Catecholaminergic region A15 in the bovine and porcine hypothalamus

Brain Research Bulletin, Vol. 37. No. 4, pp. 351-358, 1995 1995 Elsevier Science Ltd Printed in the USA. All rights resewed 0361-9230195 $9.50 + .OO

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Catecholaminergic Region Al5 in the Bovine and Porcine Hypothalamus L. S. LESHIN,*’ R. R. KRAELING,* T. E. KISER,t C. R. BARB* AND G. B. RAMPACEKt *USDA-ARS, R. B. Russell Agricultural Research Center, 950 College Station Road, Athens, GA 30673 tAnimal and Dairy Science Department, University of Georgia, Athens, GA 30602 [Received 18 April 1994; Accepted 20 December 19941 METHOD

ABSTRACT:Magnocellular pewikaryawithin the retrochiasmatic division of the supraoptic nucleus of bovine and porcine hypothalami were immunoreactive (ir) with antiseruriI against tyrosine hydroxylase (TH), but not dopamins+hydroxylase (DBH). Few cells in this region were also immunoreactlve for vasopressin (VP) or oxytocin (OT). In contrast, the main division of the supraoptic nucleus contained numerous perikarya immunoreactive for VP and 01, but not TH nor DBH. Both the retrochiasmatic and principal divisions of the supraoptic nuclei contained TH- and DBH-ir fibers and varicosities. This region in bovine and porcine hypothalami corresponds to the ventral Al5 catecholaminergic (dopamine-producing) cell group.

KEY WORDS: Catechoiamine, Tyrosine hydroxylase, Dopamine /Mydroxylase, Immunocytochemistry, Hypothalamus, Supraoptic nucleus, Pig, Cattle.

INTRODUCTION Early neuroanatomists divided the hypothalamic supraoptic nucleus (SON) into two to five subdivisions, based on the location and orientation of magnocellular neuron clusters in relationship to the optic chiasm, from examination of Nissl-, Weil-, and Gomori-stained tissue (for review: [3,24]). For the most part, the subpopulation of sizes and shapes of magnocellular neurons within each subdivision were similar [23]. With the advent of immunohistological techniques and production of antibodies against purified neurophysins, oxytocin (OT), and vasopressin (VP), the SON could be divided into separate, functional regions producing VP or OT [ 11,131. However, VP and OT neurons were not completely segregated, but often intermingled. Therefore, the original SON subdivisions based on classical histological stains did not correspond to any one particular neurohormone except for the recently identified dorsomedial extension of SON, which in the pig contain OT but not VP neurons [44]. In this study, we describe the retrochiasmatic subdivision of the bovine and porcine hypothalamic SON, which corresponds to the ventral catecholaminergic Al5 cell group.

The methodology for fixation and immunocytochemistry of bovine and porcine brain tissue was described in Leshin et al. [20] and Kineman et al. [17]. Cow (n = 3) and steer (n = 2) heads were obtained from the University of Georgia abattoir soon after death. The carotid arteries were cannulated and heads bilaterally perfused with 2 liters of cold heparinized saline or 2.9% citrate buffer, followed by Zamboni’s fixative (pH 7.3). Female calves (n = 2) and pigs (n = 4) were anesthetized with pentobarbital, the carotid arteries catheterized, and heads perfused as described. The septal-preoptic area, hypothalamus, and pituitary gland were removed as one block, further subdivided, and submerged overnight in fresh fixative at 4”C, followed by 15%, then 30% sucrose in 0.1 M phosphate buffer until submerged. Tissues were frozen with dry ice and stored at -80°C. Cryostat-cut coronal or sagittal sections (40-60 pm) were collected into 0.1 M phosphate buffer. For immunocytochemistry, free-floating sections were treated as follows: rinsed three times in Tris-saline buffer (TBS; 0.05 M Tris; 0.15 M NaCl, pH 7.6; 10 min each), incubated in 0.5% hydrogen peroxide in TBS (20 min), rinsed three times with TBS, incubated in 10% normal goat serum (Gibco, Grand Island, NY, 60 min), incubated 48 h at 4°C with rabbit antibodies against purified bovine adrenal tyrosine hydroxylase (TH). or dopamine /3 hydroxylase (DBH) (Eugene Tech International, Allendale, NJ) diluted 1:200 in TBS containing 1.0% bovine serum albumin (TBS-BSA), rinsed three times in TBS. incubated 24 h, at 4°C with biotinylated goat antirabbit serum (1:500); Vector Laboratories, Burlingame, CA) in TBS-BSA or TBS-BSA containing 1.O% normal goat serum to reduce nonspecific background staining, rinsed two times with TBS. then once with TRIS buffer without saline (TB), incubated with strepavidin-D horseradish peroxidase conjugate in TB (0.496, v/v 60 min, Vector Laboratories), rinsed three times in TB, incubated with 3,3’ diaminobenzidine tetrahydrochloride (0.5%, Sigma Chemical Co., St. Louis, MO) in TB containing hydrogen peroxide (0.01%) until the characteristic brown reaction product developed, rinsed in TB three times, mounted onto gelatin-coated slides, air dried, dehydrated in alcohol and coverslipped from xylene.

Mention of a trade name, proprietary, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. ’ Requests for reprints should be addressed to Dr. L. S. Leshin, USDA-ARS, R. B. Russell Research, P. 0. Box 5677, Athens, GA 30613.

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Control sections, adjacent to those stained for TH- or DBH immunoreactivity (it), were treated with either nonimmune rabbit serum or lacking one of the incubation reagents. For porcine tissue, preincubation of TH or DBH antisera with protein-A [41] did not alter the distribution or numbers of specifically immunostained cells. Preincubation with partially purified adrenal medulla homogenates [14]; cytoplasmic fraction for TH and chromaffin granule-derived fraction for DBH, also prevented specific TH and DBH immunostaining, respectively. Additional tissue sections were incubated with mouse monoclonal antibodies against OT (Al-28, used at 150 dilution) or VP (III D7-11-l. used at 1: 100 dilution, both generously donated by Dr. A.-J. Silverman). The VP and OT antibodies were characterized previously [12,13]. Because of the presence of lys-vasopressin rather than arg-vasopressin reported in porcine brain tissue [lo] and different fixation methods, VP and OT crossreactivity was reassessed under our immunocytochemical conditions by preincubation of antibodies with l-5 &ml of OT, arg 8-VP, lys 8-VP, lys 8-vasotocin, and arg 8-vasotocin (Bachem Inc., Torrance, CA). Immunostaining with the VP antibodies was blocked by preabsorption with either excess arg 8 or lys 8-VP but not by excess OT or forms of vasotocin. Immunostaining with OT antibodies was blocked by excess OT and partially blocked by Arg 8-vasotocin at high concentrations, but not by excess VP or lys 8-vasotocin. Control sections, adjacent to those immunostained, were treated with either nonimmune rabbit or mouse semm, or in the absence of one of the incubation reagents. Additional sections were stained with cresyl violet to identify nuclear boundaries and fiber tracts. An additional cow and pig hypothalamus were not frozen, but embedded in paraffin, sectioned coronally [20 pm], and stained for myelinated fibers and perikarya with Luxol fast blue and cresyl violet [ 191.

RESULTS In cresyl violet-stained tissue of the porcine and bovine hypothalamus, the retrochiasmatic subdivision of the supraoptic nucleus (SONr) comprised a region of intensely stained magnocellular and some parvocellular neurons located bilaterally, within the ventral optic chiasm extending posteriorly into the ventral retrochiasmatic area (Figs. lA,B, 2A, and 3A). In bovine tissue, this region was approximately 900 to 1000 firn long in the anterior-posterior, lOOO- 1300 pm long in the medial-lateral, and 300-400 pm long in the dorsal-ventral planes. In porcine tissue, a similar bilateral group of cells was located slightly more posterior, extending within the retrochiasmatic area from the medialposterior edge of the optic tract to the lateralanterior median eminence (infundibulum, Figs. 1A and 2A). The region encompassing these cells occupied a 600 to 800 pm diameter sphere in porcine tissue. In both species, these cells were similar in size, 20-50 Frn diameter, and stained intensely as neurons of the principal SON (Figs. lA-D, and 2A-C) located along the ventral edge of the hypothalamus, dorsal and anterior to the optic chiasm. However, neurons within SONr were more widely spaced than the closely compact cells of the SON. In bovine hypothalamus, interspersed magnocellular neurons connected the dorsal aspect of the SONr with SON. These neurons formed a linear arrangement around the medialdorsal curvature of the optic chiasm-optic tract transition region (Fig. 2A). This arrangement often paralleled small chiasmatic blood vessels. In sag&al and coronal sections, neurons of SONr were distinctly separate from the arcuate nucleus (infundibular nucleus) being further lateral and anterior to the arcuate nucleus in the

bovine (Figs. 1C and 3A) and further anterior and ventral in the porcine hypothalamus (Figs. 1A and 2A). Most perikaraya and their processes within the SONr were immunostained with TH antiserum, forming a dense network in both species (Figs. 2D and 3B). These perikarya were either similar in size to magnocellular cells in Nissl-stained preparations or smaller parvicellular perikarya (20-25 pm diameter) (Figs. 2E.F and 3C,D). Magnocellular THimmunoreactive (TH-ir) neurons were often multipolar with numerous branching processes. Smaller TH-ir neurons were rounded or fusiform in shape and tended to be bipolar. There was a clear separation of SONr and arcuate nucleus TH-ir neurons in both bovine and porcine hypothalamus (Fig. 2D). In addition to the more ventral and anterior location of SONr with respect to the arcuate nucleus, TH-ir perikarya of the arcuate nucleus were all parvicellular (Fig. 2E,F). In bovine tissue, TH-ir perikarya and fibers were additionally interspersed between the SON and SONr, similar to those magnocellular neurons observed in Nissl-stained tissue. These perikarya tended to be fusiform shape (Fig. 3D), orientated parallel along this pathway. In one sagittally sectioned porcine SONr, magnocellular neurons were arranged in circular fashion around a few other centrally positioned magnocellular neurons (Fig. lA,B). Control tissues showed no specific immunostaining. Incubation with nonimmune rabbit serum did darken magnocellular areas of SON and SONr but this was diffuse, ill-defined staining that lacked the clear delineation of cellular structures. Previous studies by Watkins [48], Meijer et al. [22], and Van der Beek [41] found that the Fc component of rabbit immunoglobulins nonspecifically bind to porcine magnocellular neurons in SON, resulting in false-positive staining. We have found this result to be quite variable among individual pigs and rabbit serum. Preincubation with TH and DBH antisera with protein-A did not alter specific immunostaining, although it reduced nonspecific staining slightly. Residual cells of meningeal membranes that surround the hypophysial stalk and ventral optic chiasm were nonspecifically stained, because they readily darkened by peroxidase-diaminobenzidine treatment even in the absence of initial antibody incubations. These cells were easily distinguished from neurons by their location, shape, and intensely dark black color. Further immunocytochemical comparison of the bovine and porcine SONr with SON revealed that both regions contained a dense network of TH- and DBH-ir fibers (Fig. 3E). However, only the SONr region contained TH-ir perikarya, and neither region contained DBH-ir perikarya. Both OT- and VP-ir neurons were abundant in the principal division of the supraoptic nucleus of both species, but only a few VP and OT-ir perikara were present in the retrochiasmatic region or (Fig. 4A-D) along the medial dorsal curvature of the optic chiasm-optic tract transition region. DISCUSSION These results are the first to localize the A15 catecholaminergic (dopamine-synthesizing) group in porcine and bovine hypothalamus. The Al5 group of neurons is an addition to the original description and nomenclature of catecholaminergic neurons described by Dahlstrom and Fuxe [5]. The Al5 cell group was delineated in the rat by Hokfelt et al. [l l] using immunocytochemical methods. The older histofluorescent techniques were apparently not of sufficient sensitivity to detect this Al5 cell group. Interestingly, in contrast to the identification of both a dorsal and ventral component of the Al5 group in rats, only a ventral Al5 group was detected in pigs and cattle. Also, in sheep, only a ventrally located Al5 group was described, which was

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FIG. 1. (A) Cresyl violet-stained sagittal section of the porcine ventral hypothalamus. The principal division of supraoptic nucleus (SON) consists of intensely stained cells located above the optic chiasm (OC). Similarly, magnocellular intensely stained cells form a cluster, the retrochiasmatic division of the SON (SONr, bordered by arrowheads), posterior to OC but anterior to arcuate nucleus (ARC). (B) Enlarged region of the SONr (from photograph A, delineated by arrowheads). (C) Cresyl violet-stained sagittal section of the bovine ventral hypothalamus. The SON consists of densely stained cells located above the OC. Extending ventrally and caudahy, a few similarly intensely stained cells form a band along the dorsal-caudal border of the optic chiasm (arrowheads) extending from the SON to a small nucleus of intensely stained cells similar to that of the SON, the SONr (anterior SONr enlarged in D). Abbreviations: ARC- arcuate nucleus, AH-anterior hypothalamus, FX-fomix, pt-pars tuberalis of the adenohypophysis, RC-retrochiasmatic area, VMN-ventromedial nucleus, V-infudibular recess of the third ventricle. Calibration bars: A = 500 pm, B = 100 pm, C = 1 mm, D = 100 pm.

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FIG. 2. (A) Cresyl violet-stained coronal half-section of the porcine ventral hypothalamus. Intensely darkstaining neurons of the SON and the less densely arranged SONr are enlarged in B and C, respectively. (D) TH-immunoreactive neurons of the SONr, corresponding to the Al5 catecholaminergic group, are distinctly separate from the A12 group within the arcuate nucleus (ARC). Individual neurons encircled are enlarged in

E and F, respectively. Abbreviations: OT-optic tract, V-third C= lO~m.D=5OO~m,E-F= 10pm.

both TH- and DBH-ir [38,39]. In cattle and pigs, as described also in sheep [38], this group of perikarya also contains a dense network of DBH-ir fibers and varicosities, indicative of probably catecholaminergic afferents synthesizing either norepinephrine or possibly epinephrine.

ventricle. Calibration bars: A = 500 pm, B-

Differences in the location and cytoarchitecture of SONr exist between cattle and pigs and other species. In both cattle and pigs, SONr is distinctly separated from the larger principal division of SON. A thin connecting band of elongated TH-ir neurons was observed only in cattle tissue. Because not all sections of tissue

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Also, for the pig, the SONr is distinctly posterior from the vasopressin-oxytocin nucleus (VON), a sexually dimorphic nucleus in mature pigs, described recently by Van Eerdenburg et al. [42,43]. In the rat, the SONr is located posteriomedial to the optic tract and consists of a single sheet of cells along the base of the hypothalamus, just beneath the pia mater [27]. This region contains mostly VP producing neurons, with some OT neurons centrally located within this SON division [13,28]. For the rat, it is not clear if these SONr neurons also synthesize catecholamines, which would designate them as the Al5 group described by Hokfelt et al. [ 111. The ventral Al5 group in rats, described by Hokfelt et al. [ 111 appears to include a larger region than just SONr. The Al5 neuron group included catecholaminergic perikarya of the SON and extends into the retrochiasmatic area and caudally along the ventral surface of the hypothalamus, but lateral to the Al2 group of the arcuate nucleus. In cattle, the rostral aspect of the Al5 group is clearly situated within the ventral optic chiasm, extending posterior, but ending anterior of the arcuate nucleus. In pigs, the ventral Al5 group is most similar to that described for sheep [39]. In sheep, this neuron group is located medial and ventral to the optic tract, described as posterior division of the SON by Welento et al. [49]. Similar dark staining neurons medial and ventral to the optic tract in the sheep atlas of Richard [29], plates A29 and A30, appear to be mislabeled as the suprachiasmatic nucleus (SCH) rather than the supraoptic nucleus (SON). There were no TH-ir perikarya in the principal division of the SON of cattle and they were rarely observed in pigs, although TH-ir varicosities were abundant in both cattle and pigs. This lack of catecholaminergic perikarya in the principal division SON also contrasts with their scattered presence in the rat [40] and cat [ 181 and their abundance (40% of SON neurons) in humans [21,26]. Immunoreactive VP and OT neurons and their associated neurophysins tend to be located in different regions of the principal division of the SON as previously described for cattle [7.45], pigs [44], rats [1,28,46] and humans [8]. In both porcine and bovine SON, OT-ir neurons were more abundant anteriorly in dorsal and medial SON, VP-ir neurons were more posterior in ventral and lateral regions, although there was still extensive intermingling of OT- and VP-ir neurons. In the SONr, however, mainly VP-ir neurons were detected. Unfortunately, there was no mention in these previous immunocytochemical studies, of the occurence of unstained magnocellular neurons in the SONr that would be expected to exist based on our results. The source of DBH innervation of SONr or Al5 group in cattle and pigs is not known, but similar DBH innervation was observed in sheep. It likely arises from the Al catecholamlnergic neurons of the ventrolateral medulla because this region projects to the SONr in rats [4,30]. Colocalization of TH with OT or VP in these magnocellular neurons was not apparent by examination of adjacent thick secFlG. 3. (A) Tyrosinehydmxylaseimmunostainedcoronalhalf-sectionof the tions, but conclusive evidence will require verification by double bovine rehuchiasmaticarea. On the ventral surface of the retrochiasmatic immunocytochemical techniques. Colocalization of TH and OT arealies the A15 catccholaminegroup (enlargedin B) with numemus THir perikarya(arrowheads).TypicalTH-irrounded,multipolarueumnslocated in a small population of paraventricular neurons has been rein SONr (C) or elongated shape along the tractmnnecting with SON 0). ported in rabbits [31] and in the caudal or ventromedial part of (B) Dopamine p-hydmxylasc immunomactivevaricosities(arrowheads)in SON (homologous to the retrochiasmatic division reported SONr are prevalentsurroundingunstainedcells. Abbreviations:BSin Figs. herein) of humans [21,32]. Although more abundant VP and OT 1and2.CalibmtionbatszA=1mrn,B=300pm,C-E=10~m. perikarya were observed by Van Eerdenburg et al [44] than described here, confirmation of distinctly separate sets of ‘D-I, VP, and OT neurons still requires double immunocytochemical techwere irnmunostained, it is likely we missed detection of this neuniques. The sparce occurrence of OT-ir perikarya in both pigs ron bridge. Indeed, both Ellis and Watkins [9] and Van Eerdenand cattle and absence of VP- and TH-ir colocalization in this burg [44] describe a connecting bridge of neurons in the pig that region of other species suggest this possibility is doubtful. have similar staining characteristics as SON and SONr nuclei.

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PIG. 4. Comparison of oxytocin and vasopressin immunocytochemical staining between the SON and SONr in the bovine hypothalamus. Similar tesults were obtained with porcine tissue (not shown). Oxytocin-ir is localized in numerous neurons in the principal division of SON in both bovine (A) and porcine (not shown), but only a few isolated perkarya (arrowheads) of SONr (B). Vasopmssin-ir pe&arya were abundant in SON (C), but relatively few cells (arrowheads) in SONr were immunostained (D). Calibration bar = IO pm.

Because, in cattle and pigs, this region is immunocytochemically distinct from classical supraoptic neurons (TH-ir perikarya in SONr), we suggest the nomenclature for this region as the retrochiasmatic division of SON rather than tuberal portion of SON as used by Kineman et al. [16,17]. This would maintain its anatomical continuity with the modem English name for its homologous region in the rat, the retrochiasmatic part of SON [25,33] as well with the older nomenclature for this region in cattle and pigs: nucleus postopticus magnocellularis [47], postoptischer part of nucleus supraopticus [6], nucleus supraopticus pars postchiasmatica [49], nucleus supraopticus pars postoptica [15], and nucleus supraopticus pars posterior [35,36]. The function of this group of catecholamine neurons will require further research in pigs and cattle. They may influence reproductive physiology, because, in sheep, chemical lesions of these neurons with 6-hydroxydopamine prevented estrogen-induced inhibition of pulsatile luteinizing hormone secretion [37]. Szentagothai et al. [34] suggested on the basis of their staining characteristics, that most large retrochiasmatic supraoptic neurons (described here as part of SONr) served some function other than neurohypophyseal secretion. Although some of the foundations for this conclusion has partly been dispelled [2], our immunocytochemical findings that TH-ir neurons are abundant with

few OT-ir and VP-ir neurons in bovine and porcine SONr of the hypothalamus favor his early conclusions. ACKNOWLBDGEMENTS The authors thank Dr. A.-J. Silverman for donating oxytocin and vasopressin monoclonal antibodies; Dr. L. Rund, D. Slavin, R. UtJey, and L. Tams for technical assistance; S. Silvers for use of photographic equipment facilities; J. Minor and S. Coffman for preparing this manuscript; and Drs. F. J. C. M. Van Eerdenburg and N. McArthur for their critical review.

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