Interactions of Stress and CRF in Ethanol-Withdrawal Induced Anxiety in Adolescent and Adult Rats

Repeated stress or administration of corticotropin-releasing factor (CRF) prior to ethanol exposure sensitizes anxiety-like behavior in adult rats.

Current experiments determined whether adolescent rats were more sensitive to these challenges in sensitizing ethanol withdrawal-induced anxiety and altering CRF levels in brain during withdrawal.

Male adult and adolescent Sprague–Dawley rats were restraint stressed (1 hour) twice 1 week apart prior to a single 5-day cycle of ethanol diet (ED; stress/withdrawal paradigm). Other rats received control diet (CD) and three 1-hour restraint stress sessions. Rats were then tested 5, 24, or 48 hours after the final withdrawal for anxiety-like behavior in the social interaction (SI) test. In other experiments, adolescent rats were given two microinjections of CRF icv 1 week apart followed by 5 days of either CD or ED and tested in social interaction 5 hours into withdrawal. Finally, CRF immunoreactivity was measured in the central nucleus of the amygdala (CeA) and paraventricular nucleus (PVN) after rats experienced control diet, repeated ethanol withdrawals, or stress/withdrawal.

Rats of both ages had reduced SI following the stress/withdrawal paradigm, and this effect recovered within 24 hours. Higher CRF doses were required to reduce SI in adolescents than previously reported in adults. CRF immunohistochemical levels were higher in the PVN and CeA of CD-exposed adolescents. In adolescent rats, repeated ethanol withdrawals decreased CRF in the CeA but was not associated with decreased CRF cell number. There was no change in CRF from adult treatments.

In the production of anxiety-like behavior, adolescent rats have equal sensitivity with stress and lower sensitivity with CRF compared to adults. Further, adolescents had higher basal levels of CRF within the PVN and CeA and reduced CRF levels following repeated ethanol withdrawals. This reduced CRF within the CeA could indicate increased release of CRF, and future work will determine how this change relates to behavior

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Corticotropin-releasing factor-1 receptor involvement in behavioral neuroadaptation to ethanol: A urocortin₁-independent mechanism
Published online on June 30, 2008 Proc. Natl. Acad. Sci. USA, 10.1073

A common expression of neuroadaptations induced by repeated exposure to addictive drugs is a persistent sensitized behavioral response to their stimulant properties. Neuroplasticity underlying drug-induced sensitization has been proposed to explain compulsive drug pursuit and consumption characteristic of addiction.

The hypothalamic-pituitary-adrenal (HPA) axis-activating neuropeptide, corticotropin-releasing factor (CRF), may be the keystone in drug-induced neuroadaptation. Corticosterone-activated glucocorticoid receptors (GRs) mediate the development of sensitization to ethanol (EtOH), implicating the HPA axis in this process. EtOH-induced increases in corticosterone require CRF activation of CRF₁ receptors.

We posited that CRF₁ signaling pathways are crucial for EtOH-induced sensitization. We demonstrate that mice lacking CRF₁ receptors do not show psychomotor sensitization to EtOH, a phenomenon that was also absent in CRF_{1 + 2} receptor double-knockout mice. Deletion of CRF₂ receptors alone did not prevent sensitization. A blunted endocrine response to EtOH was found only in the genotypes showing no sensitization. The CRF₁ receptor antagonist CP-154,526 attenuated the acquisition and prevented the expression of EtOH-induced psychomotor sensitization. Because CRF₁ receptors are also activated by urocortin-1 (Ucn₁), we tested Ucn₁ knockout mice for EtOH sensitization and found normal sensitization in this genotype.

Finally, we show that the GR antagonist mifepristone does not block the expression of EtOH sensitization. CRF and CRF₁ receptors, therefore, are involved in the neurobiological adaptations that underlie the development and expression of psychomotor sensitization to EtOH.

A CRF/CRF₁-mediated mechanism involving the HPA axis is proposed for acquisition, whereas an extrahypothalamic CRF/CRF₁ participation is suggested for expression of sensitization to EtOH.

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CRF-1 Antagonist and CRF-2 Agonist Decrease Binge-Like Ethanol Drinking in C57BL/6J Mice Independent of the HPA Axis

Recent evidence suggests that corticotropin-releasing factor (CRF) receptor (CRFR) signaling is involved in modulating binge-like ethanol consumption in C57BL/6J mice.

In this report, a series of experiments were performed to further characterize the role of CRFR signaling in binge-like ethanol consumption.

The role of central CRFR signaling was assessed with intracerebroventricular (i.c.v.) infusion of the nonselective CRFR antagonist, α-helical CRF_9–41 (0, 1, 5, 10 μg/1 μl). The contribution of central CRF type 2 receptor (CRF₂R) signaling was assessed with i.c.v. infusion of the selective CRF₂R agonist, urocortin (Ucn) 3 (0, 0.05, 0.1, or 0.5 μg/1 μl). The role of the hypothalamic–pituitary–adrenal (HPA) axis was assessed by pretreating mice with intraperitoneal (i.p.) injection of (1) the corticosterone synthesis inhibitor, metyrapone (0, 50, 100, 150 mg/kg) or (2) the glucocorticoid receptor antagonist, mifepristone (0, 25, 50 mg/kg), and (3) by using radioimmunoassay to determine whether binge-like ethanol intake influenced plasma corticosterone levels. Finally, we determined whether the ability of the CRF₁R antagonist, CP-154,526 (CP; 0, 10, 15 mg/kg, i.p.), to blunt binge-like drinking required normal HPA axis signaling by comparing the effectiveness of CP in adrenalectomized (ADX) and normal mice.

Results showed that i.c.v. infusion of a 1 μg dose of α-helical CRF_9–41 significantly attenuated binge-like ethanol consumption relative to vehicle treatment, and i.c.v. infusion of Ucn 3 dose-dependently blunted binge-like drinking.

On the other hand, metyrapone nonselectively reduced both ethanol and sucrose consumption, mifepristone did not alter ethanol drinking, and binge-like drinking did not correlate with plasma corticosterone levels.

Finally, i.p. injection of CP significantly attenuated binge-like ethanol intake in both ADX and normal mice.

Together, these results suggest that binge-like ethanol intake in C57BL/6J mice is modulated by CRF₁R and CRF₂R signaling, such that blockade of CRF₁R or activation of CRF₂R effectively reduces excessive ethanol intake.

Furthermore, normal HPA axis signaling is not necessary to achieve binge-like drinking behavior.


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A Corticotropin Releasing Factor Pathway for Ethanol Regulation of the Ventral Tegmental Area in the Bed Nucleus of the Stria Terminalis

A growing literature suggests that catecholamines and corticotropin-releasing factor (CRF) interact in a serial manner to activate the bed nucleus of the stria terminalis (BNST) to drive stress- or cue-induced drug- and alcohol-seeking behaviors. Data suggest that these behaviors are driven in part by BNST projections to the ventral tegmental area (VTA).

Together, these findings suggest the existence of a CRF-signaling pathway within the BNST that is engaged by catecholamines and regulates the activity of BNST neurons projecting to the VTA.

Here we test three aspects of this model to determine: (1) whether catecholamines modify CRF neuron activity in the BNST; (2) whether CRF regulates excitatory drive onto VTA-projecting BNST neurons; and (3) whether this system is altered by ethanol exposure and withdrawal.

 A CRF neuron fluorescent reporter strategy was used to identify BNST CRF neurons for whole-cell patch-clamp analysis in acutely prepared slices. Using this approach, we found that both dopamine and isoproterenol significantly depolarized BNST CRF neurons. Furthermore, using a fluorescent microsphere-based identification strategy we found that CRF enhances the frequency of spontaneous EPSCs onto VTA-projecting BNST neurons in naive mice. This action of CRF was occluded during acute withdrawal from chronic intermittent ethanol exposure.

These findings suggest that dopamine and isoproterenol may enhance CRF release from local BNST sources, leading to enhancement of excitatory neurotransmission on VTA-projecting neurons, and that this pathway is engaged by patterns of alcohol exposure and withdrawal known to drive excessive alcohol intake.


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Dopamine Enhances Fast Excitatory Synaptic Transmission in the Extended Amygdala by a CRF-R1-Dependent Process
The Journal of Neuroscience, December 17, 2008, 28(51):13856-13865


A common feature of drugs of abuse is their ability to increase extracellular dopamine levels in key brain circuits. The actions of dopamine within these circuits are thought to be important in reward and addiction-related behaviors.

Current theories of addiction also posit a central role for corticotrophin-releasing factor (CRF) and an interaction between CRF and monoaminergic signaling. One region where drugs of abuse promote robust rises in extracellular dopamine levels is the bed nucleus of the stria terminalis (BNST), a CRF-rich component of the extended amygdala.

We find that dopamine rapidly enhances glutamatergic transmission in the BNST through activation of a combination of D1- and D2-like receptors. This enhancement is activity-dependent and requires the downstream action of CRF receptor 1 (CRF-R1), suggesting that dopamine induces CRF release through a local network mechanism.

Furthermore, we found that both in vivo and ex vivo cocaine induced a dopamine receptor and CRF-R1-dependent enhancement of a form of NMDA receptor-dependent short-term potentiation in the BNST. These data highlight a direct and rapid interaction between dopamine and CRF systems that regulates excitatory transmission and plasticity in a brain region key to reinforcement and reinstatement.

Because a rise in extracellular dopamine levels in the BNST is a shared consequence of multiple classes of drugs of abuse, this suggests that the CRF-R1-dependent enhancement of glutamatergic transmission in this region may be a common key feature of substances of abuse.

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Corticotropin Releasing Factor–Induced Amygdala Gamma-Aminobutyric Acid Release Plays a Key Role in Alcohol Dependence

Corticotropin-releasing factor (CRF) and gamma-aminobutyric acid (GABA)ergic systems in the central amygdala (CeA) are implicated in the high-anxiety, high-drinking profile associated with ethanol dependence. Ethanol augments CeA GABA release in ethanol-naive rats and mice.

CRF and ethanol augmented CeA GABAergic transmission in naive rats via increased GABA release. Three CRF1 receptor (CRF₁) antagonists decreased basal GABAergic responses and abolished ethanol effects. Ethanol-dependent rats exhibited heightened sensitivity to CRF and CRF₁ antagonists on CeA GABA release. Intra-CeA CRF₁ antagonist administration reversed dependence–related elevations in GABA dialysate and blocked ethanol-induced increases in GABA dialyzate in both dependent and naive rats. Polymerase chain reaction studies indicate increased expression of CRF and CRF₁ in CeA of dependent rats. Chronic CRF₁ antagonist treatment blocked withdrawal-induced increases in alcohol drinking by dependent rats and tempered moderate increases in alcohol consumption by nondependent rats in intermittent testing.

These combined findings suggest a key role for specific presynaptic CRF-GABA interactions in CeA in the development and maintenance of ethanol dependence.

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A Corticotropin Releasing Factor Pathway for Ethanol Regulation of the Ventral Tegmental Area in the Bed Nucleus of the Stria Terminalis

A growing literature suggests that catecholamines and corticotropin-releasing factor (CRF) interact in a serial manner to activate the bed nucleus of the stria terminalis (BNST) to drive stress- or cue-induced drug- and alcohol-seeking behaviors. 

 Data suggest that these behaviors are driven in part by BNST projections to the ventral tegmental area (VTA). Together, these findings suggest the existence of a CRF-signaling pathway within the BNST that is engaged by catecholamines and regulates the activity of BNST neurons projecting to the VTA. 

Here we test three aspects of this model to determine: (1) whether catecholamines modify CRF neuron activity in the BNST; (2) whether CRF regulates excitatory drive onto VTA-projecting BNST neurons; and (3) whether this system is altered by ethanol exposure and withdrawal. 

A CRF neuron fluorescent reporter strategy was used to identify BNST CRF neurons for whole-cell patch-clamp analysis in acutely prepared slices. Using this approach, we found that both dopamine and isoproterenol significantly depolarized BNST CRF neurons. Furthermore, using a fluorescent microsphere-based identification strategy we found that CRF enhances the frequency of spontaneous EPSCs onto VTA-projecting BNST neurons in naive mice. This action of CRF was occluded during acute withdrawal from chronic intermittent ethanol exposure. 

These findings suggest that dopamine and isoproterenol may enhance CRF release from local BNST sources, leading to enhancement of excitatory neurotransmission on VTA-projecting neurons, and that this pathway is engaged by patterns of alcohol exposure and withdrawal known to drive excessive alcohol intake.


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Request Reprint E-Mail:   danny.winder@vanderbilt.edu

Urocortins: CRF's siblings and their potential role in anxiety, depression and alcohol drinking behavior

It is widely accepted that stress, anxiety, depression and alcohol abuse-related disorders are in large part controlled by corticotropin-releasing factor (CRF) receptors. However, evidence is accumulating that some of the actions on these receptors are mediated not by CRF, but by a family of related Urocortin (Ucn) peptides Ucn1, Ucn2 and Ucn3. The initial narrow focus on CRF as the potential main player acting on CRF receptors appears outdated. Instead it is suggested that CRF and the individual Ucns act in a complementary and brain region-specific fashion to regulate anxiety-related behaviors and alcohol consumption.

This review, based on a symposium held in 2011 at the research meeting on “Alcoholism and Stress” in Volterra, Italy, highlights recent evidence for regulation of these behaviors by Ucns.

In studies on stress and anxiety, the roles of Ucns, and in particular Ucn1, appear more visible in experiments analyzing adaptation to stressors rather than testing basal anxiety states. Based on these studies, we propose that the contribution of Ucn1 to regulating mood follows a U-like pattern with both high and low activity of Ucn1 contributing to high anxiety states.

In studies on alcohol use disorders, the CRF system appears to regulate not only dependence-induced drinking, but also binge drinking and even basal consumption of alcohol. While dependence-induced and binge drinking rely on the actions of CRF on CRFR1 receptors, alcohol consumption in models of these behaviors is inhibited by actions of Ucns on CRFR2.

In contrast, alcohol preference is positively influenced by actions of Ucn1, which is capable of acting on both CRFR1 and CRFR2.

Because of complex distribution of Ucns in the nervous system, advances in this field will critically depend on development of new tools allowing site-specific analyses of the roles of Ucns
and CRF.


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Intermittent access ethanol consumption dysregulates CRF function in the hypothalamus and is attenuated by the CRF-R1 antagonist, CP-376395

Corticotrophin-releasing factor (CRF) is a mediator of stress responses and a key modulator of ethanol-mediated behaviors. 

We report here that the CRF receptor 1 (CRF-R1) antagonist, CP-376395 reduces 20% ethanol consumption in animals trained to consume ethanol on an intermittent, but not a continuous, schedule. 

Furthermore, using [³⁵S]GTPγS binding assays, we demonstrate that CRF-mediated G-protein signaling in the hypothalamus of the intermittent drinkers is decreased when compared to controls suggesting that the effects of CP-376395 are mediated by extrahypothalamic mechanisms. 

The present study provides further support for the use of CRF-R1 antagonists for the treatment of alcohol use disorders and suggests that ethanol consumption
dysregulates CRF function in the hypothalamus.


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News Release - NEW COMPOUND PREVENTS ALCOHOLIC BEHAVIOR, RELAPSE IN ANIMALS BY BLOCKING STRESS RESPONSE, MAY HAVE HUMAN USE

3/06/2007	NR-08-07 (03/06/07). For more information, please contact Sara Harris at (202) 962-4000 or sharris@sfn.org. Embargoed until March 6, 2007, 5:00 p.m. U.S. Eastern time.
WASHINGTON, DC March 6, 2007 - A study of alcohol-dependent animals shows that a newly discovered compound that blocks chemical signals active during the brain's response to stress effectively stops excessive drinking and prevents relapse. The new, synthetic compound, known as MTIP, also muted the anxiety that typically develops in rats experiencing the equivalent of a hangover. Such stress is linked with higher levels of a brain chemical called corticotropin-releasing factor (CRF), which is also thought to trigger relapse in rats that have developed a long-term dependency on alcohol. The research, by Markus Heilig, MD, PhD, at the National Institute on Alcohol Abuse and Alcoholism (NIAAA), and his team, appears in the March 7 issue of The Journal of Neuroscience. "This study shows the activity of a compound that potentially could be used in human subjects," says George Koob, PhD, of the Scripps Research Institute. "It moves the field over another translational barrier, closer to the day when the dark side of addiction is treated." The new findings build on previous work showing the effectiveness of blocking certain CRF receptors -- proteins that bind CRF and make it active -- in treating alcohol dependence in animals. CRF levels in the brain rise in the short term after drinking; in subjects that are not dependent, this activation returns to normal within a day or so. The new study shows that the CRF system becomes overactive in the long term in animals with a history of alcohol dependence, increasing the risk for relapse. Heilig and his team showed that MTIP blocked the activity of CRF in stressful situations without affecting its activity under ordinary circumstances. They studied rats that had been put through several cycles of heavy alcohol consumption and withdrawal to create dependency, as well as animals selectively bred to consume more alcohol. Injections of MTIP prevented excessive drinking of alcohol in both cases and eliminated the rats' susceptibility to relapse under stress. Yet the compound did not affect their native curiosity or lower levels of drinking alcohol in rats that were not alcohol-dependent. In addition to alcoholism, the compound may prove to be useful in the treatment of depression or anxiety disorders, in which CRF levels can be especially high. The compound or similar molecules can be given orally, reach the brain in sufficient amounts to block more than 90 percent of certain brain CRF receptors, and do not accumulate in other organs, most importantly the liver, in ways that would cause concerns about potential side effects, says Heilig. The work was a collaborative project between NIAAA and Eli Lilly and Co. The Journal of Neuroscience is published by the Society for Neuroscience, an organization of more than 36,500 basic scientists and clinicians who study the brain and nervous system. Heilig can be reached at Markus.Heilig@mail.nih.gov. Source: EurekAlert March 6, 2007

The CRF-1 Receptor Antagonist, CP-154,526, Attenuates Stress-Induced Increases in Ethanol Consumption by BALB/cJ Mice
Alcoholism: Clinical and Experimental Research (OnlineEarly Articles) 21 Dec 2007

Corticotropin-releasing factor (CRF) signaling modulates neurobiological responses to stress and ethanol, and may modulate observed increases in ethanol consumption following exposure to stressful events.

The current experiment was conducted to further characterize the role of CRF₁ receptor (CRF₁R) signaling in stress-induced increases in ethanol consumption in BALB/cJ and C57BL/6N mice.

Importantly, BALB/cJ mice pretreated with the CRF₁R antagonist showed blunted stress-induced increases in ethanol intake, and the CRF₁R antagonist did not influence the ethanol drinking of non-stressed mice.

The present results provide evidence that CRF₁R signaling modulates the delayed increase of ethanol consumption stemming from repeated exposure to a stressful event in BALB/cJ mice.

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Protein kinase C epsilon mediation of CRF- and ethanol-induced GABA release in central amygdala
PNAS | June 17, 2008 | vol. 105 | no. 24 | 8410-8415


In the central amygdala (CeA), ethanol acts via corticotrophin-releasing factor (CRF) type 1 receptors to enhance GABA release. Amygdala CRF mediates anxiety associated with stress and drug dependence, and it regulates ethanol intake.

Because mutant mice that lack PKC exhibit reduced anxiety-like behavior and alcohol consumption, we investigated whether PKC lies downstream of CRF₁ receptors in the CeA.

Compared with PKC^+/+ CeA neurons, PKC^–/– neurons showed increased GABAergic tone due to enhanced GABA release. CRF and ethanol stimulated GABA release in the PKC^+/+ CeA, but not in the PKC^–/– CeA. A PKC-specific inhibitor blocked both CRF- and ethanol-induced GABA release in the PKC^+/+ CeA, confirming findings in the PKC^–/– CeA.

These results identify a PKC signaling pathway in the CeA that is activated by CRF₁ receptor stimulation, mediates GABA release at nerve terminals, and regulates anxiety and alcohol consumption.

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Corticotropin-Releasing Factor Acting on Corticotropin-Releasing Factor Receptor Type 1 is Critical for Binge Alcohol Drinking in Mice

The corticotropin-releasing factor (CRF) system has been implicated in the regulation of alcohol consumption. However, previous mouse knockout (KO) studies using continuous ethanol access have failed to conclusively confirm this. Recent studies have shown that CRF receptor type 1 (CRFR1) antagonists attenuate alcohol intake in the limited access “drinking in the dark” (DID) model of binge drinking. To avoid the potential nonspecific effects of antagonists, in this study, we tested alcohol drinking in CRFR1, CRFR2, CRF, and urocortin 1 (Ucn1) KO and corresponding wild-type (WT) littermates using the DID paradigm.

On days 1 to 3, the CRFR1, CRFR2, Ucn1, and CRF KO mice and their respective WT littermates were provided with 20% ethanol or 10% sucrose for 2 hours with water available at all other times. On day 4, access to ethanol or sucrose was increased to 4 hours. At the end of each drinking session, the volume of ethanol consumed was recorded, and at the conclusion of the last session, blood was also collected for blood ethanol concentration (BEC) analysis.

CRFR1 KO mice had lower alcohol intakes and BECs and higher intakes of sucrose compared with WTs. In contrast, CRFR2 KO mice, while having reduced intakes initially, had similar alcohol intakes on days 2 to 4 and similar BECs as the WTs. To determine the ligand responsible, Ucn1 and CRF KO and WT mice were tested next. While Ucn1 KOs had similar alcohol intakes and BECs to their WTs, CRF KO mice showed reduced alcohol consumption and lower BECs compared with WTs.

Our results confirm that CRFR1 plays a key role in binge drinking and identify CRF as the ligand critically involved in excessive alcohol consumption.

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ryabinin@ohsu.edu

Nociceptin/Orphanin FQ Blockade of Corticotropin-Releasing Factor-Induced Gamma-Aminobutyric Acid Release in Central Amygdala Is Enhanced After Chroni

The central nucleus of the amygdala (CeA) mediates stress- and addiction-related processes. Corticotropin-releasing factor (CRF) and nociceptin/orphanin FQ (nociceptin) regulate ethanol intake and anxiety-like behavior. In the rat, CRF and ethanol significantly augment CeA gamma-aminobutyric acid (GABA) release, whereas nociceptin diminishes it.

Using electrophysiologic techniques in an in vitro slice preparation, we investigated the interaction of nociceptin and CRF on evoked and spontaneous GABAergic transmission in CeA slices of naive and ethanol-dependent rats and the mechanistic role of protein kinase A.

In neurons from naive animals, nociceptin dose-dependently diminished basal-evoked GABA_A receptor-mediated inhibitory postsynaptic potentials (IPSPs) by decreasing GABA release and prevented, as well as reversed, CRF-induced augmentation of IPSPs, actions that required PKA signaling. In neurons from ethanol-dependent animals, nociceptin decreased basal GABAergic transmission and blocked the CRF-induced increase in GABA release to a greater extent than in naive controls.

These data provide new evidence for an interaction between the nociceptin and CRF systems in the CeA. Nociceptin opposes CRF effects on CeA GABAergic transmission with sensitization of this effect in dependent animals. These properties of nociceptin may underlie its anti-alcohol and anxiolytic properties and identify the nociceptin receptor as a useful therapeutic target for alcoholism.

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Nociceptin/Orphanin FQ Blockade of Corticotropin-Releasing Factor-Induced Gamma-Aminobutyric Acid Release in Central Amygdala Is Enhanced After Chroni

The central nucleus of the amygdala (CeA) mediates stress- and addiction-related processes. Corticotropin-releasing factor (CRF) and nociceptin/orphanin FQ (nociceptin) regulate ethanol intake and anxiety-like behavior. In the rat, CRF and ethanol significantly augment CeA gamma-aminobutyric acid (GABA) release, whereas nociceptin diminishes it.

Using electrophysiologic techniques in an in vitro slice preparation, we investigated the interaction of nociceptin and CRF on evoked and spontaneous GABAergic transmission in CeA slices of naive and ethanol-dependent rats and the mechanistic role of protein kinase A.

In neurons from naive animals, nociceptin dose-dependently diminished basal-evoked GABA_A receptor-mediated inhibitory postsynaptic potentials (IPSPs) by decreasing GABA release and prevented, as well as reversed, CRF-induced augmentation of IPSPs, actions that required PKA signaling. In neurons from ethanol-dependent animals, nociceptin decreased basal GABAergic transmission and blocked the CRF-induced increase in GABA release to a greater extent than in naive controls.

These data provide new evidence for an interaction between the nociceptin and CRF systems in the CeA. Nociceptin opposes CRF effects on CeA GABAergic transmission with sensitization of this effect in dependent animals. These properties of nociceptin may underlie its anti-alcohol and anxiolytic properties and identify the nociceptin receptor as a useful therapeutic target for alcoholism.

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Role of corticotropin-releasing factor in the median raphe nucleus in yohimbine-induced reinstatement of alcohol seeking in rats

The pharmacological stressor yohimbine increases ongoing alcohol self-administration and reinstates alcohol seeking in rats. This effect is attenuated by systemic injections of a corticotropin-releasing factor (CRF) antagonist. The brain sites involved in CRF's role in yohimbine-induced alcohol taking and seeking are unknown.

We report that injections of the CRF receptor antagonist d-Phe CRF into the median raphe nucleus (MRN) attenuated yohimbine-induced reinstatement of alcohol seeking but had no effect on yohimbine-induced increases in alcohol intake during ongoing self-administration.

Results indicate an important role of MRN CRF receptors in yohimbine-induced reinstatement of alcohol seeking but not yohimbine-induced increases in alcohol intake.

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A key role for corticotropin-releasing factor in alcohol dependence
Trends in Neurosciences Article in Press, Corrected Proof 16 July 2007



Recent data indicate that alcohol dependence induces long-term neuroadaptations that recruit a negative emotional state. This leads to excessive alcohol ingestion motivated by relief of negative emotionality.

A key mechanism in this transition to negative reinforcement is a recruitment of corticotropin-releasing factor (CRF) signaling within the amygdala.

Long term upregulation of CRF₁ receptors is observed in the amygdala following a history of dependence, and CRF antagonists selectively block emotionality, excessive alcohol drinking and stress-induced reinstatement of alcohol-seeking in post-dependent animals. Innate upregulation of CRF₁ receptor expression mimics the post-dependent phenotype, both with regard to emotional responses and ethanol self-administration.

Therefore, the CRF system is emerging as a key element of the neuroadaptive changes driving alcoholism and as a major target for its treatment.

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Effects of CRF₁-Receptor and Opioid-Receptor Antagonists on Dependence-Induced Increases in Alcohol Drinking by Alcohol-Preferring (P) Rats
Alcoholism: Clinical and Experimental Research Published Online: 10 Jul 2008

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 μ-opioid receptor at low doses), and the recently characterized small molecule CRF₁-receptor antagonist MPZP (N,N-bis(2-methoxyethyl)-3-(4-methoxy-2-methylphenyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidin-7-amine).

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.

These results confirm a role for brain CRF₁-receptor systems in dependence-induced changes in the reinforcing properties of alcohol, and CRF₁-receptor blockade appears to suppress dependence-induced drinking at lower doses in P rats relative to other rat lines. Therefore, brain CRF₁-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.

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Type 7 Adenylyl Cyclase is Involved in the Ethanol and CRF Sensitivity of GABAergic Synapses in Mouse Central Amygdala

The GABAergic system in the central amygdala (CeA) plays a major role in ethanol dependence and in the anxiogenic response to ethanol withdrawal. 

Previously, we found that both ethanol and corticotropin releasing factor (CRF) increase GABAergic transmission in mouse and rat CeA neurons, in part by enhancing the release of GABA via activation of presynaptic CRF1 receptors. CRF1 receptors are coupled to the enzyme adenylyl cyclase (AC), which produces the second messenger cyclic AMP. 

There are nine isoforms of AC, but we recently found that CRF1 receptors in the pituitary were coupled to the Type 7 AC (AC7). Therefore, using an in vitro electrophysiological approach in brain slices, here we have investigated a possible role of the AC7 signaling pathway in ethanol and CRF effects on CeA GABAergic synapses of genetically modified mice with diminished brain Adcy7 activity (HET) compared to their littermate male wild-type (WT) mice. 

We found no significant differences in basal membrane properties, mean baseline amplitude of evoked GABA_A receptor-mediated inhibitory postsynaptic potentials (IPSPs), or paired-pulse facilitation (PPF) of GABA_A-IPSPs between HET and WT mice. 

In CeA neurons of WT mice, ethanol superfusion significantly augmented (by 39%) GABAA-IPSPs and decreased PPF (by 25%), suggesting increased presynaptic GABA release. 

However, these effects were absent in HET mice. CRF superfusion also significantly augmented IPSPs (by 38%) and decreased PPF (by 23%) in WT CeA neurons, and still elicited a significant but smaller (by 13%) increase of IPSP amplitude, but no effect on PPF, in HET mice. 

These electrophysiological data suggest that AC7 plays an important role in ethanol and CRF modulation of presynaptic GABA release in CeA and thus may underlie ethanol-related behaviors such as anxiety and dependence.

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Corticotropin-releasing factor mediates the dysphoria-like state associated with alcohol withdrawal in rats

This study investigated the role of CRF in the dysphoria-like state associated with alcohol withdrawal in rats. The intracranial self-stimulation procedure was used to assess brain reward thresholds.

Cessation of chronic alcohol administration lead to an elevation in brain reward thresholds in the alcohol dependent rats.

The CRF receptor antagonist D-Phe CRF_(12-41) dose-dependently prevented the elevations in brain reward thresholds associated with alcohol withdrawal.

This indicates that the dysphoria associated with alcohol withdrawal is at least partly mediated by the activation of central CRF receptors.

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