Ethanol Modulates Corticotropin Releasing Hormone

Release From the Rat Hypothalamus: Does

Acetaldehyde Play a Role?

Carla Cannizzaro, Marco La Barbera, Fulvio Plescia, Silvana Cacace,and Giuseppe Tringali

Background and Methods: Ethanol (EtOH) activates hypothalamic–pituitary–adrenal (HPA)axis, resulting in adrenocorticotropin hormone, glucocorticoid release, and in modifications of theresponse of the axis to other stressors. The initial site of EtOH action within the HPA systemseems to be the hypothalamus. Thus, to determine the mechanisms responsible for these effects,we investigated: (i) whether EtOH was able to release corticotrophic releasing hormone (CRH)from incubated hypothalamic explants; (ii) whether acetaldehyde (ACD), its first metaboliteformed in the brain by catalase activity, might play a role in EtOH activity. To this aim, rathypothalamic explants were incubated with: (i) medium containing EtOH at 32.6 · 103 lM; (ii)different concentration of ACD (1, 3, 10, and 30 lM); (iii) EtOH plus 3amino-1,2,4-triazole(3AT, 32 · 103 lM) an inhibitor of cerebral catalase; (iv) ACD plus D-penicillamine (DP,50.3 · 103 lM) an ACD-trapping agent. CRH levels were evaluated by a radioimmunoassay.

Results: Incubation with EtOH induced a 7-fold increase in CRH secretion, with respect tobasal levels; ACD was able to stimulate CRH release in a dose-dependent manner; the inhibitionof cerebral catalase by 3AT blocked EtOH-induced CRH outflow; the inactivation of ACD byDP reverted the ACD-stimulating effect on CRH secretion.

Conclusions: These data show that both EtOH and acetaldehyde are able to increase hypotha-lamic CRH release from the rat hypothalamus and that acetaldehyde itself appears to be themediator of EtOH activity.

Key Words: Ethanol, Acetaldehyde, Hypothalamic CRH Release, 3-Amino-1,2,4-triazole,d-Penicillamine.

T HE CENTRAL EFFECTS that follow alcohol (EtOH,ethanol) use include anxiolytic, sedative, amnesic, hyp-

notic properties, that result from the interaction betweenEtOH and different neurotransmitters, ionic channels, mem-brane proteins, and receptors (Faingold et al., 1998). Amongseveral brain structures targeted by the EtOH activity, thehypothalamic–pituitary–adrenal (HPA) axis and its relatedhormones hold a primary relevance. Indeed, as largelyreported, EtOH administration activates the HPA axis, result-ing in adrenocorticotropin hormone (ACTH), and glucocorti-coid release (Rivier, 1996; Rivier and Vale, 1988; Reddy andSarkar, 1993; Silveri and Spear, 2004). Besides, clinical studieslink the disruption of the HPA axis with alcoholism, includinga dampened ability to cope with stress, and a negative correla-

tion between cortisol and craving and relapse in alcoholics(Adinoff et al., 2005; Kiefer & Wiedemann, 2004; Lovalloet al., 2000; O’Malley et al., 2002). The penetration of EtOHin most bodily compartments had made identifying its specificsites of action with the HPA system difficult. Nonetheless, ithas been accurately documented that EtOH activation of theHPA axis occurs primarily through central pathways, i.e.,those that involve the stimulation of the paraventricularnucleus (PVN) of the hypothalamus and ⁄or its afferents(Ogilvie et al., 1998; Rivier and Lee, 1996). Still, the mecha-nisms responsible for these effects have remained elusive andone of the matters at issue is whether it acts directly orthrough the formation of metabolites. Indeed, over the past40 years, a role for acetaldehyde (ACD), EtOH first metabo-lite, has been postulated, although not precisely established(Carpenter and MacLeod, 1952; Hunt, 1990; Stoltz et al.,1944). When ingested, EtOH is diverted into ACD by alcoholdehydrogenase in the liver, and thereafter into acetic acid byaldehyde-dehydrogenase (Glud, 1949). High plasma levels ofACD are generally considered aversive and are the basis fortreating alcoholics with disulfiram, an inhibitor of aldehyde-dehydrogenase (Eckard et al., 1998).However, some evidence suggests that ACD can have posi-

tively reinforcing effects as well as mediating some of thebehavioural and neurochemical effects induced by EtOH

From the Department of Pharmacological Sciences ‘‘P. Benigno’’(CC, MLB, FP, SC), University of Palermo, Palermo; and Instituteof Pharmacology (GT), Catholic University School Medicine, Rome,Italy.

Received for publication January 28, 2009; accepted October 26, 2009.Reprint requests: Carla Cannizzaro, MD, Department of Pharma-

cological Sciences ‘‘P. Benigno’’ University of Palermo, V. Vespro129, 90127 Palermo, Italy; Fax: +39-091-6553220; E-mail:carlacannizzaro@unipa.it; carla.cannizzaro@katamail.com

Copyright � 2010 by the Research Society on Alcoholism.

DOI: 10.1111/j.1530-0277.2009.01127.x

Alcoholism: Clinical and Experimental Research Vol. 34, No. 4April 2010

Alcohol Clin Exp Res, Vol 34, No 4, 2010: pp 1–6 1

(Correa et al., 2003; Escarabajal et al., 2003; Quertemont andTambour, 2004). Under physiological conditions, however,the ACD formed peripherally by alcohol dehydrogenase isnot likely to enter the CNS, due to the high aldehyde-dehydrogenase content together with the blood–brain barrier(Smith et al.,1997; Zimatkin, 1991). Indeed, reports describe aslow but measurable accumulation of ACD in the brain tis-sue, that can be formed by the catalase H2O2 system (Aragonand Amit, 1992; Hamby-Mason et al., 1997). This enzyme isdiscreetly present in many cerebral regions, includingthe hypothalamus, and is able to oxidize EtOH to ACD inthemicromolar range (Gill et al., 1992).Given this evidence, thepresent study was designed to further explore the mechanismsby which EtOH stimulates the HPA axis, focusing on thehypothesis that ACD can act as a primary player in EtOHeffects in the brain. To this aim the incubation of hypotha-lamic explants has been used as an in vitro model, with thepurpose of carrying out in vitro studies on the central mecha-nisms controlling the HPA axis (Grossman et al., 1993;Pozzoli et al., 2001). This procedure allows the anatomicalintegrity of corticotropin releasing hormone (CRH) neuronsfrom the PVN of the hypothalamus to the median eminenceto be maintained; this makes the experimental system suitablefor investigating the functions of central CRH-ergic pathways(Sawchenko et al., 1993), as well as the effects of test drugsacting at the level of cell bodies. Thus, CRH secretion hasbeen determined following incubation with: (i) EtOH at aconcentration known to activate ACTH release (Redei et al.,1986; Rivier, 1996); (ii) different concentrations in the micro-molar range, of ACD; (iii) EtOH plus the irreversible inhibi-tor of catalase, 3-amino-1,2,4-triazole (3AT); ACD in thepresence of its trapping agent, d-penicillamine (DP). Ourhypothesis is, indeed, that EtOH is able to activate the HPAaxis, releasing CRH from the hypothalamus and that thiseffect may be mediated, at least at some extent, by ACD.

MATERIALS AND METHODS

Hypothalamic Incubations

The study was approved by the authority at the Italian Ministry ofHealth. Animal experimentation followed the European legislation(EEC n. 86 ⁄609) and the Guide for the Care and Use of LaboratoryAnimals (National Research Council, 1996) as approved by the Soci-ety for Neuroscience. All efforts were made to minimize animal painand discomfort and to reduce the number of experimental subjects.Hypothalamic explants were performed as previously described

(Navarra et al., 1991), with modifications. Briefly, male adult Wistarrats (200 to 300 g) were decapitated between 09:00 and 10:00 am toavoid circadian variations; the brains were promptly removed andthe hypothalami dissected. The hypothalamic blocks were then longi-tudinally bisected and the 2 halves were incubated together in poly-ethylene vials containing 500 ll minimum essential medium (MEM)supplemented with 0.2% bovine serum albumin (Sigma ChemicalCo., St Louis, MO), 50 ll ⁄ml ascorbic acid and 40 IU ⁄ml aprotinin,pH 7.4, in an atmosphere of 95% O2 and 5% CO2. In the first60 min of incubation, the medium was replaced every 20 minutes towash out the tissues; during this time, CRH spontaneous outflowreached a constant rate. Therefore, in all the subsequent experiments,the first hour was taken as preincubation time. After preincubation,

2 separate 20-minute fractions were collected: a prestimulation phasein which the explants were treated with medium alone to estimate thebasal outflow; a stimulation phase in which medium containing testsubstances was applied. Where necessary in the experimental design,a poststimulation phase with medium alone and a second stimulatoryperiod were included in order to demonstrate respectively return tobasal release and the persistent responsiveness of the tissue.In this experimental model, hypothalami remain viable and func-

tional, as assessed by lactate dehydrogenase assay for cellular toxicity(Dello Russo et al., 2000). Medium samples were stored at )35�Cuntil assayed for CRH immunoreactivity.

CRH Radioimmunoassay

Corticoptropic releasing hormone release was measured by radio-immunoassay as previously described (Tringali et al., 2006). A [125I]Tyr-CRF (NEN PerkinElmer, Boston, MA) was used. The detectionlimit of the assay was 1 pg ⁄ tube (200-ll sample volume from eachincubation tube), with intraassay and interassay coefficients of varia-tion of 5 and 10%, respectively. The amounts of CRH released wereexpressed as pg ⁄ml.

Drugs

All drugs have been dissolved in MEM solution. ETOH (SigmaAldrich) was employed at 32.6 · 103 lM (150 mg%; Roddet al., 2005); ACD (Sigma Aldrich) was used at 1, 3, 10, 30 lM (Gillet al., 1992; Heap et al., 1995). 3AT and DP were used respectivelyat 32 · 103 and at 50.3 · 103 lM (Enrico et al., 2009; Rodd et al.,2005). Veratridine (Sigma Aldrich) was used at a final concentrationof 10 lM (Tringali et al., 2006).

Statistical Analysis

All data were expressed as the means ± SEM of 12 samples pergroup (3 separate experiments, 4 hypothalami for each condition),and analyzed by one-way ANOVA, followed by post hoc Student’st-test. All data were analyzed using a Prism computer software(Graph-Pad, San Diego, CA). Differences were considered significantwhen p < 0.05.

RESULTS

Effects of EtOH on CRH Release

The design of the first set of experiments originated fromthe assessed capacity of EtOH to activate the HPA axis, inparticular from the evidence that the hypothalamus can be itsprimary site of action. Thus at first, after 1 hour of preincuba-tion to equilibrate the system, CRH basal release was mea-sured in the following 20-minute incubation and it wasequivalent to 60 pg ⁄ml. When the hypothalamic halves wereexposed for 20 minutes to a solution containing EtOH at32.6 · 103 lM (EtOH 1), CRH release was significantlyincreased (F = 16.862, p = 0.0038) with respect to basal lev-els. In the poststimulus 20-minute incubation, CRH contentreturned to basal release, proving that the tissue recoveredfrom the drug effect. The administration of a second pulse ofEtOH, at the same dose (EtOH 2), for 20 minutes, induceda significant (F = 11.381, p = 0.0025), although lower,increase in CRH secretion, as a proof of the viability ofthe hypothalami (Fig. 1). Data were analyzed by one-wayANOVA followed by post hoc Student’s t-test.

2 CANNIZZARO ET AL.

Effects of ACD on CRH Release

To investigate the mechanisms underlying EtOH-inducedstimulation of CRH release, and to establish the possible roleplayed by ACD, distinct sets of hypothalami were treatedwith MEM solutions containing ACD at different concentra-tions. Thus, after the collection of basal release, 20-minuteexposure to ACD (1, 3, 10, 30 lM) exerted a dose-dependentstimulatory effect (F = 6.034, p = 0.0353; F = 7.06, p =0.041; F = 13.517, p = 0.0063; F = 14.007, p = 0.0065),that plateaued at 30 lM ACD (Fig. 2A). CRH releasereturned to basal levels, after subsequent 20-minute incuba-tion, in the poststimulation phase. When a second 20-minutepulse with ACD at 10 lM was administered to the hypotha-lamic blocks, a significant (F = 1.78, p = 0.0321), increasein CRH release was detected (Fig. 2B), although of a lesserextent with respect to the ACD 10 lM former stimulation.Data were analyzed by one-way ANOVA followed by posthoc Student’s t-test.

Effect of 3AT and of DP, Respectively, on EtOH- andACD-Induced CRH Release

The next step in this set of experiments was to establish theproper role of EtOH in releasing CRH from the hypothala-mus, and precisely whether ACD could be the mediator ofEtOH effects. For this purpose, 3AT (32 · 103 lM), an irre-versible inhibitor of catalase in the brain, was added in theincubation medium containing EtOH (32.6 · 103 lM), toinfer a catalase-mediated production of ACD. In this para-digm, 3AT was able to block EtOH-induced CRH releasefrom the hypothalamic explants (F = 10.630; p = 0.0097)(Fig. 3A). Further experiments were then conducted to verifythe specific ability of ACD in releasing CRH in our experi-mental model, and to that end, ACD-trapping agent DP

(50.3 · 103 lM) was used. Thus, when administered in associ-ation with ACD (10 lM), DP completely reversed ACD-stimulating effect on hypothalamic CRH release (F = 6.049;p = 0.0351) (Fig. 3B). Data were analyzed by one-wayANOVA followed by post hoc Student’s t-test. When testedalone, 3AT and DP did not exert any intrinsic activity onCRH release. In order to determine the specificity of 3AT andDP effects on blocking, respectively, ACD formation fromEtOH, and ACD activity, the hypothalamic tissues were trea-ted with veratridine at 10 lM, a popular tool to increase neu-rotransmitter release in vitro, in the presence of 3AT and DP.As shown in Fig. 4, veratridine induced a significant increasein CRH secretion (F = 1.320; p = 0.0361) that was notaffected either by 3-AT or by DP, at the doses used in thisstudy. Data were analyzed by one-way ANOVA followed bypost hoc Student’s t-test.

DISCUSSION

The aim of this work was to assess whether EtOH, knownto activate the HPA axis, was able to stimulate CRHrelease from incubated hypothalamic explants, but mainly toinvestigate the role of ACD, its first metabolite, in mediating

Fig. 1. Effects of ethanol (EtOH) on corticotropin releasing hormone(CRH) outflow from rat hypothalamic explants. Following 60 minutes ofequilibration period, CRH basal outflow (basal 1) was estimated at the endof 20-minute incubation with minimum essential medium (MEM). Succes-sively, the hypothalamic explants were incubated with EtOH at(32.6 · 103 lM) (EtOH 1). Incubation was then resumed with MEM in orderto demonstrate return to basal release (basal 2) and a second EtOH expo-sure (EtOH 2) followed as a proof of the viability and responsiveness of thetissue. CRH amounts are expressed as pg ⁄ ml. Results are from 3 indepen-dent experiments; 4 distinct hypothalami were used for each experimentalcondition. Each column represents the mean ± SEM of 12 hypothalami pergroup. ***p < 0.005 versus basal 1 and ���p < 0.005 versus basal 2.

µ

A

B

Fig. 2. Effects of acetaldehyde (ACD) on corticotropin releasing hormone(CRH) outflow from rat hypothalamic explants. Following 60-minuteequilibration period CRH basal outflow (basal 1) was estimated at the end of20-minute incubation with MEM. Thereafter, the hypothalamic explants wereexposed for 20 minutes to different concentrations of ACD (1, 3, 10, 30 lM).Four distinct hypothalami were used for each concentration of ACD(Fig. 2A). In another set of experiments, where ACD was used as a stimulusat 10 lM (ACD 1), incubation was then resumed with MEM in order to dem-onstrate return to basal release (basal 2) and a second 10 lM pulse ACDexposure followed (ACD 2), as a proof of the viability and responsiveness ofthe tissue (Fig. 2B). Data are expressed as pg ⁄ ml. Results are from 3independent experiments; 4 distinct hypothalami were used for eachexperimental condition. Each column represents the mean ± SEM of 12hypothalami per group. *p < 0.05; **p < 0.01 versus basal 1 and �p < 0.05versus basal 2.

ETOH INDUCES CRH RELEASE THROUGH ACD MEDIATION 3

EtOH activity. These results show, for the first time, that inour experimental model, ACD stimulates CRH release fromthe hypothalamic explants in a dose-dependent manner, andthat, notably, ACD itself appears to be the mediator ofEtOH-exerted releasing effect on hypothalamic CRH. Actu-ally, it is widely reported that EtOH increases ACTH releasefrom the pituitary and corticosterone from the rat adrenalgland, and that this action is not played directly at these lev-els, but seems to be secondary to the activation of the hypo-thalamus (Rivier et al., 1984). Indeed, EtOH is able toincrease paraventricular CRH- and vasopressin transcripts,evidences that suggest a primary role for the hypothalamicstructures in the stimulation of ACTH release (Lee andRivier, 2004; Rivier and Lee, 1996). In our study, infact EtOHwas able to increase CRH release from incubated hypotha-lamic blocks, in a range of doses shown to be active instimulating ACTH release from the rat pituitary (Redei et al.,

1986) and also able to induce activation of the HPA axis(Redei et al., 1988) that may recognize, as primum movens, thepromotion of endogenous CRH release from the hypothala-mus. Several authors have demonstrated that ACD is respon-sible for the reinforcing effects of EtOH (Brown et al., 1980;Foddai et al., 2004; Smith et al., 1997). As peripherally pro-duced ACD does not easily penetrate the brain under normalconditions of EtOH consumption, it has been suggested thatACD levels within the brain are essentially dependent uponthe local metabolism of EtOH (Aragon and Amit, 1992), thatis operated by the catalase-H2O2 system. This is the main sys-tem for the central oxidative reaction of EtOH into ACD;indeed, several reports show that both brain homogenatesand neural tissue, when they are incubated with EtOH, pro-duce ACD, and that a previous treatment with the catalaseinhibitor 3AT reduces ACD production in a dose-dependentmanner (Gill et al., 1992; Hamby-Mason et al., 1997). Cata-lase is not uniformly distributed in the brain: the hypotha-lamic regions and the substantia nigra contain the highestconcentration of this enzyme (Jamal et al., 2007; Zimatkinet al., 1998). Thus, it is likely that in the hypothalamus, ACDis produced in discreet amounts that account for functionalconsequences. In this study, we show that ACD is able tostimulate CRH release from hypothalamic explants in a con-centration-dependent manner, and that this effect is specific.Our hypothesis, indeed, is that ACD may be the mediator ofEtOH activity on the hypothalamic tissue. To evaluate thishypothesis, the oxidation of EtOH into ACD in the hypothal-amus was inhibited using the irreversible inhibitor of cerebralcatalase, 3AT. This compound completely prevented EtOH-induced CRH release, strongly indicating ACD as the pri-mary mediator for EtOH activity. 3AT has been reported notto be specific since it possesses goitrogenic activity, but in ourexperimental model, when tested alone, 3AT did not showany effect on CRH spontaneous outflow. However, to better

A

B

Fig. 3. (A) Effect of 3-amino-1,2,4-triazole (3AT) on ethanol (EtOH)-stim-ulated corticotropin release hormone (CRH) release from the rat hypothala-mus. Hypothalamic explants were stimulated for 20 minutes with EtOHalone (32.6 · 103 lM), or in the presence of 3AT (32 · 103 lM) and withmedium containing 3AT. Four distinct hypothalami were used for eachexperimental condition. Results are from 3 independent experiments; 4 dis-tinct hypothalami were used for each experimental condition. Each columnrepresents the mean ± SEM of 12 hypothalami per group. ***p < 0.005 ver-sus basal 1 and ��p < 0.01 versus EtOH alone. (B) Effect of d-penicillamine(DP) on acetaldehyde (ACD)-stimulated CRH release from the rat hypothal-amus. Hypothalamic explants were stimulated for 20 minutes with ACDalone (10 lM), or in the presence of DP (50 · 103 lM) and with mediumcontaining DP. Four distinct hypothalami were used for each experimentalcondition. Results are from 3 independent experiments. Data are expressedas pg ⁄ ml. Each column represents the mean ± SEM of 12 hypothalami pergroup. **p < 0.01 versus basal 1 and �p < 0.05 versus ACD alone.

µ

Fig. 4. Effect of 3-amino-1,2,4-triazole (3AT) and d-penicillamine (DP) onveratridine stimulated corticotropin release hormone (CRH) release from therat hypothalamus. Hypothalamic explants were stimulated for 20 minuteswith veratridine alone (10 lM), or in the presence of 3AT and DP. Fourdistinct hypothalami were used for each experimental condition. Results arefrom 3 independent experiments. Data are expressed as pg CRH ⁄ ml. Eachcolumn represents the mean ± SEM of 12 hypothalami per group. *p < 0.05versus basal.

4 CANNIZZARO ET AL.

define the specificity of ACD action on CRH release, DP,an agent able to inactivate ACD, was added. This compoundis a thiol amino acid derived from penicillin that inter-acts with ACD forming a condensation product, 2,5,5-trimethylthiazolidine-4-carboxilic acid; once this product isformed, the reactivity of ACD is lost and the stable adduct isexcreted in urine (Cohen et al., 2000). When tested in thisstudy, DP did not show intrinsic activity on endogenousCRH secretion but was able to reverse the stimulating activityof ACD on CRH release, proving that ACD itself wasresponsible for the observed effects. In this study, we intro-duced another pharmacological tool, veratridine, a com-pound able to induce depolarization-evoked CRH exocytosis,to rule out any possible interference between 3AT and DPwith the functionality of the release machinery in our experi-mental model. Indeed, when tested in the presence of veratri-dine, 3AT and DP did not exert any effect, proving thespecificity of their action on blocking either ACD formationfrom EtOH or ACD activity. These results are consistent within vivo studies aiming to the assessment of ACD properties inthe central nervous system, such as the inhibition of the LTPin the dentate gyrus as the neurochemical basis of the memoryimpairment induced by EtOH intoxication (Abe et al., 1999).Other reports show that, alcohol-preferring rats self-administer ACD in the posterior ventral tegmental area, anarea implicated in the reinforcing effects of EtOH (Rodd-Henricks et al., 2002), while the central inactivation of ACDby DP blocks EtOH intake in rats, indicating that ACD playsa key role in the motivational properties of EtOH (Font et al.,2006; Melis et al., 2007).In conclusion, the present work confirms and extends

previous results reporting that EtOH can stimulate CRHsystem in the hypothalamus (Lee et al., 2004) likely as theinitial step in the activation of HPA axis. Moreover, wehave demonstrated that ACD is able to release CRH fromthe hypothalamus and that ACD itself is the mediator ofEtOH-induced CRH release. At the moment, the identifica-tion of the mechanisms underlying EtOH- and ACD-releasing properties on hypothalamic CRH deserves furtherwork. Certainly, further speculations are needed to eluci-date the role of ACD as signaling molecule, in order topursue new strategies for the prevention and the treatmentof the alterations that EtOH consumption causes in thecentral nervous system.

ACKNOWLEDGMENTS

We would like to thank Miss Irene Diliberto and VitaBarrile for their excellent technical support. This study wassupported by a grant from PRIN (MIUR, 2006).

REFERENCES

Abe K, Sugiura M, Yamaguchi S, Shoyama Y, Saito H (1999) Saffron extract

prevents acetaldehyde-induced inhibition of long-term potentiation in the

rat dentate gyrus in vivo. Brain Res 851:287–289.

Adinoff B, Junghanns K, Kiefer F, Krishnan-Sarin S (2005) Suppression of

the HPA axis stress-response: implications for relapse. Alcohol Clin Exp

Res 29:1351–1355.

Aragon CMG, Amit Z (1992) The effect of 3-amino-1,2,4-triazole on volun-

tary ethanol consumption: evidence for brain catalase involvement in the

mechanism of action. Neuropharmacology 31:709–712.

Brown ZW, Amit Z, Smith B (1980) Intraventricular self-administration of

acetaldehyde and voluntary consumption of ethanol in rats. Behav Neural

Biol 28:150–155.

Carpenter RK, MacLeod LD (1952) The effects of ethyl alcohol and acetalde-

hyde on maze behaviour and motor co-ordination in rats. J Ment Sci

98:167–173.

Cohen JF, Elberling JA, DeMaster E, Lin RC, Nagasawa H (2000) N-

terminal dipeptides od D(-)- penicillamine as sequestration agents foe acetal-

dehyde. J Med Chem 43:1029–1033.

Correa M, Arizzi MN, Betz A, Mingote S, Salamone JD (2003) Open field

locomotor effects in rats after intraventricular injections of ethanol and the

ethanol metabolites acetaldehyde and acetate. Brain Res Bull 62:197–202.

Dello Russo C, Tringali G, Ragazzoni E, Maggiano N, Menini E, Vairano M,

Preziosi P, Navarra P (2000) Evidence that hydrogen sulphide can modulate

hypothalamo–pituitary–adrenal axis function: in vitro and in vivo studies in

the rat. J Neuroendocrinol 12:225–233.

Eckard MJ, File SE, Gessa GL, Grant KA, Guerri C, Hoffman PL, Kalant

H, Kado GF, Li TK, Tobakoff B (1998) Effects of moderate alcohol con-

sumption on the central nervous system. Alcohol Clin Exp Res 22:998–

1040.

Enrico P, Sircab D, Mereu M, Peana AT, Lintasb A, Golosiob A, Diana

M (2009) Acetaldehyde sequestering prevents ethanol-induced stimulation

of mesolimbic dopamine transmission. Drug Alcohol Depend 100:265–

271.

Escarabajal MD, De Witte P, Quertemont E (2003) Role of acetaldehyde in

ethanol-induced conditioned taste aversion in rats. Psychopharmacology

167:130–136.

Faingold CL, N’Gouemo P, Riaz A (1998) Ethanol and neurotransmitter

interactions from molecular to integrative effects. Prog Neurobiol 55:509–

535.

Foddai M, Dosia G, Spiga S, DianaM (2004) Acetaldehyde increases dopami-

nergic neuronal activity in the VTA. Neuropsychopharmacology 29:530–

536.

Font L, Aragon CMG, Miquel M (2006) Voluntary ethanol consumption

decreases after the inactivation of central acetaldehyde by D-penicillamnine.

Behav Brain Res 171:78–86.

Gill K, Menez JF, Lucas D, Deitrich RA (1992) Enzymatic production of

acetaldehyde from ethanol in rat brain tissue. Alcohol Clin Exp Res 16:910–

915.

Glud E (1949) The treatment of alcoholic patients in Denmark with antabuse:

with suggestions for its trial in the United States. Q J Stud Alcohol 10:185–

197.

Grossman A, Costa A, Navarra P, Tsagarakis S (1993) The regulation of

hypothalamic corticotropin-releasing factor release: in vitro studies. Ciba

Found Symp 172:129–143.

Hamby-Mason R, Chen JJ, Schenker S, Perez A, Henderson GI (1997) Cata-

lase mediates acetaldehyde formation from ethanol in fetal and neonatal rat

brain. Alcohol Clin Exp Res 21:1063–1072.

Heap L, Ward RJ, Abiaka C, Dexter D, Lawlor M, Pratt O, Thomson A,

Shaw K, Peters TJ (1995) The influence of brain acetaldehyde on oxidative

status, dopamine metabolism and visual discrimination task. Biochem Phar-

macol 50:263–270.

Hunt WA (1990) Biochemical bases for the reinforcing effects of ethanol, in

Why People Drink: Parameters of Alcohol as a Reinforcer (Cox WM ed.),

51–70. Gordiner Press, New York.

Jamal M, Ameno K, Uekita I, Kumihashi M, Wang W, Ijiri I (2007) Catalase

mediates acetaldehyde formation in the striatum of free-moving rats. Neuro

Toxicology 28:1245–1248.

Kiefer F, Wiedemann K (2004) Neuroendocrine pathways of addictive beha-

viour. Addict Biol 9:205–212.

ETOH INDUCES CRH RELEASE THROUGH ACD MEDIATION 5

Lee S, Rivier C (1994) Hypophysiotropic role and hypothalamic gene expres-

sion of corticotropin-releasing factor and vasopressin in rats injected with

interleukin-1b systemically or into the brain ventricles. J Neuroendocrinol

6:217–224.

Lee S, Selvage D, Hansen K, Rivier C (2004) Site of action of acute alcohol

administration in stimulating the rat hypothalamic-pituitary-adrenal axis:

comparison between the effect of systemic and intracerebroventricular injec-

tion of this drug on pituitary and hypothalamic responses. Endocrinology

145:4470–4479.

Lovallo WR, Dickensheets SL, Myers DA, Thomas TL, Nixon SJ (2000)

Blunted stress cortisol response in abstinent alcoholic and polysubstance-

abusing men. Alcohol Clin Exp Res 24:651–658.

Melis M, Enrico P, Peana AT, DianaM (2007) Acetaldehyde mediates alcohol

activation of the mesolimbic dopamine system. Eur J Neurosci 26:2824–

2833.

National Research Council (1996) Institute of Laboratory Animal Resources,

Commission on Life Sciences. Guide for the Care and Use of Laboratory

Animals. National Academy Press, Washington, DC.

Navarra P, Tsagarakis S, Faria MS, Rees LH, Besser GM, Grossman AB

(1991) Interleukins-1 and -6 stimulate the release of corticotropin-

releasing hormone-41 from rat hypothalamus in vitro via the eicosanoid

cyclooxygenase pathway. Endocrinology 128:37–44.

Ogilvie K, Lee S, Rivier C (1998) Divergence in the expression of molecular

markers of neuronal activation in the parvocellular paraventricular nucleus

of the hypothalamus evoked by alcohol administration via different routes.

J Neurosci 18:4344–4352.

O’Malley SS, Krishnan-Sarin S, Farren C, Sinha R, Kreek MJ (2002)

Naltrexone decreases craving and alcohol self-administration in alcohol-

dependent subjects and activates the hypothalamo-pituitary-adrenocortical

axis. Psychopharmacology 160:19–29.

Pozzoli G, Tringali G, Dello Russo C, Vairano M, Preziosi P, Navarra P

(2001) HIV-1 Gp120 protein modulates corticotropin releasing factor

synthesis and release via the stimulation of its mRNA from the rat

hypothalamus in vitro: involvement of inducible nitric oxide synthase.

J Neuroimmunol 118:268–276.

Quertemont E, Tambour S (2004) Is ethanol a pro-drug? The role of acetalde-

hyde in the central effects of ethanol Trends Pharmacol Sci 25:130–134.

Reddy BV, Sarkar DK (1993) Effect of alcohol, acetaldehyde, and salsolinol

on beta-endorphin secretion from the hypothalamic neurons in primary cul-

tures. Alcohol Clin Exp Res 17:1261–1267.

Redei E, Branch BJ, Gholami S, Lin EYR, Taylor AN (1988) Effects of etha-

nol on CRF release in vitro. Endocrinology 123:2736–2743.

Redei E, Branch BJ, Taylor AN (1986) Direct effect of ethanol on adrenocor-

ticotropin (ACTH) release in vitro. J Pharmacol Exp Ther 237:59–64.

Rivier C (1996) Alcohol stimulates ACTH secretion in the rat: mechanisms of

action and interactions with other stimuli. Alcohol Clin Exp Res 20:240–

254.

Rivier C, Brhun T, Vale W (1984) Effects of ethanol on the hypothalamic-

pituitary-adrenal axis in the rat: role of corticosterone releasing factor

(CRF). J Pharmacol Exp Ther 229:127–131.

Rivier C, Lee S (1996) Acute alcohol administration stimulates the activity of

hypothalamic neurons that express corticotropin-releasing factor and vaso-

pressin. Brain Res 726:1–10.

Rivier C, Vale W (1988) Interaction between ethanol and stress on ACTH and

beta-endorphin secretion. Alcoholism: Clinical and Experimental Research

12:206–210.

Rodd ZA, Bell RL, Zhang Y, Murphy JM, Goldstein A, Zaffaroni A, Li TK,

McBride JW (2005) Regional heterogeneity for the intracranial self-

administration of ethanol and acetaldehyde within the ventral tegmental

area of alcohol-preferring (P) rats: involvement of dopamine and serotonin.

Neuropsychopharmacology 30:330–338.

Rodd-Henricks ZA, Melendez RI, Zaffaroni A, Goldstein A, McBride WJ, Li

T-K (2002)The reinforcing effects of acetaldehyde in the posterior ventral teg-

mental area of alcohol-preferring rats. Pharmacol BiochemBehav 72:55–64.

Sawchenko PE, Imaki T, Potter E, Kovacs K, Imaki J, Vale W (1993) The

functional neuroanatomy of corticotropin-releasing factor. Ciba Found

Symp 172:5–21.

Silveri MS, Spear LP (2004) Characterizing the ontogeny of ethanol-associated

increases in corticosterone. Alcohol 32:145–155.

Smith BR, Aragon CM, Ami Z (1997) Catalase and the production of brain

acetaldehyde: a possible mediator for the psychopharmacological effects of

ethanol. Addict Biol 2:227–289.

Stoltz E, Westerfeld WW, Berg RL (1944) The metabolism of acetaldehyde

with acetoin formation. J Biol Chem 152:41–50.

Tringali G, Aubry JM, Navarra P, Pozzoli G (2006) Lamotrigine inhibits

basal and Na+-stimulated, but not Ca2 + -stimulated, release of cortico-

tropin-releasing hormone from the rat hypothalamus. Psychopharmacology

188:386–392.

Zimatkin SM (1991) Histochemical study of aldehyde dehydrogenase in the

rat CNS. J Neurochem 56:1–11.

Zimatkin SM, Liopo AV, Deitrich RA (1998) Distribution and kinetics of

ethanol metabolism in rat brain. Alcohol Clin Exp Res 22:1623–1627.

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