Co-localization of peptide-like immunoreactivities with glucocorticoid receptor- and Fos-like immunoreactivities in the rat parabrachial nucleus

245

Brain Research, 615 (1993) 245-251 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 18961

Co-localization of peptide-like immunoreactivities with glucocorticoid receptor- and Fos-like immunoreactivities in the rat parabrachial nucleus Tommi Kainu

a

Jari Honkaniemi a Jan-,~&e Gustafsson b, Leena Rechardt and Markku Pelto-Huikko a

a

a Department of Biomedical Sciences, Universityof Tampere, Tampere (Finland) and b Department of Medical Nutrition, Huddinge University Hospital, Stockholm (Sweden) (Accepted 26 January 1993)

Key words: Immobilization stress; Colchicine; Transcription factor

The parabrachial nucleus (PB) is a brainstem nucleus, which mediates autonomic information from the viscera to various forebrain nuclei, e.g. to the central nucleus of the amygdala (ACe) and to the medial preoptic area (MPOA). The neurons of the PB contain several neuropeptides, of which calcitonin-gene related peptide-immunoreactive (CGRP-IR) and neurotensin (NT)-IR neurons provide input to the ACe, whereas corticotropin-releasing factor-IR (CRF) neurons project to the MPOA. The aim of the present paper was to study whether the neurons containing CGRP-, NT- and CRF-like immunoreactivities (LIs) in the PB also contain glucocorticoid receptor (GR)- a n d / o r Fos-LIs after stress. No co-localization was observed with the GR-LI and peptide-LIs, suggesting that plasma glucocorticoids do not have direct effects on these neurons of the PB. After stress, the vast majority of the peptide-lR perikarya exhibited Fos-LI, suggesting that the peptidergic pathways from the PB to ACe and MPOA are activated in stress. The ACe and MPOA have been connected in various stress related responses, e.g. inhibiting the hypothalamo-pituitary-gonadal axis, raising the blood pressure and pulse, and increasing the secretion of glucocorticoids. Therefore, the activation of the peptidergic pathways between the PB and the ACe and MPOA suggests that some of these responses may be elicited by the peptidergic input from the PB. Furthermore, since Fos acts as a transcription factor, stress may affect the expression of the neuropeptides studied.

INTRODUCTION

The pontine parabrachial nucleus surrounds the superior cerebellar peduncle (SCP), which divides the PB into the medial (PBm) and lateral (PB1) subdivisions. On the basis of neuronal cytoarchitecture and the content of neurotransmitters the PB can be further divided into a total of ten different subnuclei 9. The external part of the PBI has been shown to contain calcitonin gene-related peptide (CGRP)-, neurotensin (NT)- and corticotropin-releasing factor (CRF)-immunoreactive (IR) perikarya 4'33. The PBI has extensive reciprocal connections with brain areas involved in autonomic and neuroendocrine functions and the neuropeptides present in the PB have been identified in these pathways. So far it has been demonstrated, that the CGRP- and NT-IR neurons project to the central

nucleus of the amygdala (ACe; see refs. 3, 30), whereas the CRF-IR neurons provide input to the medial preoptic area of the hypothalamus (MPOA; see ref. 18). The PB also projects to the nucleus tractus solitarius (NTS; see ref. 21) and receives input from CRF-13 and NT-IR 24 neurons of the NTS. In addition, the PB projects to the hypothalamic paraventricular nucleus (PVN; see ref. 35), but the neurotransmitters of this pathway have not yet been characterized. Stress has a well documented stimulatory effect on the secretion of glucocorticoid hormones from the adrenal gland. In the brain, the main regulatory neurons modulating the secretion of glucocorticoid hormones are the CRF-IR neurons in the PVN. Both chronic intermittent ~6 and acute stress 12 stimulate the expression of CRF in the PVN resulting in enhanced glucocorticoid secretion. The effects of stress on gene

Correspondence: T. Kainu, Department of Biomedical Sciences, Box 607, SF-33101 Tampere, Finland. Fax: (358) (31) 156 170.

246 expression are mediated by transcription factors, which are capable of stimulating or inhibiting the expression of target genes. The neurons of the PVN contain large amounts of two extensively studied transcription factors: the glucocorticoid receptor (GR; see ref. 7) and after various types of stimuli the protein product of the proto-oncogene c-fos (Fos; see refs. 6, 26). The G R belongs to the steroid-thyroid hormone receptor superfamily, whereas c-los is an inducible immediate early gene (lEG). The glucocorticoid receptor is activated by binding to glucocorticoid hormones whereafter it binds to the target gene and affects its transcription 2. Nearly all the CRF-IR neurons in the PVN contain GR-like immunoreactivity (LI; ref. 7), suggesting that glucocorticoid hormones may affect CRF gene expression in the PVN through the G R 17.

The Fos protein is a transcription factor, which has a relatively low basal expression in the brain. However, it can be induced by a wide variety of extracellular stimuli, including stress and colchicine injections 6'28. In addition to the role of Fos as a transcription factor, it has proved to be a useful marker of an increased intracellular activity 26. Fos affects gene expression by dimerizing with members of the Jun family of protooncogenes, and the formed heterodimers bind to the target gene affecting its transcription (for review see ref. 29). After colchicine injections, nearly all of the CRF-IR neurons of the PVN have been reported to exhibit Fos-LI 6. Since both G R 16'17 and F o s 29 a r e able to modify neurotransmitter gene expression and they are both present in the PB ~'6 the aim of our study was to

Fig. 1. An immunofluoresence micrograph of CGRP-LI in the PB. The CGRP-IR neurons (arrows) and nerve terminals are concentrated in the external part of the PB1. SCP, superior cerebellar peduncle. Bar = 150 tzm. Figs. 2-4. Demonstration of peptide-positive somata (arrows) and nerve terminals in the PBI after the colchicine treatment. The CGRP- and NT-IR neurons and nerve terminals are situated in the external part of the PBI (Fig. 2, Fig. 3), whereas the CRF-IR neurons (Fig. 4) are located in the dorsal part of the PBI. Bar = 150/xm.

247 co-localize GR- and stress induced Fos-LI with CGRP-, NT- and CRF-LI in the PB. Furthermore, we studied whether the number or distribution of Fos-IR cells in the PB differs between colchicine injected and immobilized animals. MATERIALS AND METHODS Adult male Sprague-Dawley rats (n = 15) were used. Five of the rats were injected intracerebroventricularly with colchicine (120 p.g in 20 /zl of 0.9% saline, Fluka AG. Chemical Fabrik, Switzerland) under chloralhydrate anaesthesia (250 mg/kg, intraperitoneally) 48 h prior to death. Five of the rats were immobilized in plastic tubes for 3 h before death. The remaining five intact rats were used as controls. The rats were perfused through the ascending aorta under chloralhydrate anaesthesia first with 100 ml of saline and then for 3 min with an ice-cold fixative containing 2% paraformaldehyde in 0.1 M phosphate buffered saline (PBS). Subsequently, the brains were excised and further fixed by immersion at 4°C in the same fixative for 60 min. The samples were cryoprotected (20% sucrose in PBS), frozen with carbon dioxide, and coronal sections (10 tzm thick) were cut with a Microm HM 500 cryostat. Several sections representing different levels in the antero-posterior orientation of the PB were processed to demonstrate Fos-, GRand peptide-like immunoreactivities. For the demonstration of Fos-LI with ABC method, a rabbit polyclonal antibody was used (dilution 1:6,000; see ref. 37). This antibody recognizes a conserved region

common to the different members of the Fos family, including c-Fos and other transcription factors that contain leucine zipper structures such as LRF-1 is and A T F / C R E B protein familiesn. Therefore, the labelling observed using this antibody may result from a variety of AP-1 binding proteins. For demonstrating GR-LI the sections were incubated with a mouse monoclonal GR antibody (1/zg/ml; see ref. 22). After incubating the samples with the Fos or GR antiserum for 24 h at 4°C, the sections were rinsed and incubated with biotinylated goat anti-rabbit antibody (1:200; Vector Labs., Burlingame, USA) for Fos and with a biotinylated sheep anti-mouse antibody (1:200; Amersham, Buckinghamshire, England) for GR followed by ABCcomplex (Vector Labs., Burlingame, USA) for 30 min each. Diaminobenzidine was used as a chromogen to visualize Fos-LIs/GRLIs. After the F o s / G R staining the sections were incubated for 12-24 h at 4°C with either rabbit antisera to NT (1:800; Peninsula Labs., Belmont, CA, USA), CGRP (1 : 800; Peninsula Labs.) or CRF (1:400; see ref. 27). After two washes the sections were incubated with a rhodamine conjugated goat anti-rabbit antibody (1:200; Boehringer-Mannheim Chemicals, Mannheim, Germany). All the antibodies were diluted in PBS containing 1% BSA and 0.3% TritonX 100. After staining the sections were embedded in a mixture of glycerol and PBS (3:1) containing 0.1% paraphenylenediamine. The sections were photographed with a Nikon Microphot FXA microscope. Controls included the omission of the primary and secondary antibodies, staining with non-immunized rabbit serum (1:6,000 for the ABC-metbod and 1 : 400 for fluorescence), non-immunized mouse IgG(l:l,000; 1 ~ g / m l for the ABC method) and peptide antisera preabsorbed with the appropriate, commercially available peptides. None of the immunoreactivities described below were observed in

~L

8b Fig. 5. A light micrograph of the distribution of GR-LI in the PB. Most of the GR-IR neurons are situated in the PBm. Bar = 150/zm. Figs. 6-8. Co-localization of the peptide-positive cells (arrows) and GR-IR cells (arrowheads) after the colchicine treatment. The CGRP- (Fig. 6), NT- (Fig. 8a,b) are mainly situated in the external PBI and the CRF-IR neurons (Fig. 7) in the dorsal PBI. No co-localization can be observed between the peptide-IR neurons and GR-LI. Bar = 100/zm.

4~ O~

249 the controls. The specificity of the Fos antiserum was controlled by incubating the antibody with corresponding M-peptide, C-terminal peptide (amino acids 246-266) and N-terminal peptide (amino acids 9-28) at 1 /.tM concentration before incubating the sections with antibody. Preabsorbtion with the corresponding peptide abolished all labelling, whereas preabsoption with either C-terminal or N-terminal peptide did not influence the staining. Fos- and GR-antisera did not stain any of the structures labelled by the peptide antisera and vice versa, showing that there was no cross-reactivity between the primary or the secondary antibodies used in the first and the second staining sequences. RESULTS

Distribution of CGRP-, NT- and CRF-IR somata and nerve terminals In intact animals the cells exhibiting C G R P - L I were clustered a r o u n d the ventrolateral tip of the SCP especially in the external PB1 (Fig. 1). N o NT- or C R F - I R cells were seen. C G R P - I R - , as well as N T - I R nerve terminals were f o u n d a r o u n d the ventral tip of SCP (data not shown). Immobilization stress did not affect the distribution or n u m b e r of any of the p e p t i d e - I R structures. A f t e r colchicine injection C G R P - p o s i t i v e cells were f o u n d in the same areas as in the intact rat, although in larger n u m b e r s (Fig. 2). F u r t h e r m o r e , several N T - I R cells were observed in the external lateral nucleus (Fig. 3) and some C R F - I R cells in the dorsal lateral nucleus (Fig. 4). T h e C G R P - and N T - I R nerve terminals surr o u n d e d the ventrolateral tip of the SCP in a similar m a n n e r as in an intact rat.

Co-localization of GR-LI with CGRP-, CRF- and NT-LIs T h e G R - I R cells were mostly f o u n d in the PBm, and only a few G R - I R cells were seen in the external or dorsal parts o f the PBI (Fig. 5). Immobilization stress or the colchicine t r e a t m e n t did not alter the n u m b e r or the distribution of the G R - I R neurons. Only an occasional n e u r o n exhibiting both C G R P (Fig. 6), C R F - (Fig. 7) or N T - L I (Fig. 8a,b) and G R - L I were seen.

Co-localization of CGRP-, NT- and CRF-LI with FOS-LI Some F o s - I R n e u r o n s were observed in intact animals (Fig. 9). Both immobilization stress (Fig. 10) and the colchicine t r e a t m e n t (Fig. 11) increased the n u m ber of F o s - I R neurons. T h e distribution o f Fos-LI was similar after the colchicine injections and immobilization stress, but the n u m b e r o f F o s - I R cells was clearly higher after the colchicine t r e a t m e n t w h e n c o m p a r e d to that caused by immobilization stress. Cells containing Fos-LI were located on both the medial and lateral sides of the SCP, although the majority of the Fos-LI n e u r o n s were c o n c e n t r a t e d on the lateral side. T h e vast majority of the NT- (Fig. 12), and C G R P - I R (Fig. 13) cells in the external PBI and of the C R F - I R cells in the dorsal PBI (Fig. 14) exhibited Fos-LI after the colchicine treatment. In addition, the C G R P - I R neurons in the external PB1 were co-localized with the F o s - L I cells after immobilization stress (Fig. 15).

DISCUSSION T h e present p a p e r describes the localization o f Fosand G R - L I in the C G R P - , NT- and C R F - I R n e u r o n s in the PB. Both o f these transcription factors were found in the PB, but only Fos-LI was co-localized with the studied peptides. G R - I R n e u r o n s were concentrated in the PBm, which thus appears to be the major target in the PB for glucocorticoid hormones. T h e lack of G R - I R in the studied p e p t i d e - I R n e u r o n s in the PB implies that glucocorticoids do not have direct effects on the peptides studied in the PB. It should be noted, however, that n e u r o n s in the PB contain o t h e r neuropeptides (for review see ref. 23), and G R - L I may be co-localized with some of these neuropeptides. It has b e e n previously d e m o n s t r a t e d that colchicine injections constitute a strong stressful stimulus that results in increased a m o u n t s of F o s - L I in various brain nuclei 3 - 6 h after the colchicine injection, followed by a m o d e r a t e decrease in Fos-LI n e u r o n s 1 2 - 2 4 h after

Figs. 9-11. Distribution of Fos-IR neurons in the PB in an intact rat (Fig. 9), after immobilization stress (Fig. 10) and after the colchicine treatment (Fig. 11). The immobilization stress increases the amount of Fos-IR neurons similarly as the colchicine treatment. However, the induction of Fos is clearly stronger after the colchicine treatment when compared to that caused by immobilization stress. Bar = 150/xm. Figs. 12-14. Co-localization of NT- (Fig. 12), CGRP- (Fig. 13) and CRF-IR (Fig. 14) neurons with Fos-LI after the colchicine treatment. Nearly all the peptide-IR neurons contain also Fos-LI. Bar = 50/zm. Fig. 15. Co-localization of the CGRP- and Fos-LI in the PB after immobilization stress. The vast majority of the CGRP-IR neurons contain also Fos-LI. Bar = 50/zm.

250 the injection 6. Our observation of large numbers of Fos-LI neurons still after 48 h may result from the fact that we used an antiserum, which recognizes several Fos-related transcription factors 37. The trauma caused by the needle penetrating the brain tissue and the cytotoxic effect of colchicine cause an intense expression of Fos around the injection site (unpublished observations). However, in the PB the distribution of Fos-LI produced by the colchicine injection was similar to that after stress, although the number of Fos-LI neurons was higher. This suggests that the expression of Fos in the PB and possibly in other brainstem nuclei, is caused by intense stressful stimuli, not by the direct cytotoxic effect of colchicine or the trauma caused by the injection p e r se. The fact that Fos-LI was localized in the CGRP-, NT- and C R F - L I cells suggests that Fos may affect the synthesis of these peptides. However, the Fos antibody used in this study is not totally specific for Fos and thus further studies must be carried out to determine which Fos- a n d / o r Fos-related proteins are activated. Furthermore, the precise changes caused by the Fos protein in the synthesis of neurotransmitters have not yet been clarified. In addition, since we observed many Fos-LI neurons that did not contain any of the peptides studied, it seems likely that also neurons that contain other neuropeptides or transmitters are activated after both the immobilization stress and the colchicine treatment. After stress and colchicine injections Fos-LI has been observed in a large number of neurons in the parvocellular portion of the PVN 6, which contains most of the C R F - I R neurons that project to the median eminence 34. Thus, it is possible that Fos directly stimulates the expression of C R F in the PVN. This effect may also occur in the CRF, NT and C G R P - I R neurons of the PB, but so far there are no reports describing such an effect. NT- and C G R P - L I s have been demonstrated to coexist partly in the same neurons of the PB131. These peptidergic neurons of the PB project to the ACe 3'3°, where these peptides have been shown to cause various stress related responses. Injections of C G R P into the ACe raise the blood pressure 5'2°, injections of NT decrease the formation of gastric stress ulcers ~°, and injections of C G R P increase norepinephrine release from the adrenal medulla 5. The PB receives substantial input linked with cardiovascular regulation 8 and gastric acid secretion 36. Since we observed a significant co-localization of the C G R P - and N T - I R cells with Fos-LI, it is likely that the pathway from the PB to the ACe is activated in stress and may relay autonomic input from the viscera to this limbic system structure. The C G R P and NT-positive terminals have been shown to inner-

vate cells exhibiting Fos-LI after stress in the ACe 14, providing further evidence that the pathway from the PB that contains C G R P and N T activates the ACe under stressful conditions. Therefore, the PB is likely to have a major role in relaying stress related information to the ACe and thus promoting the changes mediated by the ACe. The CRF-positive cells of the dorsal PB1 project to the M P O A ~8, which contains luteinizing hormone-releasing hormone (LHRH)-positive neurons that project to the median eminence 32 controlling the hypophysialgonadal axis. Stress has been shown to decrease the secretion of L H R H (for review see ref. 25). It has been suggested that the mechanism by which stress affects the secretion of L H R H could be mediated by CRF, since CRF-positive nerve terminals synapse with the L H R H cells in the M P O A 19 and it has been suggested that injections of C R F into the M P O A decrease the secretion of L H R H 25. Thus, the C R F - I R neurons in the PB, which were shown to be activated in stress, may in part control the secretion of L H R H in the median eminence and decrease it during stress. In conclusion, our study demonstrates that stress induced Fos-LI but not G R - L I is localized in NT-, C G R P - and C R F - I R neurons in the parabrachial nucleus. Therefore, stress appears to activate the neurons containing these neuropeptides and they are likely to mediate stress related autonomic information to the ACe and MPOA. Furthermore, Fos, but not GR, may affect the gene expression of the peptides studied under stressful conditions. Acknowledgements. We thank Drs. M. Iadarola, NIDR, NIH, USA,

and W. Vale, the Salk Institute, La Jolla, CA, USA, for the generous supply of the antisera. The skilful technical assistance of Ms. Hannele Ylitie, Mrs. Marketta Vuorinen and Mr. Harri Nyfors is gratefully appreciated. REFERENCES 1 Ahima, R.S. and Harlan, R.E., Charting of type II glucocorticoid receptor-like immunoreactivityin the rat central nervous system, Neuroscience, 39 (1990) 579-604. 2 Beato, M., Gene regulation by steroid hormones, Cell, 56 (1989) 335-344. 3 Block, C.H., Hoffman, G. and Kapp, B.S., Peptide-containing pathways from the parabrachial complex to the central nucleus of the amygdala, Peptides, 10 (1989) 465-71. 4 Block, C.H. and Hoffman, G.E., Neuropeptide and monoamine components of the parabrachial pontine complex, Peptides, 8 (1987) 267-283. 5 Brown, M.R. and Gray, T.S., Peptide injections into the amygdala of conscious rats: effects on blood pressure, heart rate and plasma catecholamines, Reg. Pept., 21 (1988) 95-106. 6 Ceccatelli, S., Villar, M.J., Goldstein, M. and H6kfelt, T., Expression of c-Fos immunoreactivityin transmitter-characterized neurons after stress, Proc. Natl. Acad. Sci. USA, 86 (1989) 9569-9573. 7 Ceccatelli, S., Cintra, A., H6kfelt, T., Fuxe, K., Wikstr6m, A.-C. and Gustafsson, J.-A., Coexistence of glucocorticoid receptor-like

251 immunoreactivity with neuropeptides in the hypothalamic paraventricular nucleus, Exp. Brain Res., 78 (1989) 33-42. 8 Darlington, D.N. and Ward, D.G., Rostral pontine and caudal mesencephalic control of arterial pressure and iliac, celiac and renal vascular resistance. II. Separate control and topographic organization, Brain Res., 361 (1985) 301-308. 9 Fulwiler, C.E. and Saper, C.B., Subnuclear organization of the efferent connections of the parabrachial nucleus in the rat, Brain Res. Rev., 7 (1984) 229-259. 10 Glavin, G.B., Murison, R., Overmier, J.B., Pare, W.P., Bakke, H.K., Henke, P.G. and Hernandez, D.E., The neurobiology of stress ulcers, Brain Res. Rev., 16 (1991) 301-343. 11 Hai, T. and Curran, T., Cross-family dimerization of transcription factors Fos/Jun and A T F / C R E B alters DNA binding specificity, Proc. Natl. Acad. Sci. USA, 88 (1991) 3720-3724. 12 Harbuz, M.S. and Lightman, S.L., Responses of hypothalamic and pituitary mRNA to physical and psychological stress in the rat, J. Endocrinol., 122 (1989) 697-704. 13 Herbert, H. and Saper, C.B., Cholecystokinin-, galanin-, and corticotropin releasing factor-like immunoreactive projections from the nucleus of the solitary tract to the parabrachial nucleus in the rat, J. Comp. Neurol., 293 (1990) 581-598. 14 Honkaniemi, J., Co-localization of peptide- and tyrosine hydroxylase-like immunoreactivities with Fos-immunoreactive neurons in rat central amygdaloid nucleus after stress, Brain, Res., 598 (1993) 107-113. 15 Hsu, J.-C., Laz, T., Mohn, K.L. and Taub, R., Identification of LRF-1, a leucine-zipper protein that is rapidly and highly induced in regenerating liver, Proc. Natl. Acad. Sci. USA, 88 (1991) 3511-3515. 16 Imaki, T., Nahan, J.-L., Rivier, C., Sawchenko, P.E. and Vale, W., Differential regulation of corticotropin-releasing factor mRNA in rat brain regions by glucocorticoids and stress, J. Neurosci., 1l (1991) 585-599. 17 Jingami, H., Matsukara, S., Numa, S. and Imura, H. Effects of adrenalectomy and dexamethasone administration on the level of prepro-corticotropin-releasing factor messenger ribonucleic acid (mRNA) in the hypothalamus and adrenocorticotropin//3-1ipotropin precursor mRNA in the pituitary in rats, Endocrinology, 117 (1985) 1314-1320 18 Lind, R.W. and Swanson, L.W., Evidence for corticotropin releasing factor and leu-enkephalin in the neural projection from the lateral parabrachial nucleus to the median preoptic nucleus: a retrograde transport, immunocytochemical double labeling study in the rat, Brain Res., 321 (1984) 217-224. 19 Mac Lusky, N.J., Naftolin, F. and Leranth, C., Immunocytochemical evidence for direct synaptic connections between corticotropin-releasing factor (CRF) and gonadotrophin-releasing hormone (GnRH)-containing neurons in the preoptic area of the rat, Brain Res., 439 (1988) 391-395. 20 Nguyen, K.Q., Sills, M.A. and Jacobwitz, D.M., Cardiovascular effects produced by microinjection of calcitonin gene-related peptide into the rat central amygdaloid nucleus, Peptides, 7 (1986) 337-339. 21 Norgren, R., Projections from the nucleus of the solitary tract in the rat, Neuroscience, 3 (1978) 207-218.

22 Okret, S., Wikstr6m, A.-C., Wrange, t), Andersson, B. and Gustafsson, J.-A., Monoclonal antibodies against the rat liver glucocorticoid receptor, Proc. Natl. Acad. Sci. USA, 81 (1984) 1609-1613. 23 Palkovits, M., Neuropeptides in the brain. In L. Martini and W.F. Ganong (Eds.), Frontiers in Neuroendocrinology, Raven, 1988, 1-44.

24 Riche, D., De Pommery, J. and Menetrey, D., Neuropeptides and catecholamines in efferent projections of the nuclei of the solitary tract in the rat, J. Comp. Neurol., 293 (1990) 399-424. 25 Rivier, C. and Rivest, S., Effect of stress on the activity of the hypothalamic-pituitary-gonadal axis: peripheral and central mechanisms, Biol. Reprod., 45 (1991) 523-532. 26 Sagar, S.M., Sharp, F.R. and Curran, T., Expression of c-Fos protein in brain: metabolic mapping at the cellular level, Science, 240 (1988) 1328-1330. 27 Sawchenko, P.E., Swanson, L.W. and Vale, W.W., Co-expression of corticotropin-releasing factor and vasopressin immunoreactivity in parvocellular neurosecretory neurons of the adrenalectomized rat, Proc. Natl. Acad. Sci. USA, 81 (1984) 1883-1887. 28 Sharp, F.R., Sagar, S.M., Hicks, K., Lowenstein, D. and Hisanaga, K., c-fos mRNA, Fos, and Fos-related antigen induction by hypertonic saline and stress, J. Neurosci., 11 (8) (1991) 2321-2331. 29 Sheng, M. and Greenberg, M.E., The regulation and function of c-Fos and other immediate early genes in the central nervous system, Neuron, 4 (1990) 477-485. 30 Shimada, S., Shiosaka, S., Emson, P.C., Hillyard, C.J., Girgis, S., Maclntyre, L. and Tohyama, M., Calcitonin gene-related peptidergic projection from the parabrachial area to the forebrain and diencephalon in the rat: an immunohistochemical analysis, Neuroscience, 16 (1985) 607-616. 31 Shinohara, Y., Yamano, M., Matsuzaki, T. and Tohyama, M., Evidences for the coexistence of substance P, neurotensin and calcitonin gene-related peptide in single neurons of the external subdivision of the lateral parabrachial nucleus of the rat, Brain Res. Bull., 20 (1988) 257-260. 32 Silverman, A.-J., Jhamandas, J. and Renaud, L.P., Localization of luteneizing hormone-releasing hormone (LHRH) neurons that project to the median eminence, J. Neurosci., 7 (1987) 2312-2319. 33 Sutin, E.L. and Jacobowitz, D.M., Immunocytochemical localization of peptides and other neurochemicals in the rat laterodorsal tegmental nucleus and adjacent area, J. Comp. NeuroL, 270 (1988) 243-270. 34 Swanson, L.W., Sawchenko, P.E., Rivier, J. and Vale, W.W., Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunocytochemical sudy, Neuroendocrinology, 36 (1983) 165-186. 35 Tribollet, E. and Dreifuss, J.J., Localization of neurones projecting to the hypothalamic paraventricular nucleus area of the rat: a horseradish peroxidase study, Neuroscience, 6 (1981) 1315-1328. 36 Yuan, C.S. and Barber, W.D., Parabrachial nucleus - neuronal evoked responses to gastric vagal and greater splanchnic nerve stimulation, Brain Res. Bull., 27 (1991) 797-803. 37 Young, S.T., Porrino, L.J. and Iadarola, M.J., Cocaine induces striatal c-Fos-immunoreactive proteins via dopaminergic D1 receptors, Proc. Natl. Acad. Sci. USA, 88 (1991) 1291-1295.