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The distribution pattern of adrenocorticotropin-like immunoreactivity in the cat central nervous system
48
Neuroseience Letters, 71 (1986)48 52 Elsevier Scientific Publishers Ireland Ltd.
NSL 04220
The distribution pattern of adrenocorticotropin-like immunoreactivity in the cat central nervous system J a y a s h r e e K. R a o l, H a i t a o
Hu 2, C h a n d a n P r a s a d 3 a n d A. J a y a r a m a n 2
Departments ~?/ ;Pediatrics (Division o/Endocrinology). :Neurolo~o' and -~Medicine (Section ~1' E3uto~'rinnlogy ), Louisiana State ~hliversity School q/" Medicine, New Orleans. I,A 70112 ( ~T.S. A. J (Received 9 June 1986: Accepted 16 July 1986) Key w o r d s
Adrenocorticotropin Immunocytochemistry tin peroxidase complex Cat
Hypothalamtls
ACTH
Avidin bio-
The distribution pattern of adrenocorticotropin-like immunoreactivity (ACTH-LI) in cats using the avidin biotin modification of an immunocytochemical method shows cell bodies containing ACTH-LI in the medial basal hypothalamus, especially in the infundibular nucleus. The libers from these neurons extended beyond the hypothalamus, into the paraventricular nucleus of the thalamus, rostral amygdala, periaqueductal gray, locus coeruleus, parabrachial nucleus and medial nuclcus of the nucleus tractus solitarius Thc distribution panern of the cell bodies and fibers containing ACTH-LI bears several similarities to that seen in rats. The pattern differs from that of rats in the fact that the termination in the amygdala is more extensive and that ACTH-LI was not observed in cell bodies in any location othcr than the medial basal hypothalamus.
Adrenocorticotropin hormone (ACTH) is derived t¥om proopiomelanocortin [8] and is known to be produced in the pituitary gland and to regulate glucocorticoid metabolism. Recent studies in rats, however, have shown that in addition to the pituitary gland, A C T H is produced in the medial basal hypothalamus, and axons from these cells reach several nuclei of the forebrain, amygdala and brainstem [5 9, 1 I 13]. The precise role of A C T H produced by the hypothalamic neurons is not known. The nerve terminals from these A C T H neurons overlap with the terminal zones of cells that demonstrate tyrosine hydroxylase-like immunoreactivity (LI) [2]. A C T H has been speculated to play a significant role in stress response [i]. Although immunocytochemical and radioimmunoassay methods have shown the distribution pattern of A C T H - L I extensively in the rat brain, the distribution pattern of this peptide within the cat, monkey and human brain has not been studied methodically. A study was undertaken to map A C T H - L I in the cat central nervous system. For this study several adult cats, some of which received intraventricular injection of" 2% colchicine, were used. After a survival of 24 h the animals were deeply anesthetized and sacrificed with a transcardiac perfusion of one liter of normal saline and two liters of 4% paraformaldehyde (pH 7.4). The brains were removed from the skull, blocked and stored in 20% sucrose solution overnight. Then 40 Itm thick sections were processed to demonstrate A C T H - L I using the avidin biotin complex modifica0304-3940/86/$ 03.50 @ 1986 Elsevier Scientiiic Publishers Ireland lad.
49 tion of the immunocytochemical method of Hsu et al. [4]. The sections were incubated for 48 h in i:500 to 1:1000 concentration of antiserum to A C T H (Immunonuclear Corporation, Stillwater, MN, U.S.A.). For analyzing the cytoarchitectonic details of the various nuclei, alternate sections were counterstained with Neutral red. The hypothalamic nuclei were identified by means of Bleier's cytoarchitectonic atlas of the cat hypothalamus [3]. The sections were studied using bright- and darkfield microscopy, and projection drawings were made using a camera lucida. Controls were done by preincubating the sections with synthetic ACTH, and by deleting the primary antibody. Cell bodies containing A C T H - L ! were located strictly within the medial basal hypothalamus in these cases. The most abundant number of cells were seen throughout the rostrocaudal extent of the medial and ventral areas of the infundibular nucleus (Figs. I D and 2A-C). At rostral levels a few scattered cells were also seen in the periventricular area adjacent to the suprachiasmatic region. The fibers with ACTH-LI were seen most profusely throughout the rostrocaudal extent of the hypothalamus. At rostral levels ACTH-LI was prominent in the anterior hypothalamus (Fig. I A) and caudally in the paraventricular nucleus and the periventricular area of the hypothalamus (Fig. 1B, C). At the level of anterior commissure~ fibers extended dorsally from the anterior hypothalamus and terminated in the bed nucleus of the stria terminalis and the ventrolateral areas of the lateral septal nucleus (Fig. I A). The nucleus accumbens and the striatum were free of fibers with ACTH-LI. Dorsally a prominent fiber bundle traversed the midline thalamic nuclei and terminated profusely in the nucleus rhomboideus and the paraventricular nucleus of the thalamus (Figs. 1B-D and 2D). A small number of fibers were seen to cross the dorsal areas of the dorsomedial nucleus of the thalamus and to extend to the medial division of the lateral posterior thalamic nucleus (Fig. I D). ACTH-immunoreactive fibers extended lateral from the hypothalamus and coursed dorsal to the optic tract and turned medially to terminate in the anterior amygdaloid area (Fig. IB). More caudally within the amygdala, fibers with ACTH-LI terminated prominently within the medial division of the central nucleus (Fig. 1C), medial nucleus of the amygdaloid complex and the amygdalohippocampal area. Some fibers traversed laterally through the basolateral nucleus to enter the deep layers of the dorsolateral entorhinal cortex, but clearly defined terminations were not seen (Fig. 1C). At collicular levels, prominent terminations were seen in lateral and ventral regions of the periaqueductal gray area (Fig. IF, G). Fibers from periaqueductal and periventricular regions of the brainstem were noted to descend ventrally in the midline to terminate in the raphe nucleus and the nucleus interfascicularis as well as ventromedially in the tegmental reticular formation at collicular, pontine and medullary levels (Fig. 1F I). Some fibers coursed laterally to terminate in the cuneiform nucleus, nucleus of the lateral lemniscus, locus coeruleus and parabrachial nucleus (Fig. 1G-I). The density of terminations in the medial parabrachial nucleus was more profuse than those in the lateral division of this nucleus (Fig. 2E). At the lower medullary level a small but significant fiber system terminated within the medial division of the nucleus tractus solitarii (Fig. I J), but fibers with ACTH-LI could not be detected caudal to the medullary level.
D
LPm
PV Rh
J
Fig.
I. This diagram represents the location of cell bodies (closed squares) and fibers containing
and their sites of termination. basolateral nucleus
AAA,
nucleus of amygdala;
of amygdala:
CNF,
anterior
area; AHA.
BSt. bed nucleus of stria terminalis;
cuneiform
raphe: ENT, entorhinal
cortex:
nucleus interfascicularis:
IN. infundibular
medial division
amygdaloid
nucleus;
Fw. fornix;
of the lateritl posterior
CS, nucleus
Haa. anterior
CEm, medial division
centralis
hypothalamic
nucleus; IP, interpeduncular thalamic
nucleus;
amygdalohippocampal
LS. lateral
superior;
DRM.
area; Hb, habenular nucleus;
ACTHLI area; BL,
of the central nucleus
LC. locus coeruleus:
septal nucleus;
dorsal
nucleus;
IF.
LPm.
MD. medial dorsal
nucleus; MM. mamillary nucleus; mnTS. medial division of the nucleus of solitary tract; NGC, nucleus gtgantocellularis: nLL, nucleus of the lateral lemniscus; NPB, parabrachial nucleus; PAG, periaqueductal gray: Pea. periventricular
area of the hypothalamus;
paraventricular
nucleus of the hypothalamus;
taenia thalami:
V, ventricle.
PV, paraventricular
Rh. nucleus rhomboideus;
nucleus
of the thalamus:
SC, suprachiasmatic
PVH.
nucleus; TT.
The distribution pattern of ACTH-LI within the central nervous system of the cat has several similarities to that described in rats as well as some differences. In rats, the cell bodies containing ACTH-LI were noted in the arcuate and the periarcuate nuclei of the medial basal hypothalamus [2, 5. 9. 131. In cats the location of the neurons with ACTH-LI is similar. but the term infundibular nucleus is used to denote
51
D
Fig. 2. Photomicrographs of the cell bodies with ACTH-LI within the rostral (A) and caudal (B) infundibular nucleus of the hypothalamus. A neuron with ACTH-LI typically consisted of a fusiform, bipolar or round cell body (C) with an average diameter of 1:%20tim. Photomicrographs of terminals within the paraventricular nucleus (PV) of the thalamus (D) and in the medial (NPMm) and lateral (NPBIa)divisions of the nucleus parabrachialis (E). BC, Brachium conjunctivum.
this area o f the h y p o t h a l a m u s . The d i s t r i b u t i o n p a t t e r n o f the A C T H - p o s i t i v e fibers are similar in rats a n d cats. In cats, similar to rats, p r o m i n e n t fibers are seen in the p a r a v e n t r i c u l a r nucleus o f the t h a l a m u s a n d in the a n t e r i o r a m y g d a l o i d area. In cats, the p a t t e r n o f t e r m i n a t i o n s in the periaqueductal gray, locus coeruleus, c u n e i f o r m nucleus, nucleus of the lateral lemniscus, p a r a b r a c h i a l nucleus a n d the medial division of the nucleus tractus solitarius is similar to that of rats. The d i s t r i b u t i o n p a t t e r n o f A C T H - L I within the a m y g d a l o i d complex in cats is
52
more extensive than what has been reported so far in rats. In rats these fibers were seen within the medial division of the central nucleus [2, 9, 11], whereas in cats, in addition to the medial division of the central nucleus, the terminals were also seen in the medial nucleus of the amygdala and the amygdalohippocampal area. Some fibers were also present within the deep layers of the entorhinal cortex, but definite terminations could not be seen. In rats, a second population o f neurons located within the commissural nucleus at the spinomedullary junction has also been recognized to contain ACTH-LI l1 l, 13]. In our study, even in those cats which received intraventricular injection of colchicine, cell bodies of this nucleus did not contain ACTH-LI. We also did not observe fibers with ACTH-LI in the spinal cord as reported by others in rats. These differences could potentially reflect a variation between two animal species. In rats, the ACTH fibers appear to terminate in those areas where monoaminergic fibers also terminate [2]. The termination sites of A C T H fibers in cats closely resemble those in rats. In rats ACTH has been shown to cause excitation o f neurons of the locus coeruleus and results in an enhanced turnover rate of norepinephrine [10], thereby suggesting that the monoaminergic response to stress may be regulated in part by ACTH. We thank Bonnie LeBlanc and Virginia Howard for their assistance. I Axelrod, J. and Reisine, T.D., Stress hormones: their interaction and regulation, Science. 224 (1984) 452 459. 2 Baker, H., Ruggiero, D.A., Alden, S., Anwar, M. and Reis, D.J., Anatomical evidence for interactions between catecholamine- and adrenocorticotropin-containing neurons, Neuroscience, 17 (1986) 469 484.
3 Bleier, R., The Hypothalamus of the Cat: A Cytoarchitectonic Atlas with Horsley-Clark Co-ordinates, The Johns Hopkins Press, Baltimore, 1961, 109 pp. 4 Hsu, S.M., Rane, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577 580. 5 Joseph, S.A., lmmunoreactive adrenocorticotropin m rat brain: a neuroanatomical study using antiserum generated against synthetic ACTH~ 39, Am. J. Anat., 158 (1980) 533 54g. 6 Joseph, S.A., Pilcher, W.H. and Bennett-Clarke, C., lmmunocytochemical localization of A C T H perikarya m nucleus tractus solitarius: evidence for a second opiocortin neuronal system, Neurosci. Lett.. 38(1983) 221 225. 7 Joseph, S.A. and Piekut. D.T., Dual immunostaining procedure demonstrating neurotransmittcr and neuropeptide codistribution in the same brain section. Am. J. Anat., 175 (1986) "~31 342 S Khachaturian, H., Lewis, M.E., Sharer, M.K.-H. and Watson, S.J., A n a t o m y of the CNS opioid system. TrendsNeurosci.,8(1985) Ill 119. 0 Knigge, K.M., Joseph. S.A. and Nocton, J., Topography of the A(~THqmnmnorcactivc neurons m the basal hypothalamus of the rat brain, Brain Res., 216 ( 1981 ) 333 34 I. l0 Olpe, H . R and Joncs, R.S.G., Excitatory effects of A C T H on noradrenergic neurons of the locus cerulcus in the rat, Brain Rcs.. 251 (1982) 177 179. I I Romagnano, M.A. and Joseph, S.A., hnmunohistochemical localization ofACTH~ ~,, in the brain stem of the rat, Brain Res., 276 (t983) I 16. I2 Schwartzberg, D.G. and Nakanc, P.K., ACTH-related peptide containing neurons within the medulla obhmgata of the rat, Brain Res., 276 (t983) 351 356. 13 Watson, S.J., Richard. C.W. and Barchas, J.D., Adrenocorticotropin in rat brain: immunocytochenfical localization in cells and axons, Science, 200 (1978) 1180 1181.
Neuroseience Letters, 71 (1986)48 52 Elsevier Scientific Publishers Ireland Ltd.
NSL 04220
The distribution pattern of adrenocorticotropin-like immunoreactivity in the cat central nervous system J a y a s h r e e K. R a o l, H a i t a o
Hu 2, C h a n d a n P r a s a d 3 a n d A. J a y a r a m a n 2
Departments ~?/ ;Pediatrics (Division o/Endocrinology). :Neurolo~o' and -~Medicine (Section ~1' E3uto~'rinnlogy ), Louisiana State ~hliversity School q/" Medicine, New Orleans. I,A 70112 ( ~T.S. A. J (Received 9 June 1986: Accepted 16 July 1986) Key w o r d s
Adrenocorticotropin Immunocytochemistry tin peroxidase complex Cat
Hypothalamtls
ACTH
Avidin bio-
The distribution pattern of adrenocorticotropin-like immunoreactivity (ACTH-LI) in cats using the avidin biotin modification of an immunocytochemical method shows cell bodies containing ACTH-LI in the medial basal hypothalamus, especially in the infundibular nucleus. The libers from these neurons extended beyond the hypothalamus, into the paraventricular nucleus of the thalamus, rostral amygdala, periaqueductal gray, locus coeruleus, parabrachial nucleus and medial nuclcus of the nucleus tractus solitarius Thc distribution panern of the cell bodies and fibers containing ACTH-LI bears several similarities to that seen in rats. The pattern differs from that of rats in the fact that the termination in the amygdala is more extensive and that ACTH-LI was not observed in cell bodies in any location othcr than the medial basal hypothalamus.
Adrenocorticotropin hormone (ACTH) is derived t¥om proopiomelanocortin [8] and is known to be produced in the pituitary gland and to regulate glucocorticoid metabolism. Recent studies in rats, however, have shown that in addition to the pituitary gland, A C T H is produced in the medial basal hypothalamus, and axons from these cells reach several nuclei of the forebrain, amygdala and brainstem [5 9, 1 I 13]. The precise role of A C T H produced by the hypothalamic neurons is not known. The nerve terminals from these A C T H neurons overlap with the terminal zones of cells that demonstrate tyrosine hydroxylase-like immunoreactivity (LI) [2]. A C T H has been speculated to play a significant role in stress response [i]. Although immunocytochemical and radioimmunoassay methods have shown the distribution pattern of A C T H - L I extensively in the rat brain, the distribution pattern of this peptide within the cat, monkey and human brain has not been studied methodically. A study was undertaken to map A C T H - L I in the cat central nervous system. For this study several adult cats, some of which received intraventricular injection of" 2% colchicine, were used. After a survival of 24 h the animals were deeply anesthetized and sacrificed with a transcardiac perfusion of one liter of normal saline and two liters of 4% paraformaldehyde (pH 7.4). The brains were removed from the skull, blocked and stored in 20% sucrose solution overnight. Then 40 Itm thick sections were processed to demonstrate A C T H - L I using the avidin biotin complex modifica0304-3940/86/$ 03.50 @ 1986 Elsevier Scientiiic Publishers Ireland lad.
49 tion of the immunocytochemical method of Hsu et al. [4]. The sections were incubated for 48 h in i:500 to 1:1000 concentration of antiserum to A C T H (Immunonuclear Corporation, Stillwater, MN, U.S.A.). For analyzing the cytoarchitectonic details of the various nuclei, alternate sections were counterstained with Neutral red. The hypothalamic nuclei were identified by means of Bleier's cytoarchitectonic atlas of the cat hypothalamus [3]. The sections were studied using bright- and darkfield microscopy, and projection drawings were made using a camera lucida. Controls were done by preincubating the sections with synthetic ACTH, and by deleting the primary antibody. Cell bodies containing A C T H - L ! were located strictly within the medial basal hypothalamus in these cases. The most abundant number of cells were seen throughout the rostrocaudal extent of the medial and ventral areas of the infundibular nucleus (Figs. I D and 2A-C). At rostral levels a few scattered cells were also seen in the periventricular area adjacent to the suprachiasmatic region. The fibers with ACTH-LI were seen most profusely throughout the rostrocaudal extent of the hypothalamus. At rostral levels ACTH-LI was prominent in the anterior hypothalamus (Fig. I A) and caudally in the paraventricular nucleus and the periventricular area of the hypothalamus (Fig. 1B, C). At the level of anterior commissure~ fibers extended dorsally from the anterior hypothalamus and terminated in the bed nucleus of the stria terminalis and the ventrolateral areas of the lateral septal nucleus (Fig. I A). The nucleus accumbens and the striatum were free of fibers with ACTH-LI. Dorsally a prominent fiber bundle traversed the midline thalamic nuclei and terminated profusely in the nucleus rhomboideus and the paraventricular nucleus of the thalamus (Figs. 1B-D and 2D). A small number of fibers were seen to cross the dorsal areas of the dorsomedial nucleus of the thalamus and to extend to the medial division of the lateral posterior thalamic nucleus (Fig. I D). ACTH-immunoreactive fibers extended lateral from the hypothalamus and coursed dorsal to the optic tract and turned medially to terminate in the anterior amygdaloid area (Fig. IB). More caudally within the amygdala, fibers with ACTH-LI terminated prominently within the medial division of the central nucleus (Fig. 1C), medial nucleus of the amygdaloid complex and the amygdalohippocampal area. Some fibers traversed laterally through the basolateral nucleus to enter the deep layers of the dorsolateral entorhinal cortex, but clearly defined terminations were not seen (Fig. 1C). At collicular levels, prominent terminations were seen in lateral and ventral regions of the periaqueductal gray area (Fig. IF, G). Fibers from periaqueductal and periventricular regions of the brainstem were noted to descend ventrally in the midline to terminate in the raphe nucleus and the nucleus interfascicularis as well as ventromedially in the tegmental reticular formation at collicular, pontine and medullary levels (Fig. 1F I). Some fibers coursed laterally to terminate in the cuneiform nucleus, nucleus of the lateral lemniscus, locus coeruleus and parabrachial nucleus (Fig. 1G-I). The density of terminations in the medial parabrachial nucleus was more profuse than those in the lateral division of this nucleus (Fig. 2E). At the lower medullary level a small but significant fiber system terminated within the medial division of the nucleus tractus solitarii (Fig. I J), but fibers with ACTH-LI could not be detected caudal to the medullary level.
D
LPm
PV Rh
J
Fig.
I. This diagram represents the location of cell bodies (closed squares) and fibers containing
and their sites of termination. basolateral nucleus
AAA,
nucleus of amygdala;
of amygdala:
CNF,
anterior
area; AHA.
BSt. bed nucleus of stria terminalis;
cuneiform
raphe: ENT, entorhinal
cortex:
nucleus interfascicularis:
IN. infundibular
medial division
amygdaloid
nucleus;
Fw. fornix;
of the lateritl posterior
CS, nucleus
Haa. anterior
CEm, medial division
centralis
hypothalamic
nucleus; IP, interpeduncular thalamic
nucleus;
amygdalohippocampal
LS. lateral
superior;
DRM.
area; Hb, habenular nucleus;
ACTHLI area; BL,
of the central nucleus
LC. locus coeruleus:
septal nucleus;
dorsal
nucleus;
IF.
LPm.
MD. medial dorsal
nucleus; MM. mamillary nucleus; mnTS. medial division of the nucleus of solitary tract; NGC, nucleus gtgantocellularis: nLL, nucleus of the lateral lemniscus; NPB, parabrachial nucleus; PAG, periaqueductal gray: Pea. periventricular
area of the hypothalamus;
paraventricular
nucleus of the hypothalamus;
taenia thalami:
V, ventricle.
PV, paraventricular
Rh. nucleus rhomboideus;
nucleus
of the thalamus:
SC, suprachiasmatic
PVH.
nucleus; TT.
The distribution pattern of ACTH-LI within the central nervous system of the cat has several similarities to that described in rats as well as some differences. In rats, the cell bodies containing ACTH-LI were noted in the arcuate and the periarcuate nuclei of the medial basal hypothalamus [2, 5. 9. 131. In cats the location of the neurons with ACTH-LI is similar. but the term infundibular nucleus is used to denote
51
D
Fig. 2. Photomicrographs of the cell bodies with ACTH-LI within the rostral (A) and caudal (B) infundibular nucleus of the hypothalamus. A neuron with ACTH-LI typically consisted of a fusiform, bipolar or round cell body (C) with an average diameter of 1:%20tim. Photomicrographs of terminals within the paraventricular nucleus (PV) of the thalamus (D) and in the medial (NPMm) and lateral (NPBIa)divisions of the nucleus parabrachialis (E). BC, Brachium conjunctivum.
this area o f the h y p o t h a l a m u s . The d i s t r i b u t i o n p a t t e r n o f the A C T H - p o s i t i v e fibers are similar in rats a n d cats. In cats, similar to rats, p r o m i n e n t fibers are seen in the p a r a v e n t r i c u l a r nucleus o f the t h a l a m u s a n d in the a n t e r i o r a m y g d a l o i d area. In cats, the p a t t e r n o f t e r m i n a t i o n s in the periaqueductal gray, locus coeruleus, c u n e i f o r m nucleus, nucleus of the lateral lemniscus, p a r a b r a c h i a l nucleus a n d the medial division of the nucleus tractus solitarius is similar to that of rats. The d i s t r i b u t i o n p a t t e r n o f A C T H - L I within the a m y g d a l o i d complex in cats is
52
more extensive than what has been reported so far in rats. In rats these fibers were seen within the medial division of the central nucleus [2, 9, 11], whereas in cats, in addition to the medial division of the central nucleus, the terminals were also seen in the medial nucleus of the amygdala and the amygdalohippocampal area. Some fibers were also present within the deep layers of the entorhinal cortex, but definite terminations could not be seen. In rats, a second population o f neurons located within the commissural nucleus at the spinomedullary junction has also been recognized to contain ACTH-LI l1 l, 13]. In our study, even in those cats which received intraventricular injection of colchicine, cell bodies of this nucleus did not contain ACTH-LI. We also did not observe fibers with ACTH-LI in the spinal cord as reported by others in rats. These differences could potentially reflect a variation between two animal species. In rats, the ACTH fibers appear to terminate in those areas where monoaminergic fibers also terminate [2]. The termination sites of A C T H fibers in cats closely resemble those in rats. In rats ACTH has been shown to cause excitation o f neurons of the locus coeruleus and results in an enhanced turnover rate of norepinephrine [10], thereby suggesting that the monoaminergic response to stress may be regulated in part by ACTH. We thank Bonnie LeBlanc and Virginia Howard for their assistance. I Axelrod, J. and Reisine, T.D., Stress hormones: their interaction and regulation, Science. 224 (1984) 452 459. 2 Baker, H., Ruggiero, D.A., Alden, S., Anwar, M. and Reis, D.J., Anatomical evidence for interactions between catecholamine- and adrenocorticotropin-containing neurons, Neuroscience, 17 (1986) 469 484.
3 Bleier, R., The Hypothalamus of the Cat: A Cytoarchitectonic Atlas with Horsley-Clark Co-ordinates, The Johns Hopkins Press, Baltimore, 1961, 109 pp. 4 Hsu, S.M., Rane, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577 580. 5 Joseph, S.A., lmmunoreactive adrenocorticotropin m rat brain: a neuroanatomical study using antiserum generated against synthetic ACTH~ 39, Am. J. Anat., 158 (1980) 533 54g. 6 Joseph, S.A., Pilcher, W.H. and Bennett-Clarke, C., lmmunocytochemical localization of A C T H perikarya m nucleus tractus solitarius: evidence for a second opiocortin neuronal system, Neurosci. Lett.. 38(1983) 221 225. 7 Joseph, S.A. and Piekut. D.T., Dual immunostaining procedure demonstrating neurotransmittcr and neuropeptide codistribution in the same brain section. Am. J. Anat., 175 (1986) "~31 342 S Khachaturian, H., Lewis, M.E., Sharer, M.K.-H. and Watson, S.J., A n a t o m y of the CNS opioid system. TrendsNeurosci.,8(1985) Ill 119. 0 Knigge, K.M., Joseph. S.A. and Nocton, J., Topography of the A(~THqmnmnorcactivc neurons m the basal hypothalamus of the rat brain, Brain Res., 216 ( 1981 ) 333 34 I. l0 Olpe, H . R and Joncs, R.S.G., Excitatory effects of A C T H on noradrenergic neurons of the locus cerulcus in the rat, Brain Rcs.. 251 (1982) 177 179. I I Romagnano, M.A. and Joseph, S.A., hnmunohistochemical localization ofACTH~ ~,, in the brain stem of the rat, Brain Res., 276 (t983) I 16. I2 Schwartzberg, D.G. and Nakanc, P.K., ACTH-related peptide containing neurons within the medulla obhmgata of the rat, Brain Res., 276 (t983) 351 356. 13 Watson, S.J., Richard. C.W. and Barchas, J.D., Adrenocorticotropin in rat brain: immunocytochenfical localization in cells and axons, Science, 200 (1978) 1180 1181.