- Email: [email protected]
Acute ethanol induces c-fos immunoreactivity in GABAergic neurons of the central nucleus of the amygdala1
Brain Research 798 Ž1998. 333–336
Interactive report
Acute ethanol induces c-fos immunoreactivity in GABAergic neurons of the central nucleus of the amygdala 1 Marisela Morales ) , Jose R. Criado, Pietro Paolo Sanna, Steven J. Henriksen, Floyd E. Bloom The Scripps Research Institute, Department of Neuropharmacology, Alcohol Research Center, North Torrey Pines Road, La Jolla, CA 92037, USA Accepted 13 April 1998
Abstract The central nucleus of the amygdala ŽCNA. is a component of the brain reward pathway which is believed to represent an anatomical substrate for drugs of abuse. Previous studies have shown that acute ethanol administration induces the expression of c-fos in the CNA of rat brains. We report here, that over 70% of these c-fos immunoreactive neurons are GABAergic. This observation provides the first anatomical evidence that GABAergic neurons of the CNA are responsive to acute ethanol exposure and suggest that the GABAergic system of the CNA is a key neuronal substrate for ethanol actions on the central nervous system. q 1998 Elsevier Science B.V. All rights reserved. Keywords: GAD; Interneuron; Drug addiction; Extended amygdala
Expression of the immediate early gene c-fos is commonly utilized as a high resolution histological marker of trans-synaptic neuronal stimulation w4,19,20x. Previous studies have shown that acute ethanol administration induces expression of c-Fos immunoreactivity in the CNA w3,18x. In the present study, we used a combination of in situ hybridization and immunohistochemistry to demonstrate that ethanol-responsive neurons within the CNA are mainly GABAergic. Our anatomical data support the participation of GABAergic cells of the CNA in the acute effects of ethanol. Twelve male Sprague–Dawley rats ŽCharles River Laboratory, Hollister, CA. weighting 300–380 g were housed in groups on a 12r12 h lightrdark cycle. Rats received daily intraperitoneal ŽIP. injections of saline for at least seven days to facilitate adaptation to the injection and handling procedures. Following such adaptation protocol, animals were randomly assigned to receive IP injections of 2.0 grkg of ethanol w16% in saline Žwrv., REMET Corp., La Mirada, CAx or control saline. Two hours after injection, animals were anesthetized by CO 2 narcosis, and )
Corresponding author. Present address: National Institute on Drug Abuse, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA. Fax: q1 Ž410. 550 1645; E-mail: [email protected]. 1 Published on the World Wide Web on 27 May 1998. 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 4 5 7 - 0
serum was collected for blood ethanol determination ŽSigma Chemicals, St. Louis, MO.. Mean blood ethanol levels were 161.2 mg% " 11.82 ŽS.E.M... Anesthetized rats were perfused transcardially with a solution of 4% paraformaldehyde in 0.1 M phosphate buffer ŽPB., pH 7.3. Brains were then removed from the skull, postfixed from 2–15 h at 48C, rinsed with PB and sequentially transferred to 10%, 12% and 18% sucrose solutions. Brains were then frozen on dry ice and sectioned on a cryostat to obtain coronal sections of 40 m m in thickness. To detect both mRNA and protein in the same sections, we performed in situ hybridization followed by immunohistochemistry as previously described w10x. After pre-hybridization, cryosections were hybridized at 558C for 16 h with 10 7 cpmrml of w 35 Sx-RNA probes corresponding to the two isoforms of rat glutamic acid decarboxylase, GAD65 and GAD67 Žgenerously provided by Dr. Allan Tobin, University of California Los Angeles, Los Angeles, CA.. Sections were treated with RNase A at 4 m grml at 378C for 1 h, washed in 1 = SSC, 50% formamide at 558C for 2 h, and in 0.1 = SSC at 688C for 1 h. Sections were rinsed with PB, blocked in 1% bovine serum albumin with 0.3% Triton X-100 for 1 h, incubated with a polyclonal anti-c-fos antibody ŽSanta Cruz Biotechnology, Santa Cruz, CA. at dilution 1:1000 for 24 h at 48C. Sections were processed with an ABC kit ŽVector. and peroxidase activ-
334
M. Morales et al.r Brain Research 798 (1998) 333–336
ity was revealed with 0.003% H 2 O 2 and 0.05% 3, 3-diaminobenzidine-4 HCl ŽDAB., which results in a brown precipitate in specifically immunolabelled structures. Sections were mounted on coated slides, air dried, dipped in nuclear track emulsion and exposed for several weeks prior to development. Sections were analyzed and photographed with bright field microscopy. Two populations of cells Žc-Fos single immunolabelled cells and double labelled c-Fos im m unolabelled cells containing GAD65 rGAD67 transcripts. were counted in random sections of the CNA. The percentage of neurons expressing the GAD65 rGAD67 mRNA was calculated from the total population of c-Fos immunoreactive neurons with data collected from 6 randomly selected sections of 6 different alcohol-injected animals. Neurons were considered
double-labelled if their nuclei appeared brown Ži.e. c-Fos immunoreactive. and more than ten silver grains were visible over the nuclei themselves or in their immediate vicinity. Such level of hybridization signal was found to be above background noise. In preliminary experiments, we found that ethanol concentrations as low as 0.25 grkg induced c-Fos immunoreactivity in the CNA, 1–2 h after ethanol administration Ždata not shown.. However, while low concentrations of ethanol Ž0.25–1.0 grkg. showed variable levels of c-Fos induction, consistent results were obtained with ethanol doses of 2.0 grkg. Based on these observations, the latter dose was used in the study. Acute administration of ethanol Ž2.0 grkg. produced a remarkable induction of c-Fos immunoreactivity in many
Fig. 1. Simultaneous detection of GAD65 rGAD67 transcripts and c-Fos immunoreactivity in the lateral subdivision of the CNA 2 h after IP injection of ethanol ŽA. or saline ŽB.. Co-localization of c-fos immunoreactivity Žbrown. with GAD65 rGAD67 trancripts Žsilver grains, seen as black dots. is observed in most neurons Žarrows.. Note the very low levels of c-fos immunoreactivity in saline treated controls. Scale bar s 15 m m.
M. Morales et al.r Brain Research 798 (1998) 333–336
neurons of CNA ŽFig. 1A.. In contrast, the CNA from control animals contained few c-Fos immunoreactive neurons ŽFig. 1B.. The CNA has a high density of GABAergic neurons w13,22x. Thus, we used a combination of in situ hybridization and immunocytochemical methods to investigate if c-Fos immunoreactive neurons, responsive to ethanol administration, were GABAergic. Brain sections were initially hybridized with radioactive riboprobes specific for transcripts of the two isoforms of rat glutamic acid decarboxylase, GAD65 and GAD67 . Then, the same sections were incubated with an anti-c-Fos antibody to detect c-Fos immunoreactive neurons. We found a striking co-localization of GAD65 rGAD67 transcripts with c-Fos immunoreactivity in the lateral subdivision of the CNA in ethanol treated rats. Quantitative analysis indicated that 72% " 3 ŽS.E.M.. of c-Fos immunoreactive cells Ž n s 443. colocalized with GAD65 rGAD67 transcripts. The CNA together with the bed nucleus of the stria terminalis and regions of the nucleus accumbens form a forebrain structure known as the extended amygdala w1x. This structure is highly inter-reactive w22x. It is likely that ethanol-activation of GABAergic neurons of the lateral part of the CNA will affect other components of the extended amygdala as well as its projecting areas. Within the extended amygdala, the medial subdivision of the CNA and the bed nucleus of the stria terminalis receive abundant GABAergic inputs from the lateral part of the CNA w15,16,22x. In turn, the medial subdivision of the CNA projects to the lateral hypothalamus w9,21x, ventral tegmental area w11,24x and brainstem w6,23x. Many of the GABAergic neurons of the lateral part of the CNA contain neuropeptides w2,5x. Recent experimental evidences indicate that GABAergic as well as neuropeptidergic systems of the CNA participate in ethanol-related behaviors w7,12,14x. The participation of the CNA in ethanol reinforcement is also supported by the observation that microinjection of GABA antagonists into this nucleus produce a dose-dependent reduction of oral ethanol selfadministration in rats w7x. Furthermore, microinjection of a GABA agonist into the CNA decreases ethanol self-administration in ethanol dependent animals w17x, these observations have lead to the suggestion that the GABAergic system of the CNA is altered during the course of ethanol dependence w17x. According to current neuroadaptative theories, compensatory changes within neuronal systems that are acutely activated by alcohol or other drugs of abuse underlie vulnerability to relapse as well as symptoms of withdrawal after chronic administration w8x. Our results provide anatomical evidence indicating that GABAergic neurons of the CNA are responsive to acute ethanol exposure. The GABAergic system of the CNA has been implicated in ethanol reinforcement w7x, and adaptive changes in this neuronal system appear to be brought about by ethanol dependence w17x. Taken together, these observations suggest that the GABAergic system of the CNA is a
335
key neuronal substrate of ethanol actions on the central nervous system.
Acknowledgements The GAD65 and GAD67 plasmids were generously provided by Dr. Allan Tobin, University of California Los Angeles, Los Angeles, CA. Partially supported by Grant AA 06420. P.P.S. is the recipient of a NARSAD young investigator award.
References w1x G.F. Alheid, L. Heimer, New perspectives in basal forebrain organization of special relevance for neuropsychiatric disorders: the striatopallidal, amygdaloid, and corticopetal components of substantia innominata, Neuroscience 1 Ž1988. 1–39. w2x M.D. Cassell, T.S. Gray, J.Z. Kiss, Neuronal architecture in the rat central nucleus of the amygdala: a cytological, hodological, and immunocytochemical study, J. Comp. Neurol. 246 Ž1986. 478–499. w3x S.L. Chang, N.A. Patel, A.A. Romero, Activation and desensitization of Fos immunoreactivity in the rat brain following ethanol administration, Brain Res. 679 Ž1995. 89–98. w4x T. Curran, J.I. Morgan, Fos: An immediate-early transcription factor in neurons, J. Neurobiol. 26 Ž1995. 403–412. w5x T.S. Gray, M.E. Carney, D.J. Magnuson, Direct projections from the central amygdaloid nucleus to the hypothalamic paraventricular nucleus. Possible role in stress-induced adrenocorticotropin release, Neuroendocrinology 50 Ž1989. 433–446. w6x D.A. Hopkins, D. Holstege, Amygdaloid projections to the mesencephalon, pons, and medulla oblongata in the cat, Exp. Brain Res. 32 Ž1978. 529–547. w7x P. Hyttia, G.F. Koob, GABAA receptor antagonism in the extended amygdala decreases ethanol self-administration in rats, Eur. J. Pharmacol. 283 Ž1995. 151–159. w8x G.F. Koob, P.P. Sanna, F.E. Bloom, Neurobiology of Drug addiction, Neuron, in press. w9x J.E. Krettek, J.L. Price, Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat, J. Comp. Neurol. 178 Ž1978. 225–254. w10x M. Morales, F.E. Bloom, The 5-HT 3 receptor is present in different subpopulations of GABAergic neurons in the rat telencephalon, J. Neuroscience 17 Ž1997. 3157–3167. w11x O.T. Phillipson, Afferent projections to the ventral tegmental area of Tsai and intrafascicular nucleus. A horseradish peroxidase study in the rat, J. Comp. Neurol. 187 Ž1979. 117–143. w12x E.M. Pich, M. Lorang, M. Yeganeh, F.F. Rodriguez, J. Raber, G.F. Koob, F. Weiss, Increase of extracellular corticotropin-releasing factor-like immunoreactivity levels in the amygdala of awake rats during restraint stress and ethanol withdrawal as measured by microdialysis, J. Neuroscience 15 Ž1995. 5439–5447. w13x A. Pitkdnen, D.G. Amaral, The distribution of GABAergic cells, fibers and terminals in the monkey amygdaloid complex: An immunohistochemical and in situ hybridization study, J. Neuroscience 14 Ž1993. 2200–2224. w14x S. Rassnick, S.C. Heinrichs, K.T. Britton, G.F. Koob, Microinjection of a corticotropin-releasing factor antagonist into the central nucleus of the amygdala reverses anxiogenic-like effects of ethanol withdrawal, Brain. Res. 605 Ž1993. 25–32. w15x G.W. Roberts, P.L. Woodhams, J.M. Polak, T.J. Crow, Distribution of neuropeptides in the limbic system of the rat: The amygdaloid complex, Neuroscience 7 Ž1982. 99–131.
336
M. Morales et al.r Brain Research 798 (1998) 333–336
w16x G.W. Roberts, Neuropeptides: Cellular morphology, major pathways, and functional considerations, in: J.P. Aggleton ŽEd.., The Amygdala. Neurobiological Aspects of Emotion, Memory and Mental Dysfunction, Wiley-Liss, Chichester, 1992, pp. 115–142. w17x A.J. Roberts, M. Cole, G.F. Koob, Intra-amygdala muscimol decreases operant ethanol self-administration in dependent rats, Alcohol Clin. Exp. Res. 20 Ž1996. 1289–1298. w18x A.E. Ryabinin, J.R. Criado, S.J. Henriksen, F.E. Bloom, M.C. Wilson, Differential sensitivity of c-Fos expression in hippocampus and other brain regions to moderate and low doses of alcohol, Molecular Psychiatry 2 Ž1. Ž1997. 32–43. w19x S.M. Sagar, F.R. Sharp, T. Curran, Expression of c-fos protein in brain: Metabolic mapping at the cellular level, Science 240 Ž1988. 1328–1331. w20x M. Sheng, M.E. Greenberg, The regulation and function of c-fos and other immediate early genes in the nervous system, Neuron 4 Ž1990. 477–485.
w21x S. Shiosaka, M. Tokyama, H. Takagi, Y. Takahashi, T. Saitoh, H. Sakumoto, H. Nakagawa, N. Shimizu, Ascending and descending components of the medial forebrain bundle in the rat as demonstrated by the horseradish peroxidase-blue reaction. I. Forebrain and upper brainstem, Exp. Brain Res. 39 Ž1980. 377–388. w22x N. Sun, M.D. Cassell, Intrinsic GABAergic neurons in the rat central extended amygdala, J. Comp. Neurol. 330 Ž1993. 381–404. w23x J.G. Veening, L.W. Swanson, P.E. Sawchenko, The organization of projections from the central nucleus of the amygdala to brainstem sites involved in central autonomic regulation. A combined retrograde transport-immunohistochemical study, Brain Res. 303 Ž1984. 337–357. w24x D.M. Wallace, D.J. Magnuson, T.S. Gray, The amygdalo-brainstem pathway: dopaminergic, noradrenergic and adrenergic cells in the rat, Neurosci. Lett. 97 Ž1989. 252–258.
Interactive report
Acute ethanol induces c-fos immunoreactivity in GABAergic neurons of the central nucleus of the amygdala 1 Marisela Morales ) , Jose R. Criado, Pietro Paolo Sanna, Steven J. Henriksen, Floyd E. Bloom The Scripps Research Institute, Department of Neuropharmacology, Alcohol Research Center, North Torrey Pines Road, La Jolla, CA 92037, USA Accepted 13 April 1998
Abstract The central nucleus of the amygdala ŽCNA. is a component of the brain reward pathway which is believed to represent an anatomical substrate for drugs of abuse. Previous studies have shown that acute ethanol administration induces the expression of c-fos in the CNA of rat brains. We report here, that over 70% of these c-fos immunoreactive neurons are GABAergic. This observation provides the first anatomical evidence that GABAergic neurons of the CNA are responsive to acute ethanol exposure and suggest that the GABAergic system of the CNA is a key neuronal substrate for ethanol actions on the central nervous system. q 1998 Elsevier Science B.V. All rights reserved. Keywords: GAD; Interneuron; Drug addiction; Extended amygdala
Expression of the immediate early gene c-fos is commonly utilized as a high resolution histological marker of trans-synaptic neuronal stimulation w4,19,20x. Previous studies have shown that acute ethanol administration induces expression of c-Fos immunoreactivity in the CNA w3,18x. In the present study, we used a combination of in situ hybridization and immunohistochemistry to demonstrate that ethanol-responsive neurons within the CNA are mainly GABAergic. Our anatomical data support the participation of GABAergic cells of the CNA in the acute effects of ethanol. Twelve male Sprague–Dawley rats ŽCharles River Laboratory, Hollister, CA. weighting 300–380 g were housed in groups on a 12r12 h lightrdark cycle. Rats received daily intraperitoneal ŽIP. injections of saline for at least seven days to facilitate adaptation to the injection and handling procedures. Following such adaptation protocol, animals were randomly assigned to receive IP injections of 2.0 grkg of ethanol w16% in saline Žwrv., REMET Corp., La Mirada, CAx or control saline. Two hours after injection, animals were anesthetized by CO 2 narcosis, and )
Corresponding author. Present address: National Institute on Drug Abuse, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA. Fax: q1 Ž410. 550 1645; E-mail: [email protected]. 1 Published on the World Wide Web on 27 May 1998. 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 4 5 7 - 0
serum was collected for blood ethanol determination ŽSigma Chemicals, St. Louis, MO.. Mean blood ethanol levels were 161.2 mg% " 11.82 ŽS.E.M... Anesthetized rats were perfused transcardially with a solution of 4% paraformaldehyde in 0.1 M phosphate buffer ŽPB., pH 7.3. Brains were then removed from the skull, postfixed from 2–15 h at 48C, rinsed with PB and sequentially transferred to 10%, 12% and 18% sucrose solutions. Brains were then frozen on dry ice and sectioned on a cryostat to obtain coronal sections of 40 m m in thickness. To detect both mRNA and protein in the same sections, we performed in situ hybridization followed by immunohistochemistry as previously described w10x. After pre-hybridization, cryosections were hybridized at 558C for 16 h with 10 7 cpmrml of w 35 Sx-RNA probes corresponding to the two isoforms of rat glutamic acid decarboxylase, GAD65 and GAD67 Žgenerously provided by Dr. Allan Tobin, University of California Los Angeles, Los Angeles, CA.. Sections were treated with RNase A at 4 m grml at 378C for 1 h, washed in 1 = SSC, 50% formamide at 558C for 2 h, and in 0.1 = SSC at 688C for 1 h. Sections were rinsed with PB, blocked in 1% bovine serum albumin with 0.3% Triton X-100 for 1 h, incubated with a polyclonal anti-c-fos antibody ŽSanta Cruz Biotechnology, Santa Cruz, CA. at dilution 1:1000 for 24 h at 48C. Sections were processed with an ABC kit ŽVector. and peroxidase activ-
334
M. Morales et al.r Brain Research 798 (1998) 333–336
ity was revealed with 0.003% H 2 O 2 and 0.05% 3, 3-diaminobenzidine-4 HCl ŽDAB., which results in a brown precipitate in specifically immunolabelled structures. Sections were mounted on coated slides, air dried, dipped in nuclear track emulsion and exposed for several weeks prior to development. Sections were analyzed and photographed with bright field microscopy. Two populations of cells Žc-Fos single immunolabelled cells and double labelled c-Fos im m unolabelled cells containing GAD65 rGAD67 transcripts. were counted in random sections of the CNA. The percentage of neurons expressing the GAD65 rGAD67 mRNA was calculated from the total population of c-Fos immunoreactive neurons with data collected from 6 randomly selected sections of 6 different alcohol-injected animals. Neurons were considered
double-labelled if their nuclei appeared brown Ži.e. c-Fos immunoreactive. and more than ten silver grains were visible over the nuclei themselves or in their immediate vicinity. Such level of hybridization signal was found to be above background noise. In preliminary experiments, we found that ethanol concentrations as low as 0.25 grkg induced c-Fos immunoreactivity in the CNA, 1–2 h after ethanol administration Ždata not shown.. However, while low concentrations of ethanol Ž0.25–1.0 grkg. showed variable levels of c-Fos induction, consistent results were obtained with ethanol doses of 2.0 grkg. Based on these observations, the latter dose was used in the study. Acute administration of ethanol Ž2.0 grkg. produced a remarkable induction of c-Fos immunoreactivity in many
Fig. 1. Simultaneous detection of GAD65 rGAD67 transcripts and c-Fos immunoreactivity in the lateral subdivision of the CNA 2 h after IP injection of ethanol ŽA. or saline ŽB.. Co-localization of c-fos immunoreactivity Žbrown. with GAD65 rGAD67 trancripts Žsilver grains, seen as black dots. is observed in most neurons Žarrows.. Note the very low levels of c-fos immunoreactivity in saline treated controls. Scale bar s 15 m m.
M. Morales et al.r Brain Research 798 (1998) 333–336
neurons of CNA ŽFig. 1A.. In contrast, the CNA from control animals contained few c-Fos immunoreactive neurons ŽFig. 1B.. The CNA has a high density of GABAergic neurons w13,22x. Thus, we used a combination of in situ hybridization and immunocytochemical methods to investigate if c-Fos immunoreactive neurons, responsive to ethanol administration, were GABAergic. Brain sections were initially hybridized with radioactive riboprobes specific for transcripts of the two isoforms of rat glutamic acid decarboxylase, GAD65 and GAD67 . Then, the same sections were incubated with an anti-c-Fos antibody to detect c-Fos immunoreactive neurons. We found a striking co-localization of GAD65 rGAD67 transcripts with c-Fos immunoreactivity in the lateral subdivision of the CNA in ethanol treated rats. Quantitative analysis indicated that 72% " 3 ŽS.E.M.. of c-Fos immunoreactive cells Ž n s 443. colocalized with GAD65 rGAD67 transcripts. The CNA together with the bed nucleus of the stria terminalis and regions of the nucleus accumbens form a forebrain structure known as the extended amygdala w1x. This structure is highly inter-reactive w22x. It is likely that ethanol-activation of GABAergic neurons of the lateral part of the CNA will affect other components of the extended amygdala as well as its projecting areas. Within the extended amygdala, the medial subdivision of the CNA and the bed nucleus of the stria terminalis receive abundant GABAergic inputs from the lateral part of the CNA w15,16,22x. In turn, the medial subdivision of the CNA projects to the lateral hypothalamus w9,21x, ventral tegmental area w11,24x and brainstem w6,23x. Many of the GABAergic neurons of the lateral part of the CNA contain neuropeptides w2,5x. Recent experimental evidences indicate that GABAergic as well as neuropeptidergic systems of the CNA participate in ethanol-related behaviors w7,12,14x. The participation of the CNA in ethanol reinforcement is also supported by the observation that microinjection of GABA antagonists into this nucleus produce a dose-dependent reduction of oral ethanol selfadministration in rats w7x. Furthermore, microinjection of a GABA agonist into the CNA decreases ethanol self-administration in ethanol dependent animals w17x, these observations have lead to the suggestion that the GABAergic system of the CNA is altered during the course of ethanol dependence w17x. According to current neuroadaptative theories, compensatory changes within neuronal systems that are acutely activated by alcohol or other drugs of abuse underlie vulnerability to relapse as well as symptoms of withdrawal after chronic administration w8x. Our results provide anatomical evidence indicating that GABAergic neurons of the CNA are responsive to acute ethanol exposure. The GABAergic system of the CNA has been implicated in ethanol reinforcement w7x, and adaptive changes in this neuronal system appear to be brought about by ethanol dependence w17x. Taken together, these observations suggest that the GABAergic system of the CNA is a
335
key neuronal substrate of ethanol actions on the central nervous system.
Acknowledgements The GAD65 and GAD67 plasmids were generously provided by Dr. Allan Tobin, University of California Los Angeles, Los Angeles, CA. Partially supported by Grant AA 06420. P.P.S. is the recipient of a NARSAD young investigator award.
References w1x G.F. Alheid, L. Heimer, New perspectives in basal forebrain organization of special relevance for neuropsychiatric disorders: the striatopallidal, amygdaloid, and corticopetal components of substantia innominata, Neuroscience 1 Ž1988. 1–39. w2x M.D. Cassell, T.S. Gray, J.Z. Kiss, Neuronal architecture in the rat central nucleus of the amygdala: a cytological, hodological, and immunocytochemical study, J. Comp. Neurol. 246 Ž1986. 478–499. w3x S.L. Chang, N.A. Patel, A.A. Romero, Activation and desensitization of Fos immunoreactivity in the rat brain following ethanol administration, Brain Res. 679 Ž1995. 89–98. w4x T. Curran, J.I. Morgan, Fos: An immediate-early transcription factor in neurons, J. Neurobiol. 26 Ž1995. 403–412. w5x T.S. Gray, M.E. Carney, D.J. Magnuson, Direct projections from the central amygdaloid nucleus to the hypothalamic paraventricular nucleus. Possible role in stress-induced adrenocorticotropin release, Neuroendocrinology 50 Ž1989. 433–446. w6x D.A. Hopkins, D. Holstege, Amygdaloid projections to the mesencephalon, pons, and medulla oblongata in the cat, Exp. Brain Res. 32 Ž1978. 529–547. w7x P. Hyttia, G.F. Koob, GABAA receptor antagonism in the extended amygdala decreases ethanol self-administration in rats, Eur. J. Pharmacol. 283 Ž1995. 151–159. w8x G.F. Koob, P.P. Sanna, F.E. Bloom, Neurobiology of Drug addiction, Neuron, in press. w9x J.E. Krettek, J.L. Price, Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat, J. Comp. Neurol. 178 Ž1978. 225–254. w10x M. Morales, F.E. Bloom, The 5-HT 3 receptor is present in different subpopulations of GABAergic neurons in the rat telencephalon, J. Neuroscience 17 Ž1997. 3157–3167. w11x O.T. Phillipson, Afferent projections to the ventral tegmental area of Tsai and intrafascicular nucleus. A horseradish peroxidase study in the rat, J. Comp. Neurol. 187 Ž1979. 117–143. w12x E.M. Pich, M. Lorang, M. Yeganeh, F.F. Rodriguez, J. Raber, G.F. Koob, F. Weiss, Increase of extracellular corticotropin-releasing factor-like immunoreactivity levels in the amygdala of awake rats during restraint stress and ethanol withdrawal as measured by microdialysis, J. Neuroscience 15 Ž1995. 5439–5447. w13x A. Pitkdnen, D.G. Amaral, The distribution of GABAergic cells, fibers and terminals in the monkey amygdaloid complex: An immunohistochemical and in situ hybridization study, J. Neuroscience 14 Ž1993. 2200–2224. w14x S. Rassnick, S.C. Heinrichs, K.T. Britton, G.F. Koob, Microinjection of a corticotropin-releasing factor antagonist into the central nucleus of the amygdala reverses anxiogenic-like effects of ethanol withdrawal, Brain. Res. 605 Ž1993. 25–32. w15x G.W. Roberts, P.L. Woodhams, J.M. Polak, T.J. Crow, Distribution of neuropeptides in the limbic system of the rat: The amygdaloid complex, Neuroscience 7 Ž1982. 99–131.
336
M. Morales et al.r Brain Research 798 (1998) 333–336
w16x G.W. Roberts, Neuropeptides: Cellular morphology, major pathways, and functional considerations, in: J.P. Aggleton ŽEd.., The Amygdala. Neurobiological Aspects of Emotion, Memory and Mental Dysfunction, Wiley-Liss, Chichester, 1992, pp. 115–142. w17x A.J. Roberts, M. Cole, G.F. Koob, Intra-amygdala muscimol decreases operant ethanol self-administration in dependent rats, Alcohol Clin. Exp. Res. 20 Ž1996. 1289–1298. w18x A.E. Ryabinin, J.R. Criado, S.J. Henriksen, F.E. Bloom, M.C. Wilson, Differential sensitivity of c-Fos expression in hippocampus and other brain regions to moderate and low doses of alcohol, Molecular Psychiatry 2 Ž1. Ž1997. 32–43. w19x S.M. Sagar, F.R. Sharp, T. Curran, Expression of c-fos protein in brain: Metabolic mapping at the cellular level, Science 240 Ž1988. 1328–1331. w20x M. Sheng, M.E. Greenberg, The regulation and function of c-fos and other immediate early genes in the nervous system, Neuron 4 Ž1990. 477–485.
w21x S. Shiosaka, M. Tokyama, H. Takagi, Y. Takahashi, T. Saitoh, H. Sakumoto, H. Nakagawa, N. Shimizu, Ascending and descending components of the medial forebrain bundle in the rat as demonstrated by the horseradish peroxidase-blue reaction. I. Forebrain and upper brainstem, Exp. Brain Res. 39 Ž1980. 377–388. w22x N. Sun, M.D. Cassell, Intrinsic GABAergic neurons in the rat central extended amygdala, J. Comp. Neurol. 330 Ž1993. 381–404. w23x J.G. Veening, L.W. Swanson, P.E. Sawchenko, The organization of projections from the central nucleus of the amygdala to brainstem sites involved in central autonomic regulation. A combined retrograde transport-immunohistochemical study, Brain Res. 303 Ž1984. 337–357. w24x D.M. Wallace, D.J. Magnuson, T.S. Gray, The amygdalo-brainstem pathway: dopaminergic, noradrenergic and adrenergic cells in the rat, Neurosci. Lett. 97 Ž1989. 252–258.