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Alterations of corticotropin-releasing factor-like immunoreactivity in different brain regions after acute cocaine administration in rats
315
Brain Research, 616 (1993) 315-319 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25708
Alterations of corticotropin-releasing factor-like immunoreactivity in different brain regions after acute cocaine administration in rats Zoltfin Sarnyai
a,,
I~va Bir6
a Jfinos
Gardi b Mikl6s Vecserny6s and Gyula Telegdy a
b Jfinos J u l e s z
b
a Department of Pathophysiology and b Endocrine Unit of First Department of Medicine, Albert Szent-Gy6rgyi Medical University, Szeged (Hungary) (Accepted 16 March 1993)
Key words: Cocaine; Corticotropin-releasing factor; Hypothalamus; Extrahypothalamic-limbic brain region; Radioimmunoassay
Corticotropin-releasing factor (CRF) may mediate some of the neuroendocrine and behavioral responses to cocaine. In this study, the distribution of CRF-like immunoreactivity (CRF-LI) was determined in the hypothalamus and in several extrahypothalamic brain regions after acute cocaine administration in handled rats. CRF-LI decreased dose-dependently with cocaine administration in the hypothalamus and in the basal-forebrain structures. A small dose of cocaine (7.5 mg/kg) decreased CRF-LI in the hippocampus and in the frontal cortex. A significant, selective, dose-dependent increase in CRF-LI was found in the amygdala after cocaine injection. None of the investigated doses of cocaine altered CRF-LI in the striatum. These results suggest that acute cocaine administration alters brain CRF systems to contribute behavioral and neuroendocrine responses to cocaine.
Several lines of evidence suggest that the activation of endogenous CRF may contribute to the behavioral and neuroendocrine effects of cocaine. CRF and cocaine have many similarities in their actions, for example, both CRF (see refs. 4 and 22 for reviews) and c o c a i n e 9'15'24-26'28'31"32 activate the hypothalamic-pituitary-adrenal (HPA) system to secrete ACTH and corticosterone, induce locomotor activity and stereotyped behavior, increase neuronal firing activity ultimately producing epileptiform seizures, decrease pain sensitivity, food intake, sexual activity and suppress cellular immune functions. 'Anxiety'-like behavior measured by both a defensive withdrawal test and an elevated plus maze has been observed with CRF and cocaine administration in rats 4'33. Increased activity of the brain CRF system may be involved in the pathophysiology of anxiety, depression and panic attack 2t, which are the main psychiatric consequences of chronic cocaine abuse and withdrawal in humans 5. Although these similarities do not necessarily indicate common functional mechanisms, direct involve-
ment of endogenous CRF has been demonstrated recently. Cocaine-induced ACTH and corticosterone secretion were inhibited by peripheral administration of CRF-antiserum 26. Central (intracerebroventricular, i.c.v.) injections of both CRF-antiserum and a CRF-receptor antagonist, a-helical-CRF9_41, were able to block a cocaine-induced corticosterone response 28. Locomotor hyperactivity induced by cocaine was blocked by i.c.v, administration of CRF-antiserum and by CRF-receptor antagonist, dose-dependently 3°. These data strongly suggest the critical involvement of endogenous CRF in cocaine-induced behavioral and neuroendocrine responses. Only one study demonstrated the direct stimulatory effect of cocaine on CRF release from the hypothalamus, in vitro t. We were not able to locate any data about the effects of cocaine on CRF levels in the hypothalamus and in extrahypothalamic brain regions, in vivo. The purpose of the present study was to demonstrate the effects of the acute administration of differ-
Correspondence: Z. Sarnyai, M.D. Alcohol and Drug Abuse Research Center, Harvard Medical School-McLean Hospital, 115 Mill Street, Belmont, MA 02178, USA. Fax: (1) (617) 855 2519. * Present address: Alcohol and Drug Abuse Research Center, Harvard Medical School-McLean Hospital, 115 Mill Street, Belmont, MA 02178, USA.
316 ent doses of cocaine on CRF-LI measured in the hypothalamus and in several extrahypothalamic regions, including the hippocampus, amygdala, basalforebrain structures (tuberculum olfactorium, nucleus accumbens and septal area), frontal cortex and striatum in rats. These structures have been chosen on the basis of their possible involvement in the mediation of the behavioral and neuroendocrine actions of CRF 4. Furthermore, these regions contain immunoreactive CRF, CRF cell bodies, fibers or receptors 22. Adult male Wistar rats (LATI, G6d6116, Hungary) weighing 180-220 g were used. They were housed 5 per cage with food and water available ad lib±turn in an environmentally controlled animal facility (12 h light/dark cycles with lights on at 06.00 h). Rats were handled daily (5 to 10 min) for 6 consecutive days before the experiment to accustom them to the presence of humans and to minimize the effects of nonspecific stress. Animals were sacrificed 30 min after the intraperitoneal (i.p.) injection of saline (0.9% NaCI) and of different doses of cocaine (7.5, 15 and 30 mg/kg) in a separate room. This time lag was chosen, since both the maximal behavioral and HPA axis activating effects of cocaine have been measured at this point in time 28'3°. After decapitation, the brains were quickly removed and various brain regions were isolated from the both hemispheres on ice by a modification of the technique of Glowinski and Iversen 6. Briefly, the hypothalamus was defined as tissue within 3 mm of the ventral surface of the brain within the following borders: the anterior extreme was the optic chiasm; posterior extreme was the mamillary bodies; lateral extremes were the lateral hypothalamic sulci. The hippocampus was removed as a whole. The striatum (caudate-putamen) was defined medially by the lateral
ventricles and laterally by the corpus callosum. The amygdaloid complex was dissected from below the base of the caudate-putamen excluding the adjacent cortical surface. The anterior and posterior borders of the amygdaloid complex were defined by the genu of the corpus callosum and the optic chiasm, respectively. The basal forebrain area, containing the tuberculum olfactorium, the nucleus accumbens and the septal area, were removed from the anterior-ventral part of the telencephalon as a trapezoid form tissue slice by razor cuts. This complex was bordered by the frontal cortex, anteriorly and by a razor cut at the level of the anterior commissure, posteriorly. The dorsal border of the basal forebrain complex was the corpus callosum and the lateral borders were running along with the internal walls of the lateral ventricles laterally to the ventral surface of the anterior telencephalon. Frontal cortex was obtained by a razor cut at the level of the genu of the corpus callosum. For the radioimmunoassay (RIA) to determine CRF-LI, each area was homogenized with ultrasound (Son±prep 150 MSE, Great Britain) in HCI (100 mM) containing 1 mM ascorbic acid and an aliquot was sampled for protein measurement 17. The residual homogenates were centrifuged at 6,000 × g for 20 min, 4°C, aliquots were taken and liophilized for RIA. The CRF antiserum (kindly donated by Paul Vecsei, Dept. of Pharmacology, University of Heidelberg, Germany) was obtained from a rabbit immunized with hCRF. The CRF antibody 18 was specific for the C-terminal region of CRF41 molecule since it did not cross react with fragments of CRFI_20 and CRF6_33. The CRF tracer was prepared by using a modified iodogen method in order to minimize the demage of the iodinated peptide 16. The labeled material was purified via two steps of reverse
TABLE I
Effects of cocaine on corticotropin-releasing factor-like immunoreacticity (CRF-L1 pg / mg proteine ± S.E.M.) in different rat brain regions Each g r o u p c o n t a i n s 8 - 1 0 animals. N u m b e r s in p a r e n t h e s i s r e p r e s e n t the c o c a i n e - i n d u c e d c h a n g e s of C R F - L I in p e r c e n t a g e of s a l i n e - t r e a t e d (0 m g / k g of cocaine) control.
Brain regions
Cocaine (rng / kg body weight) 0
7.5
15
30
Hypothalamus
2 427 + 711
Basal forebrain
593.7 + 42
866.4 ± 156 * ( - 64%) 294.5 + 25 * ( - 50%) 12.7 ± 3.1 * ( - 52%) 22.8 ± 3.4 * (-40%) 1 5 1 8 ± 185 * ( + 229%) 25.3 ± 2.7 ( - 7%)
648 ± 131 * ( - 73%) 273.3 + 28 * ( - 54%) 19.8 _+ 1.7 ( - 27%) 31.8 ± 2 ( - 16%) 2559±311 * ( + 455%) 32.5 ± 7 ( + 20%)
624 + 75 * ( - 74%) 188 _+ 22 * ( - 68%) 19.5 ± 2 ( - 27%) 31.8 ± 2 ( - 16%) 2785±597 * ( + 504%) 40 ± 4 ( + 4O%)
Hippocampus
26.8 ± 3.3
Frontal cortex
37.7 ± 4
Amygdala
461 ± 8 4
Striatum
27 ± 4
* P < 0.05 vs. control (0 m g / k g of cocaine) by A N O V A followed by D u n n e t t ' s test.
317 phase chromatography 11, applying a gradient HPLC system in the second step. The specific radioactivity of the purified tracer was 1700-1900 Ci/mmol. The freeze-dried residues were redissolved in a 1 ml assay buffer (50 mM phosphate, pH 7.4, containing 0.25% human serum albumin, 0.1% Triton X-100) and 200 ml aliquots were subjected to RIA. The assay standard was a synthetic h / r C R F preparation (Bachem, Budendorf, Switzerland). The procedure involved a nonequilibrium system: a 16 h preincubation of the samples or standards with antiserum (100 /xl, working dilution 1:10,000 was followed by a 24 h incubation with CRF tracer 100/xl, 10,000 cpm). The immunologically bound and free fractions were separated with a second antibody (raised in sheep against whole rabbit IgG in our laboratory) and subsequently with polyethylenglycol 6000 precipitation (FERAK Laborat. Gmbh, Berlin, Germany) by the D A B / P E G method. The lower limit of assay detection was 7-8 pg/tube. The intra- and interassay coefficients of variation were 4.0 and 13.8%, respectively. CRF immunoreactivity in the brain extracts subjected to HPLC has been shown to cocromatograph with synthetic r / h CRFI_41 (Gardi et al., unpublished observation). The CRF-LI is expressed in pg/mg protein. All data are presented as mean _+S.E.M. Data were analyzed by one-way ANOVA followed by Dunnett's test. Probability level of 0.05 was accepted as indicating statistically significant difference. As is shown in Table I, acute administration of cocaine resulted in a significant, dose-dependent decrease in CRF-LI in the hypothalamus (F3,30 = 6.66, P < 0.002; 7.5 mg/kg cocaine: P < 0.05 vs. saline, -64%; 15 mg/kg cocaine: P < 0.05 vs. saline, - 7 3 % and 30 mg/kg cocaine: P < 0.05 vs. saline, - 74%) and in the basal forebrain (F3,29--18.09, P < 0.0001; 7.5 mg/kg cocaine: P < 0.05 vs. saline, -50%; 15 mg/kg cocaine: P < 0.05 vs. saline, - 5 0 % and 30 mg/kg cocaine: P < 0.05 vs. saline, - 68%). 7.5 mg/kg dose of cocaine decreased the CRF-LI in the hippocampus (F3.33 = 5.47, P < 0.005; - 52%) and in the frontal cortex (Faro = 4.54, P < 0.01; -40%). Cocaine administration increased CRF-LI in the amygdala, dose-dependently (F3,32 = 8.41, P < 0.0005; 7.5 mg/kg cocaine: P < 0.05 vs. saline, + 229%; 15 mg/kg cocaine: P < 0.05 vs. saline, + 455% and 30 mg/kg cocaine: P < 0.05 vs. saline, +504%). Striatal CRF-LI not altered significantly with cocaine administration (F3,30 = 2.40, p = 0.09; - 7%, + 20% and + 40%, respectively, p > 0.05 vs. saline). The results of the present study with regard to the distribution of basal CRF-LI in saline-treated, nonstressed, handled rats are similar, but not identical to
previous RIA studies 2'm. Chappell et al. 2 measured CRF-LI by using antisera against ovine CRF in rat brain nuclei, dissected out according to Palkovits and Brownstein's micropunch method 23. They have found 5054 + 674 pg CRF-LI/mg protein in the median eminence/arcuate nucleus complex. Measurements in several other hypothalamic nuclei equaled approximately 1200 pg CRF-L1/mg protein. The hypothalamus of the saline-treated control rats contained 2427 + 711 pg/mg CRF-LI in our present study. In limbic forebrain structures, tuberculum olfactorium, septum (lateral and medial) and nucleus accumbens, approximately 350 pg CRF-LI/mg protein were detected. In the present study the CRF-LI in basal forebrain, containing tuberculum olfactorium, medial septum and nucleus accumbens, was approximately 600 pg/mg protein. Chappell et al. 2 reported approximately 600 pg CRF-LI/mg protein as a sum of the results of distinct nuclei of the amygdala, which was 461 _+84 pg/mg protein in the whole amygdaloid complex in the present experiment. There were 31 and 34 pg/mg protein CRF-LI in the ventral and dorsal hippocampus, respectively, as measured by Chappell et al. 2, while we measured 26.8 + 3.3 pg CRF-LI/mg protein from the whole hippocampus. They found 22 pg/mg protein CRF-LI in the medial prefrontal cortex, while in our present experiment 37.7 pg/mg protein CRF-LI were detected in the frontal cortex. More recently, Inoue et al. 1° determined CRFLI in the rat brain dissected out according to Palkovits and Brownstein 23 by using a double antibody RIA specific for rat CRF. Levels of CRF-LI in the hypothalamus, basal forebrain (septum and nucleus accumbens) and in the amygdala of the control, non-stressed animals were very close to our present data. Frontal cortex CRF-LI has been reported to be much higher compared to data from the same structure presented here and by others 2. Such differences in CRF-LI obtained by different experiments may be due to differences in strains of animal, dissection techniques, antisera and RIA procedures used. Several lines of evidence suggest that stress and psychostimulants may interact in the central nervous system. Daily cocaine injection and stress exposure produce similar effects on mesocorticolimbic dopamine neurotransmission m. Both stress and cocaine activate the release of stress hormones, ACTH and CORT, through CRF 4'26'28. Stress m and CRF (Sarnyai et al., unpublished observation) potentiate the cocaine-induced stereotyped behavior in rats. Stress activates the hypothalamic and extrahypothalamic CRF in the rat brain. It was demonstrated that the acute stress increases the synthesis and release of CRF in the hypothalamus 8. Our report of dose-dependent decrease of
318 CRF-LI in the hypothalamus after acute cocaine administration supports the hypothesis of the stress-like effect of cocaine on the H P A axis. Effects of acute stress on CRF-LI in the extrahypothalamic brain regions are much less consistent 2'm'2°. Chappell et al. 2 found an increase in CRF-LI in the locus coeruleus, but not in other limbic and midbrain regions after acute immobilization and cold stress. Inoue et al. m demonstrated that single immobilization stress did not change CRF-LI in discrete brain regions. In contrast, it was demonstrated recently by in vivo microdialysis that acute immobilization stress produces a six-fold release of CRF from the amygdala in freely moving rats 2°. Our present findings show a pronounced decrease of CRF-LI in several extrahypothalamic regions, such as basal forebrain, hippocampus and frontal cortex. A dramatic, dose-dependent increase in CRF concentration was also demonstrated in the amygdala. One possible explanation of these inconsistencies between the effects of stress and cocaine on brain CRF could be that stress may activate some divergent neurochemical mechanisms (e.g. norepinephrine, dopamine, endogenous opiates or GABA) unspecifically, which can increase or decrease the functional activity of brain CRF producing no overall changes in CRF levels. In contrast, cocaine could act as a specific pharmacological stressor activating well-defined neurochemical pathways (DA-ergic, NE-ergic and 5-HT-ergic) which are important in the stimulation of CRF secretion. Present results show that acute cocaine administration decreased the levels of CRF-LI in the hypothalamus, basal forebrain, hippocampus and frontal cortex. Although, the measurement of peptide concentration alone cannot distinguish between synthesis, release or degradation, a decrease in the immunoreactive peptide level in a certain brain area usually reflects an increased release and subsequent degradation of the neuropeptide 29. Our present data may have some functional implications in terms of the mediation of the central nervous system effects of cocaine. Cocaine-induced reduction of hypothalamic CRF-LI was probably due to the release of CRF into the hypothalamic-pituitary portal circulation which activates the pituitaryadrenal axis. This interpretation is supported by the data that cocaine stimulates A C T H and corticosterone secretion in the same doses and time interval that have been reported previously 26'28. Furthermore, in vitro experiments on a hypothalamic slice also demonstrated an increased release of CRF induced by cocaine ~. Thus we can hypothesize that the CRF released from the hypothalamus may be at least one of the major endogenous substrates which can mediate cocaine's action on the HPA axis. Acute cocaine administration produced
a significant reduction in CRF-LI in the basal forebrain structures and in the hippocampus. Increased locomotor activity has been demonstrated by local microinfusion of both CRF and cocaine into the basal forebrain. Bilateral microinjection of CRF into the hippocampus also produced an intense locomotor activity 13. Cocaine-induced locomotor hyperactivity was either inhibited or ultimately completely abolished by different doses of CRF-antiserum and CRF-receptor antagonist 3°. It could be hypothesized that CRF release in limbic-basal forebrain structures may contribute to the expression of cocaine-induced locomotor response. CRF-LI was also decreased by cocaine in the frontal cortex. CRF receptor labeling 7 and CRF-L127 were decreased in the rat frontal cortex after chronic cocaine administration leading to anxiety in both experimental animals 27 and humans 5. This raises the possibility that endogenous CRF in the frontal cortex may be implicated in cocaine-induced emotional changes. The most dramatic changes in CRF-LI were detected in the amygdala after acute cocaine treatment. The amygdala has a critical importance in the development of fear and anxiety. CRF produces a behavioral state that resembles fear or anxiety 3. Lesion of central nucleus of the amygdala blocked this effect of CRF indicating that both the amygdala and CRF are critically involved in fear and anxiety 14. Cocaine also induces the behavioral symptoms of anxiety in rats 33. Anxiety and depression are the most important psychiatric consequences of chronic use a n d / o r withdrawal of cocaine in humans 5. Our present data on the cocaine-induced activation of intra-amygdaloid CRF, in lights of the behavioral evidences mentioned above, raise the possibility that CRF release in the amygdala could be an important mediator of the neurobiological processes of cocaine-induced anxiety and depression. In conclusion, this is the first report of the differential action of cocaine on CRF-LI in different brain structures of the rat. Cocaine decreases the CRF-LI in the hypothalamus, in the basal-forebrain structures, in the hippocampus and in the frontal cortex, whereas in the amygdala an increase in CRF-LI is found. These data suggest that cocaine activates brain CRF systems which may be one of the important endogenous substrates to mediate neuroendocrine, behavioral and emotional responses to cocaine.
1 Calogero, A.E., Gallucci, W.T., Kling, M.A., Chrousos, G.P. and Gold, P.W., Cocaine stimulates rat hypothalamiccorticotropin-releasing hormone secretion in vitro, Brain Res., 505 (1989) 7-11. 2 Chappell, P.B., Smith, M.A., Kilts, C.D., Bisette, G., Ritchie, J., Anderson, C and Nemeroff, C.B., Alterations in corticotropin-releasing factor-like immunoreactivityin discrete rat brain regions after acute and chronic stress. J. Neurosci., 6 (1986) 2908-2914.
319 3 Davis, M., The role of the amygdala in conditioned fear. In: J. Aggleton (Ed.), The amygdala: neurobiological aspects of emotion, memory and mental dysfunction, Wiley, New York, 1992, pp. 255-305. 4 Dunn, A.J. and Berridge, C.W., Physiological and behavioral responses to corticotropin-releasing factor administration: is CRF a mediator of anxiety or stress responses?, Brain Res. Rev., 15 (1990) 71-100. 5 Gawin, F.H. and Ellinwood, E.H., Cocaine dependence, Annu. ReL'. Med., 40 (1989) 149-161. 6 Glowinski, J. and Iversen, L.L., Regional studies of catecholamines in the rat brain - I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in variopus regions of the brain. J. Neurochem., 13 (1966) 655-669. 7 Goeders, N.E., Bienvenu, O.J. and De Sousa, E.B., Chronic cocaine administration alters corticotropin-releasing factor receptors in the rat brain, Brain Res., 531 (1990) 322-328. 8 Haas, D. and George S.R., Single and repeated mild stress increases synthesis and release of hypothalamic corticotropin-releasing factor. Brain Res. 461 (1988) 230-237. 9 Heffner, T.G., Zigmond, M.J. and Stricker, E.M., Effects of dopaminergic agonists and antagonists of feeding in intact and 6-hydroxydopamine-treated rats, J. Pharmacol. Exp. Ther., 201 (1977) 386-399. 10 Inoue, T., Koyama, T., Muraki, A., Yamashita, I., Effects of single and repeated immobilization stress on corticotropin-releasing factor concentration in discrete rat brain regions. Prog. Neuro-Psychopharmacol. BioL Psychiat. 17 (1993) 161-170. 11 Jan~iky, T., T6th, G., Penke, B., Kov~cs, K. and L~iszl6, F.A., lodination of peptide hormones and purification of iodinated peptides, J. Liquid Chromatography, 5 (1982) 1499-1507. 12 Kalivas, P.W. and Duffy, P., Similar effects of daily cocaine and stress on mesocorticolimbic dopamine neurotransmission in the rat. Biol. Psychiatry, 25 (1989) 913-928. 13 Lee, E.H.Y. and Tsai, M.J., The hippocampus and amygdala mediate the locomotor stimulating effects of corticotropin-releasing factor in mice. Behav. Neural Biol., 51 (1989) 412-423. 14 Liang, K.C., Melia, K.R., Campeau, S., Falls, W.A., Miserendino, M.J.D.and Davis, M., Lesions of the central nucleus of the amygdala, but not the paraventricular nucleus of the hypothalamus, block the excitatory effects of corticotropin-releasing factor on the acoustic startle reflex, J. Neurosci., 12 (1992) 2313-2320. 15 Lin, Y., Morrow, T.J., Kiritsy-Roy, J.A., Terry. L.C. and Casey, K.L., Cocaine: evidence for supraspinal, non-opiate analgesia, Brain Res, 479 (1989) 306-312. 16 Linton, E.A. and Lowry, P.J., Radioimmunoassay and chromatographic characterization of CRF-41-1ike immunoreactivity in hypothalami from several species. Neuropeptides, 3 (1982) 45-52. 17 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent. J. BioL Chem., 193 (1951) 265-275. 18 Maser-Gluth, C. and Vecsei, P., Corticotropin-releasing factorlike immunoreactivity in human 24 h urine. Clin Endocrinol., 30 (1989) 405-412. 19 MacLennan, A.J. and Maier, S.F., Coping and the stress-induced potentiation of stimulant stereotypy in the rat. Science, 219 (1983) 1091-1093.
20 Merlo-Pich, E., Koob, G.F., Sattler, S.C., Menzaghi, F., Heilig, M., Heinrichs, S.C., Vale, W., Weiss, F., Stress-induced release of corticotropin-releasing factor in the amygdala measured by in vivo microdialysis, Soc. Neurosci. Abstr., 18 (1992) 225.17. 21 Nemeroff, C.B., New vistas in neuropeptide research in neuropsychiatry: focus on corticotropin-releasing factor, Neuropsychopharmacology, 6 (1992) 69-75. 22 Owens, M.J, and Nemeroff, C.B., Physiology and pharmacology of corticotropin-releasing factor, Pharmacol. Rev., 43 (1991) 425473. 23 Palkovits, M. and Brownstein, M.J., Microdissection of brain areas by the punch technique. In A.C. Cuello, (Ed.), Brain Microdissection Techniques, Wiley, New York, 1983, pp. 1-36. 24 Post, R.M., Weiss, S.R.B., Pert, A. and Uhde, T.W., Chronic cocaine administration: sensitization and kindling effects. In S. Fischer, A. Raskin and E.H. Uhlenhuth (Eds.) Cocaine: Clinical and Biobehavioral Aspects, Oxford University Press, New York, 1987, pp. 109-173. 25 Raum, W.J., McGivern, R.F., Peterson, M.A., Shryne, J.H. and Gorski, R.A., Prenatal inhibition of hypothalamic sexual differentiation by cocaine: effects on neurobehavioral sexual differentiaton in male rats, Dev. Brain Res., 53 (1990) 230-236. 26 Rivier, C. and Vale, W., Cocaine stimulates adrenocorticotropin (ACTH) secretion throuhg a corticotropin-releasing factor (CRF)-mediated mechanism, Brain Res., 422 (1987) 403-406. 27 Sarnyai, Z., B~r6, t~., Gardi, J., Vecserny~s, M., Julesz, J., Penke, B. and Telegdy, G., Corticotropin-releasing factor (CRF) as an endogenous mediator of the behavioral and neuroendocrine actions of cocaine in rats., Abstracts of the Hans Selye Symposia on Neuroendocrinology and Stress, CRF and Cytokines: [{ole in Stress Response. Montreal, Canada, 1992, p. 13. 28 Sarnyai, Z., Bir6, E., Penke, B. and Telegdy, G., The cocaine-induced elevation of plasma corticosterone is mediated by endogenous corticotropin-releasing factor (CRF) in rats, Brain Res., 589 (1992) 154-156. 29 Sarnyai, Z., B~r6, IE., Vecserny~s, M., Laczi, F., Szab6, G. and Kov~cs, G.L., Effects of cocaine on the contents of neurohypophyseal hormones in the plasma and in different brain structures in rats. Neuropeptides, 23 (1992) 27-31. 30 Sarnyai, Z., H6hn, J., Szab6, G. and Penke, B., Critical role of endogenous corticotropin-releasing factor (CRF) in the mediation of the behavioral action of cocaine in rats. Life Sci., 51 (1992) 2019-2024. 31 Scheel-Kriiger, J., Baestrup, C., Nielson, M., Golembiowska, K. and Mogilnicka, F., Cocaine: discussion on the role of dopamine in the biochemical mechanism of action. In H.H. Ellinwood and M.M. Kilbey (Eds.), Advances in Behavioral Biology, Vol. 21, Cocaine and Other Stimulants, Plenum Press, New York, NY, 1977, pp. 373-408. 32 Watzl, B. and Watson, R.R., Minireview: Immunomodulation by cocaine-A neuroendocrine mediated response, Life Sci., 46 (1990) 1319-1329. 33 Yang, X.M., Gorman, A.L., Dunn, A.J. and Goeders, N.E., Anxiogenic effects of acute and chronic cocaine administration: neurochemical and behavioral studies, Pharmacol. Biochem. Behay., 41 (1992) 643-650.
Brain Research, 616 (1993) 315-319 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25708
Alterations of corticotropin-releasing factor-like immunoreactivity in different brain regions after acute cocaine administration in rats Zoltfin Sarnyai
a,,
I~va Bir6
a Jfinos
Gardi b Mikl6s Vecserny6s and Gyula Telegdy a
b Jfinos J u l e s z
b
a Department of Pathophysiology and b Endocrine Unit of First Department of Medicine, Albert Szent-Gy6rgyi Medical University, Szeged (Hungary) (Accepted 16 March 1993)
Key words: Cocaine; Corticotropin-releasing factor; Hypothalamus; Extrahypothalamic-limbic brain region; Radioimmunoassay
Corticotropin-releasing factor (CRF) may mediate some of the neuroendocrine and behavioral responses to cocaine. In this study, the distribution of CRF-like immunoreactivity (CRF-LI) was determined in the hypothalamus and in several extrahypothalamic brain regions after acute cocaine administration in handled rats. CRF-LI decreased dose-dependently with cocaine administration in the hypothalamus and in the basal-forebrain structures. A small dose of cocaine (7.5 mg/kg) decreased CRF-LI in the hippocampus and in the frontal cortex. A significant, selective, dose-dependent increase in CRF-LI was found in the amygdala after cocaine injection. None of the investigated doses of cocaine altered CRF-LI in the striatum. These results suggest that acute cocaine administration alters brain CRF systems to contribute behavioral and neuroendocrine responses to cocaine.
Several lines of evidence suggest that the activation of endogenous CRF may contribute to the behavioral and neuroendocrine effects of cocaine. CRF and cocaine have many similarities in their actions, for example, both CRF (see refs. 4 and 22 for reviews) and c o c a i n e 9'15'24-26'28'31"32 activate the hypothalamic-pituitary-adrenal (HPA) system to secrete ACTH and corticosterone, induce locomotor activity and stereotyped behavior, increase neuronal firing activity ultimately producing epileptiform seizures, decrease pain sensitivity, food intake, sexual activity and suppress cellular immune functions. 'Anxiety'-like behavior measured by both a defensive withdrawal test and an elevated plus maze has been observed with CRF and cocaine administration in rats 4'33. Increased activity of the brain CRF system may be involved in the pathophysiology of anxiety, depression and panic attack 2t, which are the main psychiatric consequences of chronic cocaine abuse and withdrawal in humans 5. Although these similarities do not necessarily indicate common functional mechanisms, direct involve-
ment of endogenous CRF has been demonstrated recently. Cocaine-induced ACTH and corticosterone secretion were inhibited by peripheral administration of CRF-antiserum 26. Central (intracerebroventricular, i.c.v.) injections of both CRF-antiserum and a CRF-receptor antagonist, a-helical-CRF9_41, were able to block a cocaine-induced corticosterone response 28. Locomotor hyperactivity induced by cocaine was blocked by i.c.v, administration of CRF-antiserum and by CRF-receptor antagonist, dose-dependently 3°. These data strongly suggest the critical involvement of endogenous CRF in cocaine-induced behavioral and neuroendocrine responses. Only one study demonstrated the direct stimulatory effect of cocaine on CRF release from the hypothalamus, in vitro t. We were not able to locate any data about the effects of cocaine on CRF levels in the hypothalamus and in extrahypothalamic brain regions, in vivo. The purpose of the present study was to demonstrate the effects of the acute administration of differ-
Correspondence: Z. Sarnyai, M.D. Alcohol and Drug Abuse Research Center, Harvard Medical School-McLean Hospital, 115 Mill Street, Belmont, MA 02178, USA. Fax: (1) (617) 855 2519. * Present address: Alcohol and Drug Abuse Research Center, Harvard Medical School-McLean Hospital, 115 Mill Street, Belmont, MA 02178, USA.
316 ent doses of cocaine on CRF-LI measured in the hypothalamus and in several extrahypothalamic regions, including the hippocampus, amygdala, basalforebrain structures (tuberculum olfactorium, nucleus accumbens and septal area), frontal cortex and striatum in rats. These structures have been chosen on the basis of their possible involvement in the mediation of the behavioral and neuroendocrine actions of CRF 4. Furthermore, these regions contain immunoreactive CRF, CRF cell bodies, fibers or receptors 22. Adult male Wistar rats (LATI, G6d6116, Hungary) weighing 180-220 g were used. They were housed 5 per cage with food and water available ad lib±turn in an environmentally controlled animal facility (12 h light/dark cycles with lights on at 06.00 h). Rats were handled daily (5 to 10 min) for 6 consecutive days before the experiment to accustom them to the presence of humans and to minimize the effects of nonspecific stress. Animals were sacrificed 30 min after the intraperitoneal (i.p.) injection of saline (0.9% NaCI) and of different doses of cocaine (7.5, 15 and 30 mg/kg) in a separate room. This time lag was chosen, since both the maximal behavioral and HPA axis activating effects of cocaine have been measured at this point in time 28'3°. After decapitation, the brains were quickly removed and various brain regions were isolated from the both hemispheres on ice by a modification of the technique of Glowinski and Iversen 6. Briefly, the hypothalamus was defined as tissue within 3 mm of the ventral surface of the brain within the following borders: the anterior extreme was the optic chiasm; posterior extreme was the mamillary bodies; lateral extremes were the lateral hypothalamic sulci. The hippocampus was removed as a whole. The striatum (caudate-putamen) was defined medially by the lateral
ventricles and laterally by the corpus callosum. The amygdaloid complex was dissected from below the base of the caudate-putamen excluding the adjacent cortical surface. The anterior and posterior borders of the amygdaloid complex were defined by the genu of the corpus callosum and the optic chiasm, respectively. The basal forebrain area, containing the tuberculum olfactorium, the nucleus accumbens and the septal area, were removed from the anterior-ventral part of the telencephalon as a trapezoid form tissue slice by razor cuts. This complex was bordered by the frontal cortex, anteriorly and by a razor cut at the level of the anterior commissure, posteriorly. The dorsal border of the basal forebrain complex was the corpus callosum and the lateral borders were running along with the internal walls of the lateral ventricles laterally to the ventral surface of the anterior telencephalon. Frontal cortex was obtained by a razor cut at the level of the genu of the corpus callosum. For the radioimmunoassay (RIA) to determine CRF-LI, each area was homogenized with ultrasound (Son±prep 150 MSE, Great Britain) in HCI (100 mM) containing 1 mM ascorbic acid and an aliquot was sampled for protein measurement 17. The residual homogenates were centrifuged at 6,000 × g for 20 min, 4°C, aliquots were taken and liophilized for RIA. The CRF antiserum (kindly donated by Paul Vecsei, Dept. of Pharmacology, University of Heidelberg, Germany) was obtained from a rabbit immunized with hCRF. The CRF antibody 18 was specific for the C-terminal region of CRF41 molecule since it did not cross react with fragments of CRFI_20 and CRF6_33. The CRF tracer was prepared by using a modified iodogen method in order to minimize the demage of the iodinated peptide 16. The labeled material was purified via two steps of reverse
TABLE I
Effects of cocaine on corticotropin-releasing factor-like immunoreacticity (CRF-L1 pg / mg proteine ± S.E.M.) in different rat brain regions Each g r o u p c o n t a i n s 8 - 1 0 animals. N u m b e r s in p a r e n t h e s i s r e p r e s e n t the c o c a i n e - i n d u c e d c h a n g e s of C R F - L I in p e r c e n t a g e of s a l i n e - t r e a t e d (0 m g / k g of cocaine) control.
Brain regions
Cocaine (rng / kg body weight) 0
7.5
15
30
Hypothalamus
2 427 + 711
Basal forebrain
593.7 + 42
866.4 ± 156 * ( - 64%) 294.5 + 25 * ( - 50%) 12.7 ± 3.1 * ( - 52%) 22.8 ± 3.4 * (-40%) 1 5 1 8 ± 185 * ( + 229%) 25.3 ± 2.7 ( - 7%)
648 ± 131 * ( - 73%) 273.3 + 28 * ( - 54%) 19.8 _+ 1.7 ( - 27%) 31.8 ± 2 ( - 16%) 2559±311 * ( + 455%) 32.5 ± 7 ( + 20%)
624 + 75 * ( - 74%) 188 _+ 22 * ( - 68%) 19.5 ± 2 ( - 27%) 31.8 ± 2 ( - 16%) 2785±597 * ( + 504%) 40 ± 4 ( + 4O%)
Hippocampus
26.8 ± 3.3
Frontal cortex
37.7 ± 4
Amygdala
461 ± 8 4
Striatum
27 ± 4
* P < 0.05 vs. control (0 m g / k g of cocaine) by A N O V A followed by D u n n e t t ' s test.
317 phase chromatography 11, applying a gradient HPLC system in the second step. The specific radioactivity of the purified tracer was 1700-1900 Ci/mmol. The freeze-dried residues were redissolved in a 1 ml assay buffer (50 mM phosphate, pH 7.4, containing 0.25% human serum albumin, 0.1% Triton X-100) and 200 ml aliquots were subjected to RIA. The assay standard was a synthetic h / r C R F preparation (Bachem, Budendorf, Switzerland). The procedure involved a nonequilibrium system: a 16 h preincubation of the samples or standards with antiserum (100 /xl, working dilution 1:10,000 was followed by a 24 h incubation with CRF tracer 100/xl, 10,000 cpm). The immunologically bound and free fractions were separated with a second antibody (raised in sheep against whole rabbit IgG in our laboratory) and subsequently with polyethylenglycol 6000 precipitation (FERAK Laborat. Gmbh, Berlin, Germany) by the D A B / P E G method. The lower limit of assay detection was 7-8 pg/tube. The intra- and interassay coefficients of variation were 4.0 and 13.8%, respectively. CRF immunoreactivity in the brain extracts subjected to HPLC has been shown to cocromatograph with synthetic r / h CRFI_41 (Gardi et al., unpublished observation). The CRF-LI is expressed in pg/mg protein. All data are presented as mean _+S.E.M. Data were analyzed by one-way ANOVA followed by Dunnett's test. Probability level of 0.05 was accepted as indicating statistically significant difference. As is shown in Table I, acute administration of cocaine resulted in a significant, dose-dependent decrease in CRF-LI in the hypothalamus (F3,30 = 6.66, P < 0.002; 7.5 mg/kg cocaine: P < 0.05 vs. saline, -64%; 15 mg/kg cocaine: P < 0.05 vs. saline, - 7 3 % and 30 mg/kg cocaine: P < 0.05 vs. saline, - 74%) and in the basal forebrain (F3,29--18.09, P < 0.0001; 7.5 mg/kg cocaine: P < 0.05 vs. saline, -50%; 15 mg/kg cocaine: P < 0.05 vs. saline, - 5 0 % and 30 mg/kg cocaine: P < 0.05 vs. saline, - 68%). 7.5 mg/kg dose of cocaine decreased the CRF-LI in the hippocampus (F3.33 = 5.47, P < 0.005; - 52%) and in the frontal cortex (Faro = 4.54, P < 0.01; -40%). Cocaine administration increased CRF-LI in the amygdala, dose-dependently (F3,32 = 8.41, P < 0.0005; 7.5 mg/kg cocaine: P < 0.05 vs. saline, + 229%; 15 mg/kg cocaine: P < 0.05 vs. saline, + 455% and 30 mg/kg cocaine: P < 0.05 vs. saline, +504%). Striatal CRF-LI not altered significantly with cocaine administration (F3,30 = 2.40, p = 0.09; - 7%, + 20% and + 40%, respectively, p > 0.05 vs. saline). The results of the present study with regard to the distribution of basal CRF-LI in saline-treated, nonstressed, handled rats are similar, but not identical to
previous RIA studies 2'm. Chappell et al. 2 measured CRF-LI by using antisera against ovine CRF in rat brain nuclei, dissected out according to Palkovits and Brownstein's micropunch method 23. They have found 5054 + 674 pg CRF-LI/mg protein in the median eminence/arcuate nucleus complex. Measurements in several other hypothalamic nuclei equaled approximately 1200 pg CRF-L1/mg protein. The hypothalamus of the saline-treated control rats contained 2427 + 711 pg/mg CRF-LI in our present study. In limbic forebrain structures, tuberculum olfactorium, septum (lateral and medial) and nucleus accumbens, approximately 350 pg CRF-LI/mg protein were detected. In the present study the CRF-LI in basal forebrain, containing tuberculum olfactorium, medial septum and nucleus accumbens, was approximately 600 pg/mg protein. Chappell et al. 2 reported approximately 600 pg CRF-LI/mg protein as a sum of the results of distinct nuclei of the amygdala, which was 461 _+84 pg/mg protein in the whole amygdaloid complex in the present experiment. There were 31 and 34 pg/mg protein CRF-LI in the ventral and dorsal hippocampus, respectively, as measured by Chappell et al. 2, while we measured 26.8 + 3.3 pg CRF-LI/mg protein from the whole hippocampus. They found 22 pg/mg protein CRF-LI in the medial prefrontal cortex, while in our present experiment 37.7 pg/mg protein CRF-LI were detected in the frontal cortex. More recently, Inoue et al. 1° determined CRFLI in the rat brain dissected out according to Palkovits and Brownstein 23 by using a double antibody RIA specific for rat CRF. Levels of CRF-LI in the hypothalamus, basal forebrain (septum and nucleus accumbens) and in the amygdala of the control, non-stressed animals were very close to our present data. Frontal cortex CRF-LI has been reported to be much higher compared to data from the same structure presented here and by others 2. Such differences in CRF-LI obtained by different experiments may be due to differences in strains of animal, dissection techniques, antisera and RIA procedures used. Several lines of evidence suggest that stress and psychostimulants may interact in the central nervous system. Daily cocaine injection and stress exposure produce similar effects on mesocorticolimbic dopamine neurotransmission m. Both stress and cocaine activate the release of stress hormones, ACTH and CORT, through CRF 4'26'28. Stress m and CRF (Sarnyai et al., unpublished observation) potentiate the cocaine-induced stereotyped behavior in rats. Stress activates the hypothalamic and extrahypothalamic CRF in the rat brain. It was demonstrated that the acute stress increases the synthesis and release of CRF in the hypothalamus 8. Our report of dose-dependent decrease of
318 CRF-LI in the hypothalamus after acute cocaine administration supports the hypothesis of the stress-like effect of cocaine on the H P A axis. Effects of acute stress on CRF-LI in the extrahypothalamic brain regions are much less consistent 2'm'2°. Chappell et al. 2 found an increase in CRF-LI in the locus coeruleus, but not in other limbic and midbrain regions after acute immobilization and cold stress. Inoue et al. m demonstrated that single immobilization stress did not change CRF-LI in discrete brain regions. In contrast, it was demonstrated recently by in vivo microdialysis that acute immobilization stress produces a six-fold release of CRF from the amygdala in freely moving rats 2°. Our present findings show a pronounced decrease of CRF-LI in several extrahypothalamic regions, such as basal forebrain, hippocampus and frontal cortex. A dramatic, dose-dependent increase in CRF concentration was also demonstrated in the amygdala. One possible explanation of these inconsistencies between the effects of stress and cocaine on brain CRF could be that stress may activate some divergent neurochemical mechanisms (e.g. norepinephrine, dopamine, endogenous opiates or GABA) unspecifically, which can increase or decrease the functional activity of brain CRF producing no overall changes in CRF levels. In contrast, cocaine could act as a specific pharmacological stressor activating well-defined neurochemical pathways (DA-ergic, NE-ergic and 5-HT-ergic) which are important in the stimulation of CRF secretion. Present results show that acute cocaine administration decreased the levels of CRF-LI in the hypothalamus, basal forebrain, hippocampus and frontal cortex. Although, the measurement of peptide concentration alone cannot distinguish between synthesis, release or degradation, a decrease in the immunoreactive peptide level in a certain brain area usually reflects an increased release and subsequent degradation of the neuropeptide 29. Our present data may have some functional implications in terms of the mediation of the central nervous system effects of cocaine. Cocaine-induced reduction of hypothalamic CRF-LI was probably due to the release of CRF into the hypothalamic-pituitary portal circulation which activates the pituitaryadrenal axis. This interpretation is supported by the data that cocaine stimulates A C T H and corticosterone secretion in the same doses and time interval that have been reported previously 26'28. Furthermore, in vitro experiments on a hypothalamic slice also demonstrated an increased release of CRF induced by cocaine ~. Thus we can hypothesize that the CRF released from the hypothalamus may be at least one of the major endogenous substrates which can mediate cocaine's action on the HPA axis. Acute cocaine administration produced
a significant reduction in CRF-LI in the basal forebrain structures and in the hippocampus. Increased locomotor activity has been demonstrated by local microinfusion of both CRF and cocaine into the basal forebrain. Bilateral microinjection of CRF into the hippocampus also produced an intense locomotor activity 13. Cocaine-induced locomotor hyperactivity was either inhibited or ultimately completely abolished by different doses of CRF-antiserum and CRF-receptor antagonist 3°. It could be hypothesized that CRF release in limbic-basal forebrain structures may contribute to the expression of cocaine-induced locomotor response. CRF-LI was also decreased by cocaine in the frontal cortex. CRF receptor labeling 7 and CRF-L127 were decreased in the rat frontal cortex after chronic cocaine administration leading to anxiety in both experimental animals 27 and humans 5. This raises the possibility that endogenous CRF in the frontal cortex may be implicated in cocaine-induced emotional changes. The most dramatic changes in CRF-LI were detected in the amygdala after acute cocaine treatment. The amygdala has a critical importance in the development of fear and anxiety. CRF produces a behavioral state that resembles fear or anxiety 3. Lesion of central nucleus of the amygdala blocked this effect of CRF indicating that both the amygdala and CRF are critically involved in fear and anxiety 14. Cocaine also induces the behavioral symptoms of anxiety in rats 33. Anxiety and depression are the most important psychiatric consequences of chronic use a n d / o r withdrawal of cocaine in humans 5. Our present data on the cocaine-induced activation of intra-amygdaloid CRF, in lights of the behavioral evidences mentioned above, raise the possibility that CRF release in the amygdala could be an important mediator of the neurobiological processes of cocaine-induced anxiety and depression. In conclusion, this is the first report of the differential action of cocaine on CRF-LI in different brain structures of the rat. Cocaine decreases the CRF-LI in the hypothalamus, in the basal-forebrain structures, in the hippocampus and in the frontal cortex, whereas in the amygdala an increase in CRF-LI is found. These data suggest that cocaine activates brain CRF systems which may be one of the important endogenous substrates to mediate neuroendocrine, behavioral and emotional responses to cocaine.
1 Calogero, A.E., Gallucci, W.T., Kling, M.A., Chrousos, G.P. and Gold, P.W., Cocaine stimulates rat hypothalamiccorticotropin-releasing hormone secretion in vitro, Brain Res., 505 (1989) 7-11. 2 Chappell, P.B., Smith, M.A., Kilts, C.D., Bisette, G., Ritchie, J., Anderson, C and Nemeroff, C.B., Alterations in corticotropin-releasing factor-like immunoreactivityin discrete rat brain regions after acute and chronic stress. J. Neurosci., 6 (1986) 2908-2914.
319 3 Davis, M., The role of the amygdala in conditioned fear. In: J. Aggleton (Ed.), The amygdala: neurobiological aspects of emotion, memory and mental dysfunction, Wiley, New York, 1992, pp. 255-305. 4 Dunn, A.J. and Berridge, C.W., Physiological and behavioral responses to corticotropin-releasing factor administration: is CRF a mediator of anxiety or stress responses?, Brain Res. Rev., 15 (1990) 71-100. 5 Gawin, F.H. and Ellinwood, E.H., Cocaine dependence, Annu. ReL'. Med., 40 (1989) 149-161. 6 Glowinski, J. and Iversen, L.L., Regional studies of catecholamines in the rat brain - I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in variopus regions of the brain. J. Neurochem., 13 (1966) 655-669. 7 Goeders, N.E., Bienvenu, O.J. and De Sousa, E.B., Chronic cocaine administration alters corticotropin-releasing factor receptors in the rat brain, Brain Res., 531 (1990) 322-328. 8 Haas, D. and George S.R., Single and repeated mild stress increases synthesis and release of hypothalamic corticotropin-releasing factor. Brain Res. 461 (1988) 230-237. 9 Heffner, T.G., Zigmond, M.J. and Stricker, E.M., Effects of dopaminergic agonists and antagonists of feeding in intact and 6-hydroxydopamine-treated rats, J. Pharmacol. Exp. Ther., 201 (1977) 386-399. 10 Inoue, T., Koyama, T., Muraki, A., Yamashita, I., Effects of single and repeated immobilization stress on corticotropin-releasing factor concentration in discrete rat brain regions. Prog. Neuro-Psychopharmacol. BioL Psychiat. 17 (1993) 161-170. 11 Jan~iky, T., T6th, G., Penke, B., Kov~cs, K. and L~iszl6, F.A., lodination of peptide hormones and purification of iodinated peptides, J. Liquid Chromatography, 5 (1982) 1499-1507. 12 Kalivas, P.W. and Duffy, P., Similar effects of daily cocaine and stress on mesocorticolimbic dopamine neurotransmission in the rat. Biol. Psychiatry, 25 (1989) 913-928. 13 Lee, E.H.Y. and Tsai, M.J., The hippocampus and amygdala mediate the locomotor stimulating effects of corticotropin-releasing factor in mice. Behav. Neural Biol., 51 (1989) 412-423. 14 Liang, K.C., Melia, K.R., Campeau, S., Falls, W.A., Miserendino, M.J.D.and Davis, M., Lesions of the central nucleus of the amygdala, but not the paraventricular nucleus of the hypothalamus, block the excitatory effects of corticotropin-releasing factor on the acoustic startle reflex, J. Neurosci., 12 (1992) 2313-2320. 15 Lin, Y., Morrow, T.J., Kiritsy-Roy, J.A., Terry. L.C. and Casey, K.L., Cocaine: evidence for supraspinal, non-opiate analgesia, Brain Res, 479 (1989) 306-312. 16 Linton, E.A. and Lowry, P.J., Radioimmunoassay and chromatographic characterization of CRF-41-1ike immunoreactivity in hypothalami from several species. Neuropeptides, 3 (1982) 45-52. 17 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent. J. BioL Chem., 193 (1951) 265-275. 18 Maser-Gluth, C. and Vecsei, P., Corticotropin-releasing factorlike immunoreactivity in human 24 h urine. Clin Endocrinol., 30 (1989) 405-412. 19 MacLennan, A.J. and Maier, S.F., Coping and the stress-induced potentiation of stimulant stereotypy in the rat. Science, 219 (1983) 1091-1093.
20 Merlo-Pich, E., Koob, G.F., Sattler, S.C., Menzaghi, F., Heilig, M., Heinrichs, S.C., Vale, W., Weiss, F., Stress-induced release of corticotropin-releasing factor in the amygdala measured by in vivo microdialysis, Soc. Neurosci. Abstr., 18 (1992) 225.17. 21 Nemeroff, C.B., New vistas in neuropeptide research in neuropsychiatry: focus on corticotropin-releasing factor, Neuropsychopharmacology, 6 (1992) 69-75. 22 Owens, M.J, and Nemeroff, C.B., Physiology and pharmacology of corticotropin-releasing factor, Pharmacol. Rev., 43 (1991) 425473. 23 Palkovits, M. and Brownstein, M.J., Microdissection of brain areas by the punch technique. In A.C. Cuello, (Ed.), Brain Microdissection Techniques, Wiley, New York, 1983, pp. 1-36. 24 Post, R.M., Weiss, S.R.B., Pert, A. and Uhde, T.W., Chronic cocaine administration: sensitization and kindling effects. In S. Fischer, A. Raskin and E.H. Uhlenhuth (Eds.) Cocaine: Clinical and Biobehavioral Aspects, Oxford University Press, New York, 1987, pp. 109-173. 25 Raum, W.J., McGivern, R.F., Peterson, M.A., Shryne, J.H. and Gorski, R.A., Prenatal inhibition of hypothalamic sexual differentiation by cocaine: effects on neurobehavioral sexual differentiaton in male rats, Dev. Brain Res., 53 (1990) 230-236. 26 Rivier, C. and Vale, W., Cocaine stimulates adrenocorticotropin (ACTH) secretion throuhg a corticotropin-releasing factor (CRF)-mediated mechanism, Brain Res., 422 (1987) 403-406. 27 Sarnyai, Z., B~r6, t~., Gardi, J., Vecserny~s, M., Julesz, J., Penke, B. and Telegdy, G., Corticotropin-releasing factor (CRF) as an endogenous mediator of the behavioral and neuroendocrine actions of cocaine in rats., Abstracts of the Hans Selye Symposia on Neuroendocrinology and Stress, CRF and Cytokines: [{ole in Stress Response. Montreal, Canada, 1992, p. 13. 28 Sarnyai, Z., Bir6, E., Penke, B. and Telegdy, G., The cocaine-induced elevation of plasma corticosterone is mediated by endogenous corticotropin-releasing factor (CRF) in rats, Brain Res., 589 (1992) 154-156. 29 Sarnyai, Z., B~r6, IE., Vecserny~s, M., Laczi, F., Szab6, G. and Kov~cs, G.L., Effects of cocaine on the contents of neurohypophyseal hormones in the plasma and in different brain structures in rats. Neuropeptides, 23 (1992) 27-31. 30 Sarnyai, Z., H6hn, J., Szab6, G. and Penke, B., Critical role of endogenous corticotropin-releasing factor (CRF) in the mediation of the behavioral action of cocaine in rats. Life Sci., 51 (1992) 2019-2024. 31 Scheel-Kriiger, J., Baestrup, C., Nielson, M., Golembiowska, K. and Mogilnicka, F., Cocaine: discussion on the role of dopamine in the biochemical mechanism of action. In H.H. Ellinwood and M.M. Kilbey (Eds.), Advances in Behavioral Biology, Vol. 21, Cocaine and Other Stimulants, Plenum Press, New York, NY, 1977, pp. 373-408. 32 Watzl, B. and Watson, R.R., Minireview: Immunomodulation by cocaine-A neuroendocrine mediated response, Life Sci., 46 (1990) 1319-1329. 33 Yang, X.M., Gorman, A.L., Dunn, A.J. and Goeders, N.E., Anxiogenic effects of acute and chronic cocaine administration: neurochemical and behavioral studies, Pharmacol. Biochem. Behay., 41 (1992) 643-650.