, with both stress paradigms causing robust decreases in actively proliferating cells in males and females. Several other hippocampal measures were feminized in males such as precursor kinetics, IL‐1β‐IR density, and cell death, reducing or abolishing some sex differences. The findings expand our understanding of the mechanisms underlying sex differences and highlight the critical role early stress can play on the balance between proliferation, neurogenesis, cell death, and hippocampal microenvironment in adulthood.

VTA CRF neurons mediate the aversive effects of nicotine withdrawal and promote intake escalation

SUMMARY Dopaminergic neurons in the ventral tegmental area (VTA) are well known for their role in mediating the positive reinforcing effects of drugs of abuse. Here, we identify in rodents and humans a population of VTA dopamine neurons co-expressing corticotropin releasing factor (CRF). We provide further evidence in rodents that chronic nicotine exposure upregulates CRF mRNA in dopaminergic neurons of the posterior VTA, activates local CRF1 receptors, and blocks nicotine-induced activation of transient GABAergic input to dopaminergic neurons. Local downregulation of CRF mRNA and specific pharmacological blockade of CRF1 receptors in the VTA reversed the effect of nicotine on GABAergic input to dopaminergic neurons, prevented the aversive effects of nicotine withdrawal, and limited the escalation of nicotine intake. These results link the brain reward and stress systems within the same brain region in signaling the negative motivational effects of nicotine withdrawal.Dopaminergic neurons in the ventral tegmental area (VTA) are well known for mediating the positive reinforcing effects of drugs of abuse. Here we identify in rodents and humans a population of VTA dopaminergic neurons expressing corticotropin-releasing factor (CRF). We provide further evidence in rodents that chronic nicotine exposure upregulates Crh mRNA (encoding CRF) in dopaminergic neurons of the posterior VTA, activates local CRF1 receptors and blocks nicotine-induced activation of transient GABAergic input to dopaminergic neurons. Local downregulation of Crh mRNA and specific pharmacological blockade of CRF1 receptors in the VTA reversed the effect of nicotine on GABAergic input to dopaminergic neurons, prevented the aversive effects of nicotine withdrawal and limited the escalation of nicotine intake. These results link the brain reward and stress systems in the same brain region to signaling of the negative motivational effects of nicotine withdrawal.

Long-term antagonism of κ opioid receptors prevents escalation of and increased motivation for heroin intake.

The abuse of opioid drugs, both illicit and prescription, is a persistent problem in the United States, accounting for >1.2 million users who require treatment each year. Current treatments rely on suppressing immediate withdrawal symptoms and replacing illicit drug use with long-acting opiate drugs. However, the mechanisms that lead to preventing opiate dependence are still poorly understood. We hypothesized that κ opioid receptor (KOR) activation during chronic opioid intake contributes to negative affective states associated with withdrawal and the motivation to take increasing amounts of heroin. Using a 12 h long-access model of heroin self-administration, rats showed escalation of heroin intake over several weeks. This was prevented by a single high dose (30 mg/kg) of the long-acting KOR antagonist norbinaltorphimine (nor-BNI), paralleled by reduced motivation to respond for heroin on a progressive-ratio schedule of reinforcement, a measure of compulsive-like responding. Systemic nor-BNI also significantly decreased heroin withdrawal-associated anxiety-like behavior. Immunohistochemical analysis showed prodynorphin content increased in the nucleus accumbens core in all heroin-exposed rats, but selectively increased in the nucleus accumbens shell in long-access rats. Local infusion of nor-BNI (4 μg/side) into accumbens core altered the initial intake of heroin but not the rate of escalation, while local injection into accumbens shell selectively suppressed increases in heroin intake over time without altering initial intake. These data suggest that dynorphin activity in the nucleus accumbens mediates the increasing motivation for heroin taking and compulsive-like responding for heroin, suggesting that KOR antagonists may be promising targets for the treatment of opioid addiction.

Chronic morphine treatment alters expression of N‐methyl‐D‐aspartate receptor subunits in the extended amygdala

The nucleus accumbens (NAcc) and central amygdala (CeA) are parts of the extended amygdala, a complex that plays a key role in drug abuse and dependence. Our previous studies showed that opiates and ethanol alter glutamatergic transmission in these regions. N‐methyl‐D‐aspartate (NMDA) receptors are key components of glutamatergic transmission likely involved in the development of opiate tolerance and dependence. In this study we examined the effects of chronic morphine administration on gene and protein expression of three major NMDA receptors subunits (NR1, NR2A, and NR2B) in NAcc and CeA. Real‐time PCR showed no differences in mRNA levels of any of the subunits in the whole NAcc between naïve and morphine‐dependent rats. However, at the protein level, immunoblotting revealed that chronic morphine significantly increased levels of NR1 and NR2B subunits. In contrast to the case for NAcc, in CeA we found an increased mRNA level for the NR1 subunit only but unchanged protein levels of all three subunits in morphine‐dependent rats. The altered expressions of NMDA receptor subunits, especially in NAcc, of morphine‐dependent rats may represent a neuroadaptation to chronic morphine and suggest a mechanism for the changes of glutamatergic transmission found in the extended amygdala in dependent rats. In addition, our results indicate a region‐specific response of NMDA receptor subunits to chronic morphine administration at the gene and protein levels.