Immunocytochemical distribution of catecholamine-synthesizing neurons in the hypothalamus and pituitary gland of pigs: Tyrosine hydroxylase and dopamine-?-hydroxylase

THE JOURNAL OF COMPARATIVE NEUROLOGY 364~151-168 (1'

Immunocytochemical Distribution of Catecholamine-Synthesizing Neurons

in the Hypothalamus and Pituitary Gland of Pigs: Tyrosine Hydroxylase and

Dopamine- p-Hydroxylasel

L.S. LESHIN, R.R. KRAELING, R.D. KINEMAN, C.R. BARB, AND G.B. RAMPACEK USDA-ARS, R.B. Russell Agricultural Research Center, Athens, Georgia 30604 (L.S.L.

R.R.K., C.R.B.); Animal and Dairy Science Department, University of Georgia, Athens, Georgia 30602 (R.D.K., G.B.R. )

ABSTRACT This study describes the distribution of catecholaminergic neurons in the hypothalamus

and the pituitary gland of the domestic pig, Sus scrofa, an animal that is widely used as an experimental model of human physiology in addition to its worldwide agricultural importance. Hypothalamic catecholamine neurons were identified by immunocytochemical staining for the presence of the catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine-P- hydroxylase. Tyrosine hydroxylase-immunoreactive perikarya were observed in the periventricular region throughout the extent of the third ventricle, the anterior and retrochiasmatic divisions of the supraoptic nucleus, the suprachiasmatic nucleus, the ventral and dorsolateral regions of the paraventricular nucleus and adjacent dorsal hypothalamus, the ventrolateral arcuate nucleus, and the posterior hypothalamus. Perikarya ranged from parvicellular (10-15 pm) to magnocellular (25-50 pm) and were of multiple shapes (rounded, fusiform, triangular, or multipolar) and generally had two to five processes with branched arborization. No dopamine-P-hydroxylase immunoreactive perikarya were observed within the hypothalamus or in the adjacent basal forebrain structures. Both tyrosine hydroxylase- and dopamine-p- hydroxylase-immunoreactive fibers and punctate varicosities were observed throughout areas containing tyrosine hydroxylase perikarya, but dopamine-P-hydroxylase immunoreactivity was very sparse within the median eminence. Within the pituitary gland, only tyrosine hydroxylase fibers, and not dopamine-P-hydroxylase immunoreactive fibers, were located throughout the neurohypophyseal tract and within the posterior pituitary in both pars intermedia and pars nervosa regions. Generally, the location and patterns of both catecholamine-synthesizing enzymes were similar to those reported for other mammalian species except for the absence of the A15 dorsal group and the very sparse dopamine-P-hydroxylase immunoreactive fibers and varicosities in the median eminence in the pig. These findings provide an initial framework for elucidating behavioral and neuroendocrine species differences with regard to catecholamine neurotransmitters. , 1 ~ 6 WiIey-Liss. Inc.*

Indexing terms: neuroanatomy, comparative neuroanatomy, dopamine, norepinephrine, swine

domestic pig, Sus scrofa, is widely used as an nental model system of human cardiovascular, respi- and gastrointestinal physiology (Tumbleson, 1986)

tion to its worldwide prominence in animal agricul- rom an agricultural perspective, there is an increas- ?rest in understanding neuroendocrine interactions feet reproduction, lactation, growth, stress, and

prduc- d animal welfare. The hypothalamus, as an integra-

tive center, often serves as the final link or bridge betw, the brain and the endocrine system. To facilitate fut

Accepted June 22,1995. Address reprint requests to R.R. Kraeling. USDA-ARS, R.B. Ru,

Agricultural Research Center, P.O. Box 5677, Athens, GA 30604-5677. 'Mention of a trade name, proprietary product, or specific equipment

not constitute a guarantee or warranty by the USDA and does not imp], approval to the exclusion of other products that may be suitable.

in pi@, because they affect

152 L.S. LESHIN ET AL.

physiological experiments that further our understanding of the interactions involved in behavioral and neuroendocrine mechanisms in pigs, it is essential to know the anatomical sites and the distribution of physiologically relevant neurotransmitters and neuromodulators in the hypothalamus. Previous immunocytological descriptions of neuroendocrine-related peptides in pigs included luteinizing hormone-releasing hormone in the preoptic regions, the hypothalamus (Kineman et al., 1988) and the olfactory bulb (Leshin et al., 1991), and included p-endorphin (Kineman et al., 19891, growth hormone-releasing factor, somatostatin (Leshin et al., 1994), oxytocin and vasopressin (Van Eerdenburg et al., 1992), vasoactive intestinal polypeptide (Calka, 19921, and neuropeptide Y (Calka et al., 1993) in the hypothalamus.

For pigs, similar to numerous laboratory animals and primates, the catecholamines (dopamine, norepinephrine, ar,d epinephrine) are intimately involved in the regulation of pituitary gland hormone secretion. For pigs, these include the reproductive and lactogenic hormones, luteinizing hormone, prolactin, and oxytocin (Ellendorff and Parvizi, 1982; Kraeling and Barb, 1990; Kraeling et al., 19921, cortisol as an indicator of stress and animal well being (Dantzer and Mormede, 1981; Moss, 1981; Dantzer, 19861, and cortisol in the etiology of porcine stress related syn- dromes (McLoughlin, 1987).

Only a few studies have described the anatomical sites of catecholamine neurons in the pig. In early studies during the development of monoamine histofluorescence techniques, monoamine fluorescent fibers (thought to be dopaminergic) were identified in both the neural lobe and the intermediate lobe of the porcine pituitary gland (Bjorklund, 1968). Recently, Erlanger et al. (1985) and Agarwal (1993) found regional differences in catecholamine and metabolite concentrations of porcine brain homogenates. Here, we describe the anatomical distribution of catecholaminergic neurons in the hypothalamus and adjacent regions and in the pituitary gland in pigs based on immunocytochemical methods that detect the catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-p-hydroxylase (DBH).

MATERIALS AND METHODS Tissue preparation

Mature female, crossbred, commercial pigs, 8-12 months of age, from the University of Georgia were housed and maintained according to USDA guidelines of animal care (Consortium, 1988). All procedures were previously ap- proved by the Animal Use and Care Committee of the R.B. Russell Agricultural Research Center. The methodology for fixation and immunocytochemistry of pig brain tissue was described by Kineman et al. (1988). Pigs (n = 5) were anesthetized with sodium thiopental (0.02 glkg) and were then maintained under a closed-circuit system of 4-5% halothane and oxygen. The carotid arteries were catheter- ized, and the heads were bilaterally perfused with 3.5% citrate buffer followed by Zamboni’s fixative. The septal- preoptic area-hypothalamus with the pituitary gland at- tached was removed as a large block and was incubated overnight in fresh fixative at 4°C followed by incubations in 15% and then 30% sucrose in phosphate buffer. Tissues were frozen with dry ice and were stored at -80°C. Cryostat-cut coronal or sagittal sections (40-60 pm) were collected in 0.1 M phosphate buffer.

Immunocytochemistry For immunocytochemistry, free-floating sections that

corresponded to intervals of 200-400 km were treated as follows: 1) rinsed three times in Tris-saline buffer (TBS; 0.05 M Tris, 0.15 M NaCl, pH 7.6; 10 minutes each); 2) incubated in 0.5% hydrogen peroxide in TBS (20 minutes); 3) rinsed three times with TBS; 4) incubated in 10% normal goat serum (Gibco, Grand Island, NY; 60 minutes); and 5) incubated for 48 hours at 4°C with rabbit antiserum against purified bovine adrenal TH or DBH (Eugene Tech Interna- tional, Allendale, NJ) diluted 1:200 to 1:1,000 in TBS containing 1.0% bovine serum albumin (TBS-BSA) and 0.1% Triton X-100. Adjacent serial sections were treated with each antiserum, and the locations of bound antibodies were visualized by the avidin-biotin-peroxidase method (Hsu et al., 1981). Tissues were: 6) rinsed three times in TBS with 2.0% normal goat serum in the third rinse; 7)

ac AH ARC BNST :I ZP DBh DBH DBv DMN 3X ‘X

n [P LPOA dS ,V ne VlNm blPOA VlS nt )C

anterior commissure anterior hypothalamic area arcuate nucleus bed nucleus of stria terminalis pituitary cleft cerebral peduncle diagonal band of Broca, horizontal limb dopamine-P-hydroxylase diagonal band of Broca, vertical limb dorsomedial nucleus external lamina of the median eminence fornix internal lamina of the median eminence interpeduncular nucleus lateral preoptic area lateral septum lateral ventricle median eminence medial mammillary nucleus medial preoptic area medial septum mammillothalamic tract optic chiasm

Abbreviations

ot OVLT

PeV PH

PA

PI PN Pt PVN SCN SON SONr SM SN STH TH V Vir VMN Vmr Vor 21

optic tract organum vasculosum of the lamina terminalis pars anterior periventricular nucleus posterior hypothalamus pars intermedia pars nervosa pars tuberalis paraventricular nucleus suprachiasmatic nucleus supraoptic nucleus retrochiasmatic division of the supraoptic nucleus supramammillary nucleus substantia nigra subthalamic nucleus tyrosine hydroxylase third ventricle infundibular recess of the third ventricle ventromedial nucleus mammillary recess of the third ventricle optic recess of the third ventricle zona incerta

HYPOTHALAMIC CATECHOLAMINERGIC NEURONS IN THE PIG 153

incubated for 24 hours at 4°C with biotinylated goat anti-rabbit serum ( 1500; Vector Laboratories, Burlingame, CA) in TBS-BSA or in TBS-BSA containing 2.0% normal goat serum; 8 ) rinsed two times with TBS then once with Tris buffer without saline (TB); 9) incubated with strepavi- din-D horseradish peroxidase conjugate (Vector Laborato- ries) in TB (0.4% v/v; 60 minutes); 10) rinsed three times in TB; 11 1 incubated with 3,3’-diaminobenzidine tetrahydro- chloride (0.05%,; Sigma Chemical Co., St. Louis, MO) in TB containing hydrogen peroxide (0.01%) until the characteristic brown reaction product developed; 12) rinsed in TB three times; and 13) mounted onto gelatin-coated slides, air dried, dehydrated in alcohol, and coverslipped with Xylene.

Control sections adjacent to those stained for TH or DBH immunoreactivity were treated with either nonimmune rabbit serum or with antiserum but without incubation with biotinylated goat anti-rabbit serum or stepavidin-D horseradish peroxidase conjugate. Additional immunocytochemical controls included preincubation of TH and DBH antisera with partially purified porcine adrenal medulla homogenates (Joh and Ross, 1983) before incubation with tissues. Because the Fc component of rabbit immunoglobu- lins may bind nonspecifically to porcine brain tissue (especially to magnocellular neurons of the hypothalamus; Wat- kins, 1977; Van Der Beek et al., 1992), additional TH and DBH antiserum was preincubated (24 hours at 4°C) with 1 or 2 mg/ml of protein A (Sigma) before incubation with tissue sections. Additional sections were stained with cresyl violet to identify nuclear boundaries.

Electrophoresis and Western blots The TH and DBH antisera were previously validated for

enzyme specificity in many species but not for porcine tissue (Armstrong et al., 1982; Jon et al., 1983; Reiner and Vincent, 1986). To demonstrate antiserum specificity in porcine tissue, standard Western blots (Sambrook et al., 1989) were prepared using purified bovine TH enzyme (Eugene Tech International), fresh or frozen homogenates of porcine hypothalamus and adrenal tissue, rat brain, and bovine hypothalamus. Supernatant from homogenized tissue was electrophoresed on sodium dodecyl sulfate poly- acrylamide (7.5% or 12.5%) gels using the PROTEAN I1 dual slab cell (Bio-Rad, Richmond, CA). Proteins were electroblotted to Immuobilon-P membrane (Millipore, Bed- ford, MA) using the Trans-Blot electrophoretic transfer cell (Bio-Rad). The Western blots were washed four times in TBS buffer, incubated in 10% normal goat serum for 1 hour, then incubated with 1:400 dilution of TH or DBH antiserum for 24 hours at 4°C on a tilt-top shaker. The bound antibody was visualized after treatment with the avidin-biotin-peroxidase method described above.

Analysis Tissue sections were examined and photographed with

brightfield and differential interference optics on a Zeiss microscope. Low-magnification photographs were obtained through a dark blue or green filter using Nikon Multiphot microphotography equipment. Schematic drawings of tissue sections were traced from low ( 9 ~ ) magnification of tissues upon which perikarya and fiber locations were marked. Only major nuclei and fiber tracts were labeled in order to maintain clarity. Terminology according to IANC (IANC, 1989) with English spelling is listed in a separate abbreviations table. Anatomical terms were based on the pig hypothalamic atlases of Solnitzky (19391, Welento

(1964), Szteyn et al. (1980) and on the sheep atlas (Richard, 1967). Sites of TH- and DBH-immunoreactive (ir) perikarya were also discussed in accordance with the original nomenclature of Dahlstrom and Fuxe (1964) as revised by Hokfelt et al. (1984).

RESULTS Controls

In tissues that were processed as controls, there was no specific immunostaining. In some tissues, however, background nonspecific staining of small blood vessels was prevalent in isolated regions and was not completely sup- pressed by preincubation in a 0.5% solution of hydrogen peroxide. This was probably the result of a localized obstruc- tion during perfusion of these large brains. These few areas from two pigs were excluded from further analysis. With pig brain tissue, hypothalamic magnocellular perikarya tended to acquire a homogeneous, nonspecific background color: This could be greatly attenuated by short incubation in 2% normal goat serum before use of goat biotinylated anti- rabbit immunoglobin. Preincubation of TH and DBH antiserum with protein A also reduced this nonspecific staining but did not affect specific immunostaining.

Preincubation of the TH and DBH antiserum with purified bovine recombinant TH or with isolated DBH enzymes as well as partially purified homogenates of porcine adrenal medulla tissue also blocked specific immunostaining. Western blots of tissue homogenates from pig adrenal and brain probed with TH antiserum resulted in a single band that migrated slightly faster than purified bovine TH, corresponding to an estimated size of 57,000 Daltons vs. 62,000 Daltons for bovine TH. With older, previously frozen tissue, there was also a small ( < 15,000 Dalton) immunoreactive element, which was probably a fragment of the enzyme produced upon thawing or homog- enization. When probed with DBH antiserum, a major band was detected with an estimated size of 73,000 Daltons, and another was detected with an estimated size of 150,000 Daltons: The larger was probably an aggregate of the smaller subunit (Craine et al., 1973).

Specific immunostaining The distribution of TH- and DBH-ir neurons within the

porcine hypothalamus is presented in Figure 1. There were no DBH-ir perikarya in the hypothalamus or in adjacent regons. All regions of DBH-ir fibers and varicosities were also TH-ir in adjacent sections. TH-ir fibers and varicosities often appeared more intense or prominent than DBH-ir fibers and varicosities, but this difference was not analyzed quantitatively. Whether TH-ir and DBH-ir are actually colocalized in the same neurons will need future verifica- tion using double immunocytochemical methods.

In the diagonal band of Broca, there were no TH-ir or DBH-ir perikarya. Loosely scattered TH- and DBH-ir fibers and varicosities occupied a narrow band just above the ventral edge of the forebrain and along the dorsal edge of the anterior supraoptic nucleus (Fig. 1A,B).

There were no TH-ir or DBH-ir perikarya in the bed nucleus of stria terminalis. Beneath the anterior commissure, DBH-ir fibers and varicosities were very dense and appeared more abundant than TH-ir varicosities (Fig. 1 C-F).

Diagonal band of Broca.

Red nucleus of stria terminalis.

TH DBH TH DBH A o , ,

c1 .2

OVLT

-L- Fig. 1. Distribution of tyrosine hydroxylase (TH) and dopamine-f-

hydroxylase (DBH) immunoreactivity in the porcine hypothalamus and adjacent regions. Tracings of coronal brain sections are bisected with TH immunostaining depicted on the left, and DBH immunostaining is depicted on the right. Letters ( A d ) indicate representative sections

(anterior to posterior), and numbers indicate the approximate distance (mm) from the first section (A). Circles indicate perikarya, and fine lines and dots indicate fibers and terminals. The density of circles and lines indicates the relative density of perikarya and fibers within one tissue section. See list for Abbreviations.

HYPOTHALAMIC CATECHOLAMINERGIC NEURONS IN THE PIG

TH DBH TH

7 1 H 5.5

N

155

DBH 16.7

Vm r

J 7.0

Figure 1 (Continued)

Organum vasculosum of lamina terminalis. Along the midline, the organum vasculosum of lamina terminalis and its immediately adjacent tissue contained small (5-8 pm diameter) TH-ir perikarya and both TH- and DBH-ir varicosities (Figs. lC, 2A,B). However, neither TH-ir perikarya nor varicosities were abundant.

There were no TH-ir or DBH-ir perikarya in either the medial or the lateral preoptic areas. Along the medial border of the medial preoptic area, some TH-ir perikarya were observed, but, from serial sections, these neurons were judged to be an anterior extension of the periventricular TH-ir neurons (see below). There were abundant TH-ir and DBH-ir varicosities in the medial 200-300 km region of the medial preoptic area, but their abundance was markedly sparse in other regions of the

Preoptic area.

medial preoptic area and throughout the lateral preoptic area.

Throughout the entire length of the third ventricle, TH-ir perikarya were abundant in the periventricular regon (Fig. 1 C J ) . The preoptic division of the periventricular nucleus, dorsal to the organum vasculosum of lamina terminalis, contained a few scattered TH-ir perikarya 400-500 pm from the midline, near the border with the medial preoptic area (Figs. lC, 2A-C). These cells were either oval or triangular in shape with bipolar or tripolar processes. Progressing posteriorly along the periventricular region, TH-ir perikarya were more ventrally located, and their abundance decreased substantially posterior to the arcuate nucleus. Most periventricular TH-ir perikarya were small (5 x 15-20 pm), bipolar, and spindle

Periventricular nucleus.

Fig. 2. A Cresyl violet-stained, coronal half section through the medial preoptic area (MPOA) and the anterior aspect of the organum vasculosum of lamina terminalis (OVLT). B: Magnified view of TH immunoreactivity along the anterior aspects of the preoptic periventricular area (PeV), OVLT, and MPOA illustrating the linear arrangement of TH immunoreactive (ir) perikarya (arrowheads) bordering the lateral PeV with the medial MPOA. C: Magnified view of the triangular

perikaryon that is circled in B. D: Most periventricular neurons were bipolar and spindle shaped (arrowhead). This is one of the few neurons situated within the ependymal cell layer (denoted by the dotted line). E: DBH-ir varicosities form pericellular arrays (arrowheads) surrounding unstained perikarya throughout the periventricular area and the supraoptic nucleus (SON). D,E, differential interference contrast optics. Scale bars = 500 pm in A, 100 km in B, 10 km in C,D.


Fig. 3. A: TH-immunoreactivity of the anterior part of the suprachi- region (arrowheads in A). C: Short TH-ir fibers (arrowheads; magnified asmatic nucleus. At this coronal level, the suprachiasmatic nucleus lies from circle C in A) can be seen a t the ventral demarcation between the directly on the optic chiasm (oc) and protrudes dorsally into the oc and the suprachiasmatic nucleus. D: DBH-ir in the suprachiasmatic supraoptic recess of the third ventricle. B: Magnified view of a nucleus is abundant and appears as punctate varicosities. Scale bars = spindle-shaped TH-ir neuron (from circle B in A) within the suprachias- 50 pm in A, 10 pm in B-D. matic nucleus. Other TH-ir neurons lie ventral and lateral to this

shaped. For the most part, they were spaced individually adjacent to the ependymal cell layer lining the ventricle, but, occasionally, they were found within the ependymal layer (Fig. 2D).

Both TH-ir and DBH-ir fibers and varicosities were abundant throughout most of the periventricular region. Similar to the distribution of TH-ir perikarya, progressing posteriorly, DBH-ir fiber and varicosity density decreased in dorsal aspects of the periventricular nucleus. Varicosities often formed pericellular rings around unstained or nonspecifically darkened perikarya (Fig. 2E).

Suprachiasmatic nucleus. Anterior regions of the suprachiasmatic nucleus situated on the dorsal surface of the optic chiasm and protruding into the third ventricle contained small ( 10 Fm diameter), bipolar, oval-shaped TH-ir n o v i l D m i i i P ; m c 1P-l7 2A-Pl Thncn x x r o v - 0 lno-torl hnth

within the interior of the nucleus and along its ventral border with the optic chiasm. More posteriorly, with the suprachiasmatic nucleus positioned near part of the ventral- lateral border of the third ventricle (Fig. l E ) , TH-ir perikarya were also observed, but their size and shape were more consistent with periventricular TH-ir neurons. Throughout the suprachiasmatic nucleus, numerous TH-ir and DBH-ir varicosities were observed (Fig. 3 B,D). Some TH- and DBH-ir fibers could be traced along the ventral border of the suprachiasmatic nucleus with the optic chiasm (Fig. 3 C ) . More posterior, it appeared that many of these fibers crossed the supraoptic commissure in a medial- dorsal direction to the ventral suprachiasmatic nucleus.

Scattered TH-ir perikarya were found mostly in anterior regions of the supraoptic nucleus

Supraoptic nucleus.

I l A m 1R-PI n f h n v TU.;v nnvilzov~m 3 n n m o o o t t n m r l thn

L.S. LESHIN ET AL. 158

length of the supraoptic nucleus, but, most often, they were situated just dorsal to the supraoptic nucleus along the ventral edge of the anterior or lateral hypothalamic areas. At the anterior pole of the supraoptic nucleus, these perikarya were small (8-10 pm diameter). A very intensely stained group of TH-ir perikarya was located in the retrochiasmatic subdivision of the supraoptic nucleus (Fig. 4A) and was separated from the principal division of the supraoptic nucleus by a line of scattered TH-ir neurons that followed the curvature of the medial-dorsal surface of the optic tract [Fig. 1F). Neurons in this group extended posteriorly to the level of the anterior median eminence but were situated lateral to it at this level. Along the curvature of the optic tract, TH-ir neurons had a flattened appearance (Fig. 4B) with the cell axis and processes orientated parallel to the lorsal edge of the optic chiasm. About one-half of these intensely stained TH-ir neurons in the retrochiasmatic region were magnocellular (30-50 pm diameter) with multipolar thick processes (Fig. 4C).

Both TH- and DBH-ir varicosities were prevalent through- 3ut the supraoptic nucleus, and they often outlined unstained neurons in a pericellular ring-like arrangement :Fig. 4D). The retrochiasmatic division of the supraoptic nucleus was also heavily innervated with DBH-ir varicosities. Processes from TH-ir cells in the retrochiasmatic Sivision could be followed in serial sections in three direc- tions: medial and posterior in the direction of the median 3minence, anterior and dorsal in the direction of the suprachiasmatic nucleus, and dorsolaterally over the optic :hiasm and optic tract in the direction of the principal livision of the supraoptic nucleus.

The dorsal region of the para- Jentricular nucleus was densely populated with TH-ir Derikarya (Figs. 1G,H, 5A,B). These perikarya were often nagnocellular (30-50 pn diameter), although smaller TH-ir :ells were also present. Most TH-ir neurons were multipolar with long branching processes (Fig. 5C). Some periven- zricularly located TH-ir perikarya near the paraventricular iucleus were more characteristic of paraventricular neu- pons than of periventricular neurons (Fig. 5B). Varicosities if TH-ir and DBH-ir were also prevalent, but they were nore abundant in the medial regions of the paraventricular iucleus.

The arcuate nucleus contained a large 3opulation of TH-ir perikarya (Fig. 1F-HI. Most of these ieurons tended to be located ventrally throughout the mtire length of the arcuate nucleus. They also were listinctly lateral to the TH-ir neurons of the arcuate ieriventricular region (Fig. 6B). These ventrolateral peri- tarya were medium sized (10-20 pm diameter) with a -ounded or oval bipolar shape. The arcuate nucleus and surrounding area contained a very high density of DBH-ir md TH-ir varicosities (Fig. 6B,C).

The median eminence was extensively innervated with TH-ir fibers and varicosities, but rH-ir perikarya were absent (Figs. 1G,H, 6B). The lateral nedian eminence contained the greatest density of TH-ir raricosities, occupying both the internal and the external amina but not penetrating into pars tuberalis (Fig. 6B). In :ontrast to the abundance of TH-ir fibers and varicosities, IBH-ir fibers and varicosities in the median eminence were sparse and were found only in anterior-dorsal regions of the nedian eminence. Except for these anterior-dorsal regions, ircuate nucleus DBH-ir abruptly ended at the ventral )order with the median eminence. In the median eminence.

Paraventricular nucleus.

Arcuate nucleus.

Median eminence.

TH-ir varicosities were clustered around capillaries in both the internal and the external lamina. However, the sparse DBH-ir varicosities did not seem to cluster around capillaries.

Ventromedial and dorsomedial nuclei. At posterior lev- els of the arcuate nucleus, a group of TH-ir perikarya extended laterally from the third ventricle around the ventromedial nucleus (Fig. 1 G,H). These perikarya were small to medium sized (10-20 pm diameter) with a rounded bipolar shape. The dorsomedial nucleus and the surrounding area also contained sparsely scattered TH-ir perikarya similar in size and shape to those around the ventromedial nucleus. In these regions, there was low abundance of TH-ir and DBH-ir fibers and varicosities. Progressing laterally a few hundred microns from the third ventricle, the density of both TH-ir and DBH-ir varicosities was dramati- cally reduced.

The lateral hypothalamus contained a few scattered TH-ir perikarya (Fig. 1E-H). Most of these neurons were located ventrally, just dorsal to the supraoptic nucleus. The group of TH-ir neurons that passed around the ventromedial nucleus continued to radi- ate laterally, then turned ventrally, and ended lateral to the arcuate nucleus. Except for the ventral regions, the lateral hypothalamus contained a very sparse distribution of TH-ir and DBH-ir fibers and varicosities, which were mostly localized near TH-ir perikarya.

Posterior hypothalamus. The posterior hypothalamus contained very small (5-10 pm diameter) bipolar TH-ir neurons that were situated ventral and lateral to the third ventricle (Figs. lI,J, 7B). At this posterior level of the ventricle, there were few TH-ir or DBH-ir varicosities. These were found mostly in the periventricular region of the posterior hypothalamus.

Mammillary complex. The supramammillary nucleus contained some small bipolar, oval-shaped TH-ir perikarya (Fig. 7C). A band o f similar TH-ir cells and fibers extended over the medial marnmillary nucleus to the lateral mammillary nucleus. Within the large medial mammillary nucleus, no TH-ir or DBH-ir neurons were found. Both TH-ir and DBH-ir varicosities were prevalent ventrally and laterally surrounding the medial mammillary nucleus and within the supramammillary nucleus.

Within the hypophysis or pituitary gland, TH immunoreactivity was extensive throughout the neurohypophysis but was absent in the adenohypophysis and pars tuberalis (Fig. 8A,B). Long, beaded TH-ir fibers coursed through the dorsal stem of the neural lobe (Fig. 9A). Near some of the larger capillaries of the neurohypophysis, DBH-ir fibers and varicosities were observed infre- quently (Fig 9B). No other pituitary regions contained DBH-ir. In the neural lobe, dense fibers and varicosities were grouped in long strands that were situated between unstained capillaries (Fig. 9C). Cells of the pars intermedia that were situated along the ventral edge of the neurohypophysis between the neural lobe and the hypophysial cleft were innervated by TH-ir fibers and varicosities. Located at the posterior tip of the adenohypophysis, a cluster of pars intermedia tissue also was innervated by TH-ir fibers (Fig. 9E). These fibers coursed over the dorsal edge of the anterior lobe (Fig. 9F).

DISCUSSION This is the first detailed description of catecholaminergic

neurons in the uorcine hvDothalamus. The findings of

Lateral hypothalamus.

Pituitary gland.


Fig. 4. A: Cresyl violet-stained sagittal section, approximately 1 mm lateral to sagittal midline, through the retrochiasmatic division of the supraoptic nucleus (SONr; encircled by arrowheads), the optic chiasm (oc), and the medial basal hypothalamus. B: The TH-ir neurons located along the posterior-dorsal border of the optic chiasm tend to be flattened and elongated, with processes that extend parallel to the dor-

sal surface of the optic chiasm. C: In the retrochiasmatic division of the SON, a loosely arranged cluster ofTH-ir neurons (arrowheads) are very intensely stained. D: DBH-ir varicosities in the SON often form pericellular rings (arrowheads) ARC, arcuate nucleus; me, median eminence; pt, pars tuberalis; Vir, infundibular recess of the third ventricle. Scale bars = 500 )*m in A, 10 p n in B-D.

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Fig. 6. A,B: Cresyl violet-stained coronal half-section (A) and TH-ir fibers and varicosities are found throughout. the arcuate nucleus, but, neurons tB) of the arcuate nucleus (ARC) and median eminence (me). ventral to the junction with the median eminence, DBH-ir varicosities Most TH-ir perikarya are located ventrally with a slight gap separating are sparsely distributed, located only in anterior-dorsal aspects of the them from the more medially located arcuate periventricular neurons median eminence. in, internal lamina of median eminence; PeV, (between arrows in B). Pars tuberalis (pt) tissue lies adjacent the periventricular nucleus; V, third ventricle; VMN, ventromedial nucleus. external lamina (ex) of the median eminence. C: Extensive DBH-ir Scale bars = 500 pm in A, 100 Fm in B, 10 pm in C.


Fig. 7. A Cresyl violet-stained coronal section through the posterior hypothalamus (PHI and the medial mammillary nucleus (MNn). B,C: Bipolar TH-ir neurons (arrowheads in C) and numerous fibers are scattered in the PH near the ventricle and in the supramammillary nucleus (SM). B, differential interference optics. Scale bars = 500 pm in A, 10 pm in B, 100 p m in C.

HYPOTHALAMIC CATECHOLAMINEKGIC NEURONS IN THE PIG 163

Fig. 8. Cresyl violet-stained (A) and TH iB) immunoreactivity in sagittal sections of the basal hypothalamus and pituitary gland. There is substantial TH-ir within the arcuate nucleus (ARC), the retrochiasmatic division of the supraoptic nucleus (SONr), and the median eminence (me) . Dense TH-immunoreactivity is also present along the neural (dorsal, Dart of the Dituitarv stalk or infiindihular s tem and bars = 1 0 m m in A-R

throughout pars nervosa (pNI and pars intermcdia (PI). Higher magnification photographs of areas circled (A-F) in photograph B are presented in Figure 9. cl. pituitary cleft; IP, interpeduncular nucleus; MNm, medial mammillary nucleus; oc, optic chiasm; PA, pars anterior; pt, pars tuberalis; V, infundibular recess of the third ventricle. Scale


Fig. 9. A-F: Magnified views of circled regions A-F in Figure 8B. A: Long, thin fibers (arrowheads) with large beaded varicosities of TH-ir make up the neural tissue of the dorsal pituitary stalk (infundibular stem). B: The top surface of a capillary (c; bounded by the larger ring of dots) contains DBH-ir fibers (arrowheads). The bottom surface of the capillary lumen is bounded by the smaller ring of dots. C: Dense, beaded varicosities in pars nervosa of the infundibulum form linear arrays

between clusters of unstained cells and capillaries. D: TH-ir fibers interspersed between unstained pars intermedia cells located along the ventral and lateral border of the neural lobe adjacent the pituitary cleft (cl) and at the distal tip of the anterior lohe (E). F: Along the dorsal surface of the anterior lobe, TH-ir fibers (arrowheads) course along the medial-dorsal surface of the anterior lobe. A, B, and F, differential interference contrast optics. Scale bars = 10 p.m in A-F.


particular interest for pigs compared to other species include the presence of very intensely stained TH-ir perikarya (A15 dorsal catecholamine group) in the retrochiasmatic division of the supraoptic nucleus, the absence of TH-ir perikarya ventral to the anterior commissure (A15 dorsal cell group), the very dense DBH-ir innervation of the arcuate nucleus, the dense innervation of only the medial side of the medial preoptic area along with the sparse DBH-ir innervation of the rest of the medial and the entire lateral preoptic area, and the paucity of DBH-ir in the median eminence. With these exceptions, the distribution of TH and DBH immunoreactivity in the hypothalamus was generally similar to the distribution of these enzymes observed in rat (Chan-Palay et al., 1984; Hokfelt et al., 1984; Van Den Pol et al., 19841, mouse (Ruggiero et al., 1984), cat (Luppi et al., 1986; Kitahama et al., 1987), sheep (Lehman et al., 1988; Tillet and Thibault, 1989), monkey (Thind and Goldsmith, 1986; Kohama et al., 19921, and human (Pearson et al., 1983, Spencer et al., 1985; Pearson et al., 1990). These studies localizing the catecholamine synthesizing enzymes TH and DBH closely correspond with previous studies in which the hypothalamic transmitters were detected by histofluorescent techniques in rat (Fuxe, 1965a,b; Ungerstedt, 1971; Bjorklund et al., 1973b; Lind- vall and Bjorklund, 1974), cat (Cheung and Sladek, 1975; Pointras and Parent, 1975), primates (Felten et al., 1974; Hoffman et al., 1976; Ishikawa and Tanaka, 1977; Felten and Sladek, 1983), and human (Nobin and Bjorklund, 1973).

Immunostaining for TH, which converts tyrosine to DOPA (3,4-dihydroxy-L-phenylalanine), identifies all catecholamine synthesizing cells but cannot distinguish the types of catecholamine produced or released. Similarly, DBH-containing neurons, which convert dopamine to norepinephrine, also identify those cells that produce epinephrine, the next enzymatic step catalyzed by phenylethanolamine N-methyl transferase (PNMT). Obviously, the presence of a synthetic enzyme does not necessarily mean that the resultant product is released as a neurotransmit- ter. However, immunostaining of TH and the absence of immunostaining for DBH is commonly acknowledged to identify exclusively dopaminergic neurons. Additional study directed toward localization of PNMT will be necessary to distinguish between norepinephrine- and epinephrine- synthesizing neurons.

Diencephalic catecholamine groups A l l to A15 Dahlstrom and Fuxe (1964) initiated the alphanumeric

system for delineation of catecholamine groups (A1 to A12). Additional groups (A13 and A14) were incorporated by Fuxe and Hokfelt (1969) and Bjorklund et al. (1973a), and group A15 was incorporated by Hokfelt et al. (1984). In rodents and in other small animals, catecholaminergic cell groups tend to lie close to adjacent groups, such that the groups appear to merge together smoothly (Hokfelt et al., 1984; Van Den Pol et al., 1984). In humans (Pearson et al., 1990) and in sheep (Tillet and Thibault, 1989), animals with large brains, these catecholamine groups tend to be isolated from adjacent groups. Likewise, in the porcine hypothalamus, most clusters of TH-ir neurons do not merge together but are spatially separated. Also, in pigs, like in humans, neurons within a group or a region tend to be more loosely clustered than they are in animals with smaller brains. Additional differences in the porcine hypothalamus are discussed below and are grouped according to the original nomenclature (A1 1 to A15) for hypothalamic catecholaminergic neurons.

The A1 1 ~ O U D is made UD of neurons of the Deriventricu-

hypothalamic areas in the rat (Dahlstrom et al., 1964; Bjorklund et a]., 1973b). In pigs, the A l l group is less extensive and does not extend into the caudal thalamus. These cells remain close to the dorsal region of the third ventricle and near the mammillothalamic tract as it pen- etrates the dorsal hypothalamus, similar to the description of this cell group in sheep (Tillet and Thibault, 1989).

The A12 region contains neurons of the arcuate nucleus and the periarcuate region including the adjacent arcuate periventricular regions. In the porcine brain, the perikarya that make up the nuclei tend to be loosely clustered: Thus, specific nuclear boundaries are not easily discerned. Most TH-ir cells lie ventrolaterally within the arcuate nucleus but do not extend into the median eminence as described in sheep (Tillet and Thibault, 1989) and human (Spencer et al., 1985). In contrast to rat (Hokfelt et al., 1984), ventral arcuate cells (A12v) do not seem to be less intensely stained than dorsal cells (A12d): They are approximately evenly stained. The entire arcuate nucleus is very densely innervated by DBH-ir fibers and varicosities in marked contrast to the median eminence, which is almost devoid of DBH-ir innervation. This dense DBH immunoreactivity in the arcuate also contrasts to the slight innervation in the rat (Swanson and Hartman, 1975).

The A13 cell group in the rat is composed of neurons in the dorsal paraventricular nucleus and the adjacent dorsal hypothalamic area extending laterally to the medial part of zona incerta and the internal capsule. Cells also occupy a region ventromedial to the mammillothalamic tract (Hok- felt et al., 1984). In pigs, this region is located more posterior and ventrolateral to the mammillothalamic tract and does not extend as far laterally as described for rats. Also, in pigs, it is not easy to distinguish clearly the posterior-dorsal boundary from the A l l group, but, similar to sheep (Tillet and Thibault, 1989), the A13 group lies more anteriorly. In addition, in both pigs and sheep, the paraventricular nucleus is heavily innervated by DBH-ir varicosities and fibers.

The A14 group encompasses all of the hypothalamic periventricular neurons and extends from near the level of the anterior commissure, including cells near the organum vasculosum of lamina terminalis and the suprachiasmatic nucleus, to the rostra1 border of the median eminence in rat (Hokfelt et al., 1984). However, TH-ir neurons in porcine hypothalamus extend farther posterior to the level of the mammillary nuclei. Pigs also lacked the anterior extension of the A14 group, which, in the rat, extends dorsally and laterally to merge with the dorsal A15 cell group. Also, in pigs (and in rats; Hokfelt et al., 1984), there is a very distinct group of TH-ir neurons that split laterally from the periventricular region just dorsal to the arcuate nucleus. These cells course laterally then ventrally around the ventromedial nucleus into the ventrolateral hypothalamus, becoming more dispersed. These lateral multipolar TH-ir neurons differ in morphology from the spindle-shaped periventricular neurons, although they are both classified as A14 neurons.

In the rat, some of the cells located along the lateral wall of the infundibular recess (part of the subdivision of the dorsocaudal arcuate nucleus) contain aromatic-L-amino acid decarboxylase (AADC), which converts the TH product L-DOPA into dopamine (Jaeger et al., 1984). However, in both the pig and the rat, TH-ir cells were not found around the infundibular recess (Hokfelt et al., 1984; Jaeger et al., 1984: Rumiero et al.. 1984). Cells that lack TH but that


DOPA and serotonin, but they are considered to be unable to synthesize catecholamines from tyrosine (Jaeger et al., 1984). Whether comparable infundibular TH-negative cells in the pig contain AADC is not yet known. The physiological implications and significance of cells lacking TH but containing AADC have not been extensively examined.

The A15 cell group, which is the most recently described of the hypothalamic groups, consists of both dorsal and ventral subgroups (Hokfelt et al., 1984). The ventral group lying within the retrochiasmic region corresponds closely with the retrochiasmatic division of the supraoptic nucleus in the pig, rat (Hokfelt et al., 1984), cat (Luppi et al., 1986) and sheep (Tillet and Thibault, 1989). The dorsal subgroup, which is situated far more anteriorly and lies in the ventral part of the bed nucleus of stria terminalis beneath the anterior commissure in the rat (Hokfelt et al., 19841, was not present in pigs. Also, a ventral A15 group was described only in the ovine hypothalamus (Tillet and Thibault, 1989). This ventral A15 group is innervated by DBH-ir varicosities in the pig (Tillet and Thibault, 1993).

There were three fiber tracts associated with this retrochiasmatic region: One coursed dorsally and then coursed anteriorly along the curvature of the optic chiasm-optic tract transition (retrochiasmatic region). A second fiber tract coursed dorsally and then coursed laterally along this curvature toward the supraoptic nucleus. A third fiber tract was directed posteriorly toward the median eminence. The origin and termination of these fiber tracts have not been determined but are described here from serial section reconstruction.

Inferences for porcine neuroendocrine physiology

The extensive TH-ir innervation of the neurohypophysis and median eminence and the lack of DBH immunoreactivity implicate dopamine as a hypothalamic neurohormone with hypophysiotropic functions in both the neurohypophysis and the adenohypophysis. Numerous pharmacological studies together with hypothalamic deafferentations and hypophysial stalk transection studies also implicate dopamine of hypothalamic origin as an inhibitor of prolactin secretion in pigs (Kraeling et al., 1992). The origin of the dopaminergic innervation of the neurohypophysis and the median eminence in the pig cannot be precisely determined by serial section histology without the use of anterograde or retrograde tract-tracing dyes. Because TH-ir fibers from the arcuate and periventricular divisions and, possibly, from the retrochiasmatic division of the supraoptic nucleus course ventrally and caudally toward the median eminence, it is likely that perikarya in these regions are the source of this innervation. In the rat, tract-tracing studies have demonstrated that dopaminergic median eminence afferents arise from these regions as well as from extrahypotha- lamic areas of the preoptic region, diagonal band of Broca, medial septum, and brainstem (Lechan et al., 1982). How- ever, the dopaminergic neurohypophyseal afferents arise from a more restricted area that includes only the most rostra1 arcuate and periventricular regions (Van Den Pol et al., 1984; Luppi et al., 1986). The superior cervical ganglia may be the source of norepinephrine innervation (Saave- dra, 1985).

In the pig, the locations of TH- and DBH-immunoreactive neurons in the diagonal band, preoptic areas, organum vasculosum of lamina terminalis, periventricular areas, and arcuate nucleus coincide with locations of luteinizing hormone-releasing hormone (LHRH; Kineman et al., 1988).

Initial double-labeled immunocytochemical studies of TH and LHRH immunoreactivity showed that TH-ir varicosities are closely apposed to LHRH-ir perikarya and dendrites (Leshin et al., 1989) and warrant further ultrastructural studies. In the arcuate nucleus, the abundance of TH-ir neurons and the dense innervation by DBH-ir fibers and varicosities also overlap with the distribution of at least one opioid peptide, P-endorphin (Kineman et al., 1988). Anatomi- cal overlap of TH-ir, LHRH-ir, and P-endorphin-ir terminals also occurs in the median eminence and is especially prominent in the lateral external layer. These potential sites of direct anatomical communication together with pharmacological studies with pigs suggest that catecholaminergic neurons may interact with LHRH and opioid neurons to regulate luteinizing hormone (LH) secretion and reproductive physiology (Kraeling and Barb, 1990; Kraeling et al., 1992; Changet al., 1993).

Locations of growth hormone-releasing hormone (GHRH) and somatostatin (SS)-ir neurons in the pig hypothalamus (Leshin et al., 1994) also overlap with the distribution of catecholaminergic neurons described here. Perikarya of both GHRH- and TH-ir neurons are found in similar regions of the arcuate nucleus and share a common terminal field in the lateral external layer of the median eminence. The porcine arcuate nucleus is also densely innervated by DBH-ir fibers and varicosities. Although it has not, yet been determined for porcine tissue, in the ventrolateral arcuate nucleus of rats, TH-ir was colocalized with most GHRH-ir neurons (Okamura et al., 1985; Meister et al., 1986). Hypothalamic SS neurons in the pig are located mostly in the periventricular region from the preoptic level to the caudal end of the paraventricular nucleus. These locations completely overlap with TH-ir perikarya and both TH-ir and DBH-ir fibers and varicosities, but colocalization of SS with TH immunoreactivity has not yet been observed in the mammalian hypothalamus.

To date, there are no studies with pigs concerning catecholaminergic neurotransmitters that influence GHRH and SS secretion and, consequently, that influence growth hormone (GH) secretion. Pharmacological studies in other species have demonstrated that GH secretion is responsive to both dopaminergic and noradrenergic agents (Muller, 1987). Because anterior and posterior hypothalamic deafferentation of pigs abolished episodic GH secretion (Molina et al., 1986), it is likely that catecholaminergic innervation described here in the arcuate nucleus is functionally coupled to GHRH neurons to convey the signal for GHRH secretion and, consequently, for pituitary GH secretion.

Pituitary catecholamines Our detection of catecholamine-synthesizing enzymes in

the porcine pituitary gland confirms the previous detection of pituitary catecholamines by Bjorklund et al. (1967) and Bjorklund (1968) using fluorometric and histofluorescence techniques. Their studies were some of the first to examine mammalian pituitary tissues with these techniques. Dopa- mine was the most abundant biogenic amine detected (0.3-0.5 pglg tissue), with norepinephrine concentrations that were 10- to 20-fold lower in the neurointermediate lobe and with both catecholamines similarly low (0.01-0.03 FgJg tissue) in the adenohypophysis. The observed dopamine-related fluorescence was located in nerve fibers of the infundibular stem and the proximal part of the neural lobe. TH-ir innervation of pars intermedia tissue, as described here, was extensive. In addition, TH immunoreactivity was located in an isolated group of cells in the most distal tip of


the anterior lobe, with TH-ir fibers along the dorsal surface of the anterior lobe. This probably accounts for the detect- able (but low) concentrations of dopamine in the anterior lobe (Bjorklund et al., 1967). Adrenergic receptors of p l type were demonstrated in porcine anterior pituitary tissue (Perkins et al., 19851, but their cellular location is still unknown, and their function is only speculative. In all regons of the pituitary, DBH-ir was observed only as small clusters of fibers and varicosities surrounding blood vessels. Similar fibers in the rat pituitary were partially eliminated by removal of the superior cervical ganglion: Therefore, this innervation was partly attributed to a peripheral sympa- thetic origin (Fuxe, 1964; Bjorklund, 1968). Their presence in the pig might be indicative of catecholaminergic regulation of pituitary microcirculation (Kemeny et al., 1985; Page, 1986).

The description of porcine hypothalamic neurons that are immunoreactive for the catecholamine synthesizing enzymes TH and DBH corresponds for the most part with the locations of these enzymes and their catecholamine products reported in other mammalian species using immunocytochemical or fluorescent formaldehyde condensates techniques. The important differences include the absence of the TH-ir neurons below the anterior commissure (dorsal A15 dopaminergic neurons), the very intense TH-ir neurons of the retrochiasmatic division of the supraoptic nucleus (ventral A15 dopaminergic neurons), and the sparse distribution of DBH-ir in the median eminence. Further anatomical and physiological studies of these specific re- mans in pigs compared to other mammalian species will offer a unique opportunity for elucidating behavioral and neuroendocrine species differences with regard to catecholamine neurotransmitters.

ACKNOWLEDGMENTS The authors thank D. Slavin, L. Taras for technical

assistance, and S. Silvers for the use of photographic facilities. We also thank Drs. F.J.C.M. Van Eerdenburg and M. Lakomy for critical review of the manuscript and J. Minor, S. Coffman, T. Martin, and A. Hitchcock-Watson for preparing this paper.

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Immunocytochemical distribution of catecholamine-synthesizing neurons in the hypothalamus and pituitary gland of pigs: Tyrosine hydroxylase and dopamine-?-hydroxylase