Proc. Nati. Acad. Sci. USA Vol. 85, pp. 953-957, February 1988 Physiological Sciences Evidence for a physiological role of hypothalamic -releasing peptide to suppress growth and prolactin release in the rat S. KENTROTI, W. L. DEES, AND S. M. MCCANN University of Texas Health Science Center at Dallas, Department of Physiology, Dallas, TX 75235 Contributed by S. M. McCann, October 2, 1987

ABSTRACT Gastrin-releasing peptide (GRP) is localized within the rostral (6). The anatomical distri- to hypothalamic neurons and is a potent inhibitor of basal and bution of GRP suggests a role for this peptide in the control (GH)-releasing factor-induced GH secretion of function. in the rat. It also acts similarly to inhibit opiate- and stress- Intravenous administration of GRP stimulates the secre- induced prolactin (PRL) release. To determine the physiolog- tion of several gastroenteropancreatic , among ical significance of the peptide in the control of the release of them gastrin, , cholecystokinin (9), and insulin these two hormones, a highly specific antiserum against GRP (10). Central administration of GRP and related peptides via was injected into the third ventricle to immunoneutralize the brain ventricles produces profound physiological and hypothalamic GRP. The injection ofthe antiserum initially did behavioral effects, including a significant decrease in gastric not alter levels of the hormones; however, both PRL and GH acid secretion (11), poikilothermy (12), grooming behavior levels in the plasma began to increase within 3 and 3.5 hr, (13), and decreased food intake (14). respectively. They were still significantly elevated 24 hr after In previous studies, we have demonstrated the ability of the injection. There was no change in the plasma levels of GRP to inhibit growth hormone (GH) release in vivo. Within either hormone in animals injected intraventricularly with a 10 min after intraventricular (i.v.t.) injection of minute doses similar volume of normal rabbit serum (NRS). Mean plasma (10-12-10-9 mol) of GRP, basal GH levels were significantly GH levels 24 hr after antiserum injection were more than twice depressed and all spontaneous GH pulses were abolished for those of the NRS-inJected controls, whereas the PRL concen- periods in excess of 90 min. GRP also blocked the GH surge trations were 14-fold higher in the antiserum injected as in response to GH-releasing factor (15, 16). We have further compared to the control NRS-injected animals. A second shown that similar injection of antiserum 24 hr after the first adminta- this GH release inhibitory activity resides within tion resulted in a slight and transient further increase in both the C-terminal heptapeptide by testing both amino- and GH and PRL levels so that they were both significantly (P < carboxyl-terminal fragments (17). It is within the C-terminal 0.001) higher than those of the animals given a second decapeptide that GRP resembles bombesin, the 14-amino hnjection of NRS. The anti-GRP antiserum was highly specific acid amphibian peptide, which also possesses GH inhibitory for GRP by radioimmunoassay procedures and this antiserum activity (17). produced positive immunostaining of GRP neuronal perikarya Matsushita et al. (18) reported an inhibitory action of GRP and terminals within discrete hypothalamic nuclei. Beaded on opiate-induced prolactin (PRL) secretion. We have also fibers and terminals were observed in the suprachiasmatic observed that GRP can inhibit the increase in plasma PRL nucleus (SCN) and the area lateral and dorsal to the SCN in induced by ether stress (unpublished data). We have further the region of the periventricular nucleus (PeVN). GRP- shown that somatostatin is an important mediator in the positive perikarya were observed in the parvocellular neurons inhibitory response of GRP on GH release (19) and that GRP of the paraventricular nucleus. In addition, GRP-positive cell acts via a dopaminergic mechanism to block the stress- bodies were observed in the PeVN in close proximity to the induced release of PRL.* In this study, we examined the third ventricle. Furthermore, the median eminence displayed physiologic significance of hypothalamic GRP in the control no immunostaining for GRP, and all traces of positive staining of GH and PRL release by immunoneutralizing the endoge- were abolished by preabsorption of the antiserum with GRP- nous peptide with a highly specific antiserum. This antise- 27 (30 ag/ml), confirming the specificity of the antiserum. rum was also used in immunohistochemical studies that The combined results with immunoneutralization of GRP and reveal a GRP neuronal system of GRP-like immunostaining the immunostaining of GRP neuronal elements in the hypo- in the rat hypothalamus. support the physiological role of this peptide in the inhibitory control of both GH and PRL release. MATERIALS AND METHODS Gastrin-releasing peptide (GRP) is one of the many peptides In Vivo Experiments. All experiments were performed on now found to be present in hypothalamic neurons. It is a female Sprague-Dawley rats (Simonsen Laboratories, Gil- 27-residue molecule originally isolated from porcine non- roy, CA) weighing 260-280 g. The rats were ovariectomized antral stomach (1). It is related to the amphibian skin peptide at least 3 weeks prior to each experiment so that the animals bombesin and has been localized in tissues from various would be in a comparable hormonal state to those used mammalian species including the human (2-5). The highest previously (15) and to eliminate ovarian steroid feedback detectable concentrations of GRP are found in tissue ex- tracts from brain (6) and gut (7, 8). Within the brain, the Abbreviations: GRP, gastrin-releasing peptide; GH, growth hor- highest concentrations of GRP-like peptides can be found mone; i.v.t., intraventricular(ly); PRL, prolactin; NRS, normal rabbit serum; SCN, ; PeVN, periventricular nucleus; PVN, paraventricular nucleus. The publication costs of this article were defrayed in part by page charge *Kentroti, S. & McCann, S. M., Proceedings of the International payment. This article must therefore be hereby marked "advertisement" Symposium on Bombesin-Like Peptides in Health and Disease, in accordance with 18 U.S.C. §1734 solely to indicate this fact. October 14, 1987, Rome, abstr. 217. 953 954 Physiological Sciences: Kentroti et al. Proc. Natl. Acad. Sci. USA 85 (1988) effects on pituitary hormone release. Animals were con- * Antiserum - treated Animals (n=7) scious and unrestrained during all experiments. 1- Lyophilized antiserum to porcine GRP (anti-GRP, serum E 100 o N RS - treated 7188) was obtained from Peninsula Laboratories (San Animals (n=7) Carlos, CA). This antiserum was shown to crossreact 100% CY with porcine GRP(1-27), -(14-27), -Ac(20-27), and bombe- _ 80 sin in radioimmunoassay. It cross-reacted only 0.1% with substance P and did not cross-react at all with motilin, 0go 60 secretin, vasoactive intestinal polypeptide, gastrin, or chole- E cystokinin(26-33). The lyophilized antiserum was brought to its original volume with distilled water for i.v.t. injection. All '40 F- other reagents were obtained from Sigma unless otherwise 3. _Q w1 specified. to 20 *"%t-E T In the first experiment, a 23-gauge stainless steel cannula (9 was' implanted into the third brain ventricle of each rat 1 NRS (3P1l,lVT) week prior to the experiment (20). Cannulae were implanted r-, L I I I I I I in the externaljugular vein 24 hr prior to the experiment (21). -20 -10 0 10 20 30 40 50 Animals received' a preliminary 3-kId dose of either anti-GRP or normal rabbit serum (NRS) i.v.t. at this time. On the Time (min) following day, the rats received a second injection of anti- FIG. 1. A single 3-1.l administration of GRP-specific antiserum GRP or NRS (3 1.l, i.v.t.). The protocol of antiserum into the third ventricle 24 hr prior to blood sampling resulted in a injection was the same as that used successfully in earlier 2-fold increase (P < 0.001) in basal GH levels as illustrated here. A studies.t Blood samples of 400 ,ul (heparinized) were drawn subsequent 3-sil administration of antiserum at time 0 resulted in a at 10-min intervals and replaced with an equal volume of slight transient surge in GH levels that was significant only when physiological saline throughout the procedure to maintain compared to the net decrease in GH levels observed in control blood volume. After the experiment, blood samples were animals. Values shown are means ± SEM of seven measurements. centrifuged at low speed and the plasma was decanted and frozen for RIA of GH and PRL. The protocol of the second synthetic porcine GRP 24 hr prior to incubation of control experiment was virtually the same except that the animals sections. received a single dose of anti-GRP or NRS (3 .l, i.v.t.) on the day of the experiment and plasma GH and PRL levels RESULTS were measured every 30 min over a 4.5-hr period to deter- mine the time course of action of the antiserum. GH. Twenty-four hours after the first i.v.t. injection and Plasma samples were assayed in duplicate for GH and just prior to the second injection, plasma GH levels in GRP PRL concentrations and results were expressed in terms of antiserum-injected animals were significantly (P < 0.001) the National Institute of Diabetes, Digestive and Kidney elevated above those in the NRS-injected controls (63.3 ± Diseases (NIDDK) rat-RP-1 standards. Statistical probabil- 15.9 vs. 28.4 ± 3.1 ng/ml, respectively) (Fig. 1). After the ities were calculated by analysis of variance with repeated second i.v.t. injection of anti-GRP antiserum, there was a measures (Newman-Keuls test). Student's t test was used slight but not significant further increase in plasma GH and for comparison of two means. the levels remained elevated for the rest of the sampling Immunohistochemistry. Animals were divided into two period of 50 min. In contrast, there was a slight but nonsig- groups of three animals each. The first group received nificant decline to lower levels of plasma GH in the NRS- colchicine (40 ,ug per 2 1LI, i.v.t.) 24 hr prior to sacrifice to injected control animals so that the difference between facilitate identification of neuronal perikarya. All six animals plasma GH in the two groups remained highly significant were anesthetized with Equithesin, flushed via cardiac per- (Fig. 2A). The mean increase in plasma GH in the antiserum- fusion with physiological saline, and fixed with Zamboni's fixative. A block of tissue consisting of the , A Growth B Prolactin hypothalamus, and median eminence was isolated from each 40 - Hormone brain and post-fixed in the same fixative for 24 hr. Trans- verse serial sections 50 Am thick were cut from each tissue block and stained for GRP by the indirect immunoper- 30m oxidase technique of Sternberger (22). All reagents for the peroxidase-antiperoxidase procedure were prepared in phos- phate-buffered saline' (PBS) (pH 7.4) containing 2% normal 7~20 sheep serum. The tissue was first incubated at 4°C for 48 hr in a 1:400 dilution of the same rabbit anti-porcine GRP used CP earlier followed by incubation at 25°C for 30 min in a 1:100 10 dilution of sheep anti-rabbit IgG (Miles). The tissue was then placed in a solution of 3,3'-diaminobenzidine tetrachloride (7.5 mg per 50 ml of PBS) to which 50 ,ul of 3% H202 had 0 been added. The sections were incubated at room tempera- ture for 5-10 min with gentle agitation, after which they were thoroughly washed in PBS (pH 7.4), dehydrated with alcohol and xylene, and covered with glass cover slides. To verify -10 Lantl-GRP NRS anti-GRP NRS the specificity of the primary antiserum, 1.0 ml of the FIG. 2. Within 20 min after a second 3-1.d administration of a anti-GRP (1:100 dilution) was preabsorbed with 30 ,ug of GRP-specific antiserum, mean plasma levels of both GH (A) and PRL (B) showed a significant increase (P < 0.001 and P = 0.05, tKhorram, 0., DePalatis, L. R. & McCann, S. M., 64th Annual respectively) when compared with the change observed in NRS- Meeting of the Endocrine Society, June 18, 1982, San Francisco, p. treated control animals. Values shown indicate the means ± SEM 294, abstr. 860. of seven measurements. Physiological Sciences: Kentroti et al. Proc. Natl. Acad. Sci. USA 85 (1988) 955

120 r 40 r *-* - Antiserum -treated Animals (n=7) 0-o= NRS-treated Animals (n=7) 100- 30 N071 w-lE *-* = Antiserum -treated Animals (n=7) c o-o= NRS-treated Animals (n=7) CP 80F ,, 20 0 .5 0 a- E 601- .,, 10 [ 0 NRS/Anti-GRP (3/L, IVT)

401F - I (9 -15 0 15 30 60 90 120 150 180 200 240 270 20 Time (min) FIG. 5. PRL levels monitored for 4.5 hr after a L I single 0 3-p.) -15 0 15 30 60 90 120 150 180 210 240 270 administration ofGRP antiserum into the third brain ventricle (i.v.t.) Time (min) displayed a significant (P < 0.001) increase by 3 hr after injection. Values shown are means ± SEM of seven measurements. FIG. 3. GH levels monitored for 4.5 hr after a single 3-pAl injection of GRP antiserum into the third brain ventricle (i.v.t.). GH animals (P < 0.001) (Fig. 5). Plasma PRL levels continued to levels demonstrated a significant (P < 0.001) increase 3.5 hr after increase and reached the peak at the termination of sampling injection. Values shown are means ± SEM of seven measurements. at 270 min. Values of PRL remained low and stable through- out the sampling period in the NRS-injected control animals. injected was animals significantly different from the de- Immunohistochemistry. Examination of the preoptic area crease observed in the NRS-injected controls (P < 0.001) and the entire rostrocaudal extent of the hypothalamus and (Fig. 2A). median eminence revealed that GRP-like immunoreactivity Since the plasma GH levels were already elevated 24 hr was confined to the rostral area. No GRP- after the first injection of hypothalamic antiserum, it appeared ofinterest to positive fibers or perikarya were detected in the preoptic examine the time course of the effect of a single injection of area in either colchicine-treated or antiserum. After this non-colchicine-treated single injection, plasma GH concentra- animals. Beaded fibers and terminals were observed in tions remained virtually unchanged for 150 the min and then suprachiasmatic nucleus (SCN), the area lateral to SCN, and gradually increased so that they were significantly elevated dorsally in the region of the periventricular nucleus (PeVN) by 210 min after the injection (P < 0.001) (Fig. 3). GH levels in non-colchicine-treated animals. A continued to increase throughout the remainder of the sparse population of sam- fibers was observed in the of pling period to a peak level of 102 12 ng/ml at 270 min. GRP-positive PeVN region the anterior no fibers were detected Prolactin. Results with PRL were similar to those just hypothalamus; however, described caudal to this area, nor were any fibers observed in the for GH, but the effects of the antiserum were even median eminence. more pronounced. Initial prolactin levels were highly signif- GRP-positive perikarya were restricted to icantly elevated in animals that had received anti-GRP the rostral hypothalamus, specifically to the PeVN and the antiserum i.v.t. 24 hr previously when compared to the parvocellular neurons at the ventral border of the paraven- values in the NRS-injected controls (P < 0.001) (Fig. 4). tricular nucleus (PVN) in rats injected i.v.t. with colchicine After the second injection of anti-GRP (Fig. 6). Preabsorption of the primary antiserum with GRP- i.v.t., there was a 27 slight further increase in plasma prolactin and a slight completely abolished all evidence of immunostaining in areas decrease in the NRS-injected controls so that there was a the previously described. further significant difference in the behavior of prolactin in these two groups within the 20 min following the second DISCUSSION injection (P = 0.05) (Fig. 2B). Consequently, the time course of the effect of a single Growth Hormone. Our earlier studies have established the i.v.t. injection was determined. Values remained virtually fact that GRP can inhibit GH release following its i.v.t. unchanged after i.v.t. injection of either antiserum or NRS injection via a dopaminergic mechanism that results in and began to- increase at 150 min in the antiserum-injected stimulation of somatostatin release (17, t, §). The somato- statin induces a decline in plasma GH, which is associated 100 with blockade of the response to GH-releasing factor. The present results indicate that this action is physiologically since 80 significant immunoneutralization ofhypothalamic GRP with highly specific GRP antisera resulted in an increase in plasma GH. This highly specific antiserum directed against E >' 60 the carboxyl terminus of GRP was used to immunostain neuronal structures IS o GRP-containing in the hypothalamic region. The anti-GRP-staining fibers and terminals were intermingled with fibers and perikarya of anti-somatostatin- a- staining neurons, which provides an anatomical basis for the 20 observed stimulation of somatostatin release by GRP.$ Previous immunoneutralization studies of this type have involved a double injection of the antisera because measure- -20 -10 0 10 20 30 40 50 Time (min) tKentroti, S. & McCann, S. M., Neural and Endocrine Peptides and FIG. Receptors Symposium, May 28-31, 1985, Washington, DC, abstr. 4. A single 3-.ul injection of GRP antiserum into the third 163. ventricle 24 hr prior to blood sampling resulted in a highly significant §Kentroti, S. & S. 67th Annual of the (P < 0.001) increase in basal PRL levels as shown here. Subsequent McCann, M., Meeting Endocrine Society, June 19, 1985, Baltimore, p. 37, abstr. 146. administration of 3 A.l of GRP antiserum at time 0 produced a S. & W. First International transient increase in plasma PRL levels within 20 lKentroti, Dees, L., Congress of min. Values , July 10, 1986, San Francisco, p. 70, abstr. shown are means + SEM of seven measurements. 182. 956 Physiological Sciences: Kentroti et al. Proc. Natl. Acad Sci. US-A 85 (1988) has also been shown to stimulate somatotropin- release inhibiting factor release in vitro (27, 28). Finally, we have examined the specificity of this antise- _t rum in immunohistochemical studies and found GRP- positive immunostaining in the SCN and PVN as described V by Roth et al. (2). In addition, we observed GRP-positive -."~~~~~~~~~~~~~~~~~~"cell bodies in the PeVN in close proximity to the third ventricle. Furthermore, all labeling disappeared when the antibody was preabsorbed with GRP-27, indicating the spec- ificity of the antiserum. We have noted that GRP-containing FIG. 6. A frontal section through the rostral hypothalamus neurons and perikarya in the hypothalamus occur solely in displayed positive immunostaining for GRP in periventricular struc- nuclei surrounding the third ventricle. GRP-like immu- tures. GRP-positive cell bodies in the PVN and the PeVN (indicated nostaining in the rat hypothalamus is confined to a few by arrows) in close proximity to the third ventricle (v) were stained discrete nuclei including the SCN, PeVN, and the parvocel- using the same antiserum as previously used in the in vivo proce- lular part of the PVN. The close association of these dures. elements to the third ventricle allows for rapid uptake and action ofthe antiserum in the appropriate tissues. In contra- ment of hormones shortly after injection of antisera had distinction, Kita et al. (29) reported that in the rabbit failed to reveal any effect (23). In the present study, it was hypothalamus the highest concentrations of GRP-like immu- noticed that plasma GH levels were increased 24 hr after the noreactivity are found in the ventromedial and infundibular first injection and showed little further change in response to nuclei followed by PVN, SCN, and PeVN. Their results a second injection of antisera, suggesting that the' immuno- concur with ours in the absence of GRP-like immunoreac- neutralization of hypothalamic GRP was nearly complete tivity in the median eminence (29). These findings suggest even 24 hr after the first injection. Consequently, we evalu- species variability with respect to GRP localization and ated the time course of the effect in the present experiments content in the mammalian brain. and showed that plasma GH began to increase 3.5 hr after The present study has enabled us to appreciate the pro- injection and reached the highest level at sampling 270 min found effect endogenous GRP has on both plasma GH and after injection. prolactin levels. The results presented here in association The time required for the elevation to take place probably with previous studies suggest that hypothalamic GRP is an represents the' time required for the antiserum to be ab- important neuromodulator ofanterior pituitary function with sorbed from the third ventricle either passively or by some a primary action to inhibit GH and PRL secretion. active mechanism' involving the ependymal cells. The re- maining interval probably represents the time to diffuse to 1. McDonald, T. J., Nilsson, G., Vagne, M., Ghatei, M., Bloom, the active sites of interaction of GRP with somatostatin. We S. R. & Mutt, V. (1978) Gut 19, 767-774. speculate that the longer the latency for the effect, the 2. Roth, K. A., Weber, E. & Barchas, J. D. (1982) Brain Res. 251, 277-282. the ventricle are the sites. In the case i.v.t. further from of 3. Roth, K. A., Evans, C. J., Lorenz, R. G., Weber, E., Bar- injection of antisera directed against galanin, the response chas, J. D. & Chang, J.-K. (1983) Biochem. Biophys. Res. can occur within 30 min, which suggests that the interaction Commun. 112, 428-435. is taking place very close to the ventricular wall (A. Ottlecz 4. Kulik, T. J., Johnson, D. E., Elde, R. P. & Lock, J. E. (1983) and S.M.M., unpublished data). J. Appl. Physiol. Respir. Environ. Exercise Physiol. 55, GH release is pulsatile in the rat and GRP was capable of 1093-1097. blocking the pulses, probably via stimulation of the release 5. Tsutsumi, Y., Osamura, R. Y., Watanabe, K. & Yanaihara, N. of which then blocked the response of the (1983) Lab. Invest. 48, 623-632. somatostatin, 6. Moody, T. W. & Pert, C. B. (1979) Biochem. Biophys. Res. pituitary to the release of GRF, which initiates the pulses Commun. 90, 7-14. (24). It is possible, however, that GRP may inhibit the 7. Moghimzadeh, E., Ekman, E., Hakanson, R., Yanaihara, N. release of GRF as well. We have no data to answer this & Sundler, F. (1983) Neuroscience 10, 553-563. question. 8. Costa, M., Furness, J. B., Yanaihara, N., Yanaihara, C. & PRL. The report of Matsushita et al. (18) has established Moody, T. W. (1984) Cell Tissue Res. 235, 285-293. the inhibitory action of GRP on opiate-induced PRL release 9. Inoue, K., McKay, D., Yajima, H. & Rayford, P. L. (1983) in vivo. Tache et al. similar results with Peptides 4, 153-157. (25) reported 10. Bloom, S. R., Edwards, A. V. & Ghatei, M. A. (1983) J. stress-induced PRL release with the related peptide bombe- Physiol. (London) 344, 37-48. sin. We have also seen an inhibitory response using GRP 11. Tache, Y., Marki, W., Rivier, J., Vale, W. & Brown, M. (1981) itself (unpublished observations). The effect of GRP antise- Gastroenterology 81, 298-302. rum to elevate plasma PRL levels was more rapid in onset (3 12. Tache, Y., Pittman, Q. & Brown, M. (1980) Brain Res. 188, hr) and more pronounced than that observed for GH. The 525-530. more rapid onset suggests that the structures involved may 13. Kulkosky, P. J., Gibbs, J. & Smith, G. R. (1982) Physiol. be closer to the ventricle or more sensitive to the regulatory Behav. 28, 505-512. 14. Stein, L. J. & Woods, S. C. (1980) Peptides (NY) 3 (5), effects of GRP in the case of PRL than in the case of the 833-835. GH-controlling system. These results suggest that hypotha- 15. Kentroti, S. & McCann, S. M. (1985) 117, lamic GRP plays an important role in the tonic inhibition of 1363-1367. PRL release. 16. Kabayama, Y., Kato, Y., Shimatsu, A., Ohta, H., Yanaihara, Our previous studies have implicated the N. & Imura, H. (1984) Endocrinology 115, 649-653. dopamine in the inhibitory response of GH to GRP.§ Fur- 17. Kentroti, S. & McCann, S. M. (1985) Fed. Proc. Fed. Am. thermore, Widerlov et al. report that bombesin, a GRP-like Soc. Exp. Biol. 44, 632 (abstr. 12%). 18. Matsushita, N., Kato, Y., Katakami, H., Shimatsu, A., Ya- peptide, increases dopamine turnover in vitro in the hypo- naihara, N. & Imura, H. (1983) Proc. Soc. Exp. Biol. Med. 172, thalamus (26). On the basis of this information, it seems 118-121. reasonable to suggest that GRP may act to inhibit PRL 19. Kentroti, S., Aguila, M. C. & McCann, S. M. (1988) Endocri- release via a dopaminergic mechanism. This may also pro- nology, in press. vide a mechanism whereby GRP inhibits GH release, since 20. Antunes-Rodrigues, J. & McCann, S. M. (1970) Proc. Soc. Physiological Sciences: Kentroti et al. Proc. Natl. Acad. Sci. USA 85 (1988) 957

Exp. Biol. Med. 133, 1464 1470. 26. Wilderlov, E., Mueller, R. A., Frye, G. D. & Breese, G. R. 21. Harms, P. G. & Ojeda, S. R. (1974) J. Appl. Physiol. 36, (1984) Peptides (Fayetteville, NY) 5, 523-528. 391-393. 27. Negro-Vilar, A., Ojeda, S. R., Arimura, A. & McCann, S. M. 22. Sternberger, L. A. (1979) Immunohistochemistry (Wiley, New (1978) Life Sci. 23, 1493-1498. York), 2nd Ed. 28. Wakabayashi, I., Miyazawa, Y., Kanda, M., Miki, N., De- 23. Khorram, O., Bedran de Castro, J. & McCann, S. M. (1984) mura, R., Demura, H. & Shizume, K. (1977) Endocrinol. Jpn. Proc. Nati. Acad. Sci. USA 81, 8004-8008. 24, 601-604. 24. Martin, J. B. & Renaud, L. P. (1974) Science 186, 538-540. 29. Kita, T., Chihara, K., Hiromi, A., Minamitani, N., Kaji, H., 25. Tache, Y., Brown, M. & Collu, R. (1979) Endocrinology 105, Kodama, H., Chiba, T., Fujita, T. & Yanaihara, N. (1986) 220-224. Brain Res. 398, 18-22.