Immunohistochemical Localization of Corticotropin-Releasing Factor, [Arginine 8 ]- Vasopressin and Oxytocin Neurons in the Goat Hypothalamus

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1 Immunohistochemical Localization of Corticotropin-Releasing Factor, [Arginine 8 ]- Vasopressin and Oxytocin Neurons in the Goat Hypothalamus Takefumi KIKUSUI*, Yukari TAKEUCHI, and Yuji MORI** Laboratory of Veterinary Ethology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan (Received 6 February 1997/Accepted 1 April 1997) ABSTRACT. Distribution patterns of corticotropin-releasing factor (CRF), [arginine 8 ]-vasopressin (AVP) and oxytocin (OXY) neurons were examined immunohistochemically in the female goat hypothalamus. The majority of the CRF immunoreactive (-IR) cells were located in the parvocellular part of the paraventricular nucleus (PVN) with smaller population found in the magnocellular part of the PVN. CRF-IR cells were also found in the suprachiasmatic nucleus, the preoptic area and around the fornix in the caudal part of the hypothalamus. AVP- and OXY-IR cells were similarly distributed in the hypothalamus. The majority of AVP- and OXY-IR cells were observed in the magnocellular part of PVN and the supraoptic nucleus. Smaller numbers of AVP- and OXY-IR cells were found in the parvocellular part of the PVN and lateral hypothalamic area. AVP-IR but not OXY-IR cells were located in the suprachiasmatic nucleus. CRF-IR fibers were concentrated in the external palisade zone of the median eminence (ME) with a few fibers found in the internal palisade zone of the ME, whereas AVP- and OXY-IR fibers were concentrated in the internal palisade zone of the ME with a few fibers found in the external zone. These results support the view that not only CRF but also AVP and OXY are released into the hypophysial portal blood and involved in the control of pituitary endocrine function in ruminant species. KEY WORDS: [arginine 8 ]-vasopressin, corticotropin-releasing factor, oxytocin, paraventricular hypothalamic nucleus, supraoptic nucleus. J. Vet. Med. Sci. 59(8): , 1997 Corticotropin-releasing factor (CRF) is regarded as the major neuropeptide which stimulates adrenocorticotropic hormone (ACTH) release in the hypothalamo-pituitary system [23]. In rats, for example, the distribution of this peptide in the brain is well established [24]. CRF immunoreactive (-IR) cells are located in the paraventricular nucleus (PVN) of the hypothalamus and send their fibers to the median eminence (ME) [23, 24], which is considered to be the system responsible for regulating the amount of ACTH release from the anterior pituitary. In addition to CRF, recent studies have revealed that AVP and OXY can also stimulate ACTH secretion in rats [1, 2, 9, 23, 24]. These two neuropeptides are similarly distributed in the hypothalamo-pituitary system in some species [5, 11, 12, 14, 19, 22, 28, 29]. Most of neurosecretory AVP and OXY cells are concentrated in the magnocellular part of the PVN and supraoptic nucleus (SON), and the fibers of these neuropeptides are observed in the ME. In the ruminant species, however, although AVP has been suggested to have more potency than CRF to stimulate ACTH secretion both in vivo [4, 6] and in vitro [8], available information is limited as to the role of CRF, AVP and OXY. Even the distribution pattern of neurons containing these neuropeptides and their fiber-projections in the brain are yet to be clarified. In this study, we therefore examined immunohistochemical localization of CRF, AVP and OXY neurons in the female Shiba goat brain as the first step of immuno- * PRESENT ADDRESS: KIKUSUI, T., Neuroscience Research Laboratories, Sankyo, Co., Ltd., Tokyo, Japan. ** CORRESPONDENCE TO: MORI, Y., Laboratory of Veterinary Ethology, The University of Tokyo, Yayoi, Bunkyoku, Tokyo 113, Japan. neuroendocrinological studies to reveal the intra-brain function of three neuropeptides in the manifestation of stress-related behavior. The Shiba goat, which is a Japanese indigenous breed of goat and its closed colony has been established [10], was used in this study because basic information necessary for neurophysiological studies is available [13, 25, 26, 31, 32]. MATERIALS AND METHODS Specimen: Brain samples were obtained from four adult female Shiba goats (Capra hirucus). They were deeply anesthetized with 30 mg/kg sodium pentobarbital following systemic heparinization (5,000 units/head, i.v.), and then perfused bilaterally via the carotid arteries with 3 l of 0.9% NaCl (the first 1 l of saline contained 10 units/ml heparin), followed by 2.5 l of Zamboni s fixative (4% paraformaldehyde, 7% saturated picric acid in 0.1 M phosphate buffer, ph 7.4) [30]. Following the perfusion, the head, from which the parietal bone was carefully removed, was put on the stereotaxic instrument for the goat [13]. The method for the stereotaxic approach was described in detail elsewhere [32]. After taking lateral X-ray photographs, reference needles were inserted into the brain vertically until they reached the basis cranii interna with predetermined distances horizontally from the inter-aural (IA) line according to the brain atlas for the Shiba goat [31]. The brain was carefully removed from the cranium and trimmed down to a block with sections parallel to the reference needles. The blocks of brain were then stored in the fixative containing 15% sucrose at 4 C overnight. Processing: The brain block containing the hypothalamus

2 622 T. KIKUSUI, Y, TAKEUCHI, AND Y. MORI was serially cut into coronal (n=3) or sagittal (n=1) sections at 40 µm on a freezing microtome. All free-floating sections were washed in 0.1 M phosphate buffer (ph 7.4). Four consecutive sections were taken as a group and each section of one group was processed either for CRF, OXY or AVP immunohistochemistry, or for Nissl s staining with cresyl violet. Free-floating sections were rinsed in 0.1 M phosphate buffered saline containing 0.05% Triton X-100 (PBST), and then incubated in PBST containing 1% normal goat serum overnight to minimize nonspecific staining. After being treated with 0.3% hydrogen peroxidase for 30 min to reduce endogenous peroxidase activity, sections were exposed to the primary antiserum (i475/002 Ab to ocrf, i481/003 Ab to Oxytocin, UCB-Bioproducts S.A., Brusseles, Belgium; RIN 8130 Ab to [Arg 8 ]-vasopressin, Peninsula Laboratories, Inc., Belmont, U.S.A.) at a dilution of 1: 30,000 for CRF, 1: 30,000 for OXY, and 1: 40,000 for AVP, respectively, in 0.1 M PBST for 40 hr at 4 C, and were then treated at room temperature sequentially with a biotinylated goat anti-rabbit IgG (Vector Laboratories, Inc., Burlingame, U.S.A.) for 2 hr, and with the avidin-biotin-horseradish peroxidase complex for 1 hr, using a commercial kit (Vectastain ABC Elite kit, Vector Laboratories, Inc., Burlingame, U.S.A.). Information about the specificity of each antiserum is available from providing companies as follows. The anti- [Arg 8 ]-vasopressin antiserum cross-reacts only partially with [Lys 8 ]-vasopressin (38%) but not with other neuropeptides such as oxytocin, ACTH and LHRH. Likewise the anti- CRF antiserum does not cross-react with other neuropeptides including vasopressin. As for the anti-oxytocin antiserum there is a very weak cross-reaction with vasopressin (actual figure is not available) and no cross-reaction with other known neuropeptides and pituitary hormones. The horseradish peroxidase activity was finally demonstrated using 0.05% 3,3 -diaminobenzidine tetrahydrochloride (Polysciences, Warrington, Pa, U.S.A.) and 0.01% hydrogen peroxidase as the chromogen. All sections were observed under a light microscope with a bright field. The immunoreactive neurons and fibers were localized by determining three-dimensional stereotaxic coordinates and plotting them on a series of coronal brain maps which were standardized based on the brain atlas for the Shiba goat [31]. RESULTS Gross anatomy of PVN and SON in goats: From the results of the cresyl violet staining, the PVN was identified at mm rostral to the IA line in the anterior-posterior direction and could be divided into five subdivisions (Fig. 1-A, B). Parvocellular parts located in the medial part of the PVN, and magnocellular parts located in its dorsolateral part, were intermingled, and it was rather difficult to separate them from each other. The anterior and the posterior magnocellular subdivisions were so closely located that the border between them was not recognized clearly, and the medial magnocellular subdivision, which was known Fig. 1. Schematic midsagittal (A) and horizontal (B) projective view of the Shiba goat brain. PVN is located from 25 mm to 30 mm rostal to the IA line (A, B left side). Main body of the SON is located from 28 mm to 32 mm, and retrochiasmatic division of the SON is from 25 mm to 28 mm (A, B right side). Abbreviations; ac, anterior commissure; IR, infundibular recess of the third ventricle; MB, mammillary body; ME, median eminence; MI, massa intermedia of the thalamus; och, optic chiasma; PD, pars distalis (anterior lobe); PI, pars intermedia (intermediate lobe); PN, pars nervosa (posterior lobe); PVN, paraventricular nucleus; PVNam, anterior magnocellular subdivision of the paraventricular nucleus; PVNap, anterior parvocellular subdivision of the paraventricular nucleus; PVNmp, medial parvocellular subdivision of the paraventricular nucleus; PVNpm, posterior magnocellular subdivision of the paraventricular nucleus; PVNpp, posterior parvocellular subdivision of the paraventricular nucleus; S, pituitary stalk, SON, supraoptic nucleus; SONmb, main body of the supraoptic nucleus; SONrch, retrochiasmatic subdivision of the supraoptic nucleus; SR, supraoptic recess of the third ventricle; 3V, third ventricle

3 CRF, AVP AND OXY-NEURONS IN GOAT HYPOTHALAMUS 623 in rats, was not found. The dorsal parvocellular subdivision was connected with the medial parvocellular subdivision. The SON was observed at mm rostral to the IA line and divided into two parts. The main body of the SON was located at mm, whereas the retrochiasmatic subdivision of the SON was located at mm (Fig. 1- A, B). The former appeared near the rostral end of the third ventricle and dilated widely in the lateral direction. In the caudal area where the optic chiasma became separated into optic tracts, the main body of the SON flattered out, and it could be divided into two small parts, namely the dorsolateral part located lateral to the optic tract and the medial part located medial to the optic tract. They were connected by fibers passing just dorsal to the optic tract, and there were a few scattered bipolar cells in this area. In the more caudal area, the medial part became the retrochiasmatic subdivision of the SON. Immunohistochemical localization of neuropeptides: All the immunoreactivity was observed in the perikarya granularly, but not in the nucleus. The distributions of these three immunoreactive cells and fibers are schematically illustrated in Fig. 2. <PVN> In the PVN, the majority of CRF-, AVP- and OXY-IR parvocellular neurons were bipolar in shape, orientating in the dorso-ventral direction, whereas magnocellular neurons were mainly polygonal in type with some branched dendrites (Fig. 3). The numbers of immunoreactive cells in the PVN are shown in Table 1-A with AP stereotaxic coordinates. Most CRF-IR cells existed in the limited area of the PVN (at mm rostral to the IA line). The densest cluster of CRF-IR cells was found in the medial parvocellular subdivision of the nucleus. A smaller number of CRF-IR cells were observed in other parts of the nucleus, particularly in the magnocellular subdivisions. AVP-IR cells were predominant over OXY- IR cells in the magnocellular parts of the PVN. Generally, AVP-IR cells were located rather rostrally, whereas OXY- IR cells were caudally. In the posterior magnocellular subdivision of the PVN at the level of mm rostral to the IA line, AVP- and OXY-IR cells were differently distributed. OXY-IR cells were distributed in the ventrolateral and dorsolateral parts of this subdivision, whereas AVP-IR cells were located in the central part of the subdivision surrounded by OXY-IR cells. At this area, CRF-IR cells were observed in the medial parvocellular subdivision, and intermingled with AVP- and OXY-IR cells (Fig. 4). In the anterior magnocellular subdivision of the PVN, AVP- and OXY-IR cells were similarly distributed. Both cells consisted of round and polygonal types. In the caudal part of this subdivision, AVP- and OXY-IR cells had long dendritic processes, and the broad band of both fibers, which contained some bipolar immunoreactive cells, extended laterally forming an arch to the ventral cluster of the SON, where the fibers joined the SON axons running medially along the dorsal surface of the optic tract toward the ME. The CRF-IR cells in the parvocellular part of the PVN extended their nerve fibers from the lateral edge of the nucleus to the ME passing around the fornix. The course was similar to the vasopressinergic and oxytocinergic tracts. <SON> In the main body of the SON, a large number of magnocellular AVP- and OXY-IR cells existed, but no CRF- IR cell was observed. The numbers of cells are shown in Table 1-B. In all the extension of this nucleus, AVP-IR cells were predominant over the OXY-IR cells. In general, AVP- and OXY-IR cells distributed similarly, but small difference was observed in the main body of the SON at the level of 29.8 mm rostral to the IA line, where AVP-IR cells existed in the ventral part of the main body of SON whereas OXY-IR cells in its dorsolateral part (Fig. 5). However, the border of the two parts was not clear and the both cells were intermingled. <ME> The CRF-IR fibers were concentrated in the ME. The majority of the fibers were found in the external palisade zone with a few in the internal palisade zone, and in the external palisade zone many axon terminals were observed (Fig. 6A). AVP- and OXY-IR fibers were also observed in the ME, and the fibers in the internal palisade of the ME were tightly extended into the posterior pituitary, whereas some terminals of both neurons were observed in the external palisade (Fig. 6B, C). <Other regions> CRF-IR cells were scattered in the suprachiasmatic nucleus (SCN), preoptic area (POA) and around the fornix at the level of 22 mm rostral to the IA line. A few AVP- and OXY-IR cells were observed in the lateral hypothalamic area (LHA). AVP-IR cells, but not OXY-IR cells, were located in the SCN. There were some CRF-IR fibers observed in the medial preoptic area (MPOA) and near the bed nucleus of stria terminalis (BNST). AVPand OXY-IR fibers were observed in the lateral septal area and around the anterior commissure. Generally, CRF-IR cells and fibers were scattered more widely in the hypothalamus than those of AVP and OXY. DISCUSSION In the present study, the localization of immunoreactive cells to CRF, AVP, and OXY were examined in the hypothalamus of the mature female Shiba goat. The overall distribution pattern of these neuropeptides were generally consistent with those reported previously in other mammals [5, 11, 12, 22, 24, 29]. The majority of CRF-IR cells were found in the parvocellular part of the PVN, and those of AVP- and OXY-IR cell bodies in the magnocellular parts of the PVN and SON, from which fibers were projected massively to the ME. These results further support the view that all these three neuropeptides would be involved in the regulation of ACTH secretion from the anterior pituitary lobe in ruminant species [4, 6]. In the PVN, the densest cluster of CRF-IR cells was centered in the medial parvocellular subdivision and it seemed to give rise to most of the fibers projecting to the external palisade of the ME. The densest cluster of AVPand OXY-IR cells was observed in the posterior and anterior magnocellular subdivisions of the PVN, which appeared to

4 624 T. KIKUSUI, Y, TAKEUCHI, AND Y. MORI Fig. 2. Schematic diagram illustrating sequential coronal sections from goat hypothalamus showing the distribution of CRF, AVP and OXY cells and fibers. A closed circle represents 10 IR magnocellular neurons and a closed triangle depicts 10 IR parvocellular neurons. Representative IR fibers are also shown as lines. Abbreviations; ACB, septal accumbens nucleus; AHA, anterior hypothalamic area; ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; DMH, dorsomedial hypothalamic nucleus; fx, fornix; MPOA, medial preoptic area; ot, optic tract; OVLT, organum vasculosum of the lamina terminalis; POA, preoptic area; SCN, suprachiasmatic nucleus, STN, subthalamic nucleus; VAT, ventroanterior nucleus of the thalamus; VMH, ventromedial hypothalamus (others, see Fig. 1 legend).

5 CRF, AVP AND OXY-NEURONS IN GOAT HYPOTHALAMUS 625 project their fibers to the ME. This is in accordance with those reported in other mammals [5, 7, 11, 12, 14 16, 18, 20 24, 29]. In this study, AVP- and OXY-IR cells were similarly distributed in the anterior magnocellular subdivision of the PVN. In its posterior magnocellular Fig. 3. Photomicrographs of CRF-IR (A), AVP-IR (B) and OXY-IR (C) cells in the PVN. Magnocellular type (left side) and parvocellular type (right side) are shown. Scale bars represent 20 µm. subdivision of it, OXY-IR cells were located in the dorsolateral and ventrolateral parts of the subdivision, whereas AVP-IR cells were located in the central part of the subdivision as surrounded by the OXY-IR cells (Fig. 4). This is in agreement with the observations in rats [27]. In contrast, in rabbits [16], guinea pigs [21] and cats [5], AVPand OXY-IR cells are concentrated in the lateral part and in the medial part of the nucleus, respectively. In monkeys [11], AVP-IR cells are distributed more ventrally and medially than OXY-IR cells. In the humans [7], there is no apparent difference in the distribution of the two cell types in the PVN. The functional significance of these species specific patterns is not known. As to the SON, the majority of AVP-IR cells were found in the ventral part of the SON, as previously reported in cats [5, 14]. On the other hand, OXY-IR cells mostly occurred in the dorsal part of the SON. As in cats [5] and rabbits [16], AVP-IR cells were mainly located in the rostral part of the SON while OXY-IR cells in the caudal part. In the ME, highly dense terminals of CRF-IR fibers were observed in the external palisade zone, which is supposed to regulate ACTH secretion. A few CRF-IR fibers were also observed in the internal palisade zone. On the other hand, AVP- and OXY-IR fibers were mainly observed in the internal palisade zone of the ME, which are thought to be extended into the posterior pituitary lobe as reported in rats [23]. Additionally, a few immunoreactive axon terminals were found in the external palisade zone, which seemed to be involved in the neurohemal regulation of the anterior pituitary lobe. These observations favor the view that CRF, AVP and OXY are all involved in the control of ACTH secretion from the anterior pituitary lobe in goats. Table 1. Number of immunoreactive CRF, AVP and OXY cells in the PVN (A) and SON (B) A: PVN Distance 1) 29 mm 28 mm 27 mm 26 mm 25 mm total Mg 2) ± ± ± ± ± 18.0 CRF Pv 3) ± ± ± ± ± 33.8 Mg 64.3 ± ± ± ± ± ± 28.8 AVP Pv ± ± ± ± ± 12.1 Mg 91.7 ± ± ± ± ± ± 37.0 OXY Pv ± ± ± ± ± 20.9 Mean ± SEM (n=3), 1); Distance from IA line, 2): Magnocellular type, 3): Parvocellular type B: SON Distance 1) 32 mm 31 mm 30 mm 29 mm 28 mm 27 mm 26 mm 25 mm total AVP (± 90.0) (± 35.5) (± 94.0) (± 14.8) (± 7.5) (± 6.0) (± 16.9) (± 236.0) OXY (± 13.0) (± 92.0) (± 64.5) (± 19.0) (± 4.2) (± 10.7) (± 3.8) (± 66.0) Mean ± SEM (n=3), all neurons are magnocellular type, 1); Distance from IA line, ; SEM was not calculated due to the loss of section.

6 626 T. KIKUSUI, Y, TAKEUCHI, AND Y. MORI Fig. 5. Coronal sections of AVP-IR (A) and OXY-IR (B) cells in the main body of the SON at the level of mm from IA line. Note that AVP-IR cells are located in the ventral part of the SON, whereas OXY-IR cells are in the dorsal part of the SON. och: optic chiasma. Scale bar represents 5 mm. the ME but also connect each other by fibers. In rats, some vasopressinergic neurons in the SON are shown to project their fibers to the PVN [17]. The functional relationship between the two nuclei possessing both AVP and OXY neurons is yet to be elucidated. ACKNOWLEDGEMENTS. The authors thank Drs. Tomohiro Hamada, Gustavo Zuccolilli, and Tomomi Tanaka for their kind help. This work was supported in part by grants to Y. M.( ) from the Ministry of Education, Science and Culture, Japan. REFERENCES Fig. 4. Coronal sections of CRF-IR (A), AVP-IR (B) and OXY-IR (C) cells in the PVN at the level of mm from IA line. Distribution pattern at this level of the PVN is also shown schematically (D). Note that CRF-IR cells are scattered in the medial parvocellular subdivision, whereas AVP-IR cells are located in the central part of the posterior magnocellular subdivision, and OXY-IR cells are in its ventrolateral and dorsolateral parts. Scale bar represents 1 mm. In other regions of the hypothalamus, CRF-IR cells were observed in the SCN, POA, and around the fornix at the level of 22 mm rostral to the IA line, which agreed with the results obtained in rats [24]. AVP- and OXY-IR cells were scattered in the LHA, and AVP-IR cells were also found in the SCN, as observed in some species [14, 16, 20]. In the sagittal view, some AVP- and OXY-IR cells and fibers were found in the area between the PVN and the SON. Thus these neurons appeared not only to extend their fibers into 1. Antoni, F. A Receptors mediating the CRH effects of vasopressin and oxytocin. Ann. New York Acad. Sci. 512: Antoni, F. A Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age. Front. Neuroendocrinol. 14: Antunes, J. L. and Zimmerman, E. A The hypothalamic magnocellular system of the rhesus monkey: An immunohistochemical study. J. Comp. Neurol. 181: Caraty, A., Grino, M., Locatelli, A., Guillaume, V., Boudouresque, F., Conte-Devolx, B., and Oliver, C Insulin-induced hypoglycemia stimulates corticotropin-releasing factor and arginine vasopressin secretion into hypophysial portal blood of conscious, unrestrained rams. J. Clin. Invest. 85: Caverson, M. M., Ciliello, J., Calaresu, F. R., and Krukoff, T. L Distribution and morphology of vasopressin-, neurophysin II-, and oxytocin-immunoreactive cell bodies in the forebrain of the cat. J. Comp. Neurol. 259:

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