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BOR - Papers in Press, published online ahead of print November 13, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.010595
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BIOLOGY OF REPRODUCTION 68, 1119–1133 (2003)
DOI: 10.1095/biolreprod.102.010595
© 2003 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology

Neuronal Inputs from the Hypothalamus and Brain Stem to the Medial Preoptic Area of the Ram: Neurochemical Correlates and Comparison to the Ewe1

Christopher J. Scott2,a, Iain J. Clarkeb, and Alan J. Tilbrooka

a Department of Physiology, Monash University, Victoria 3800, Australia b Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The retrograde tracer, FluoroGold, was used to trace the neuronal inputs from the septum, hypothalamus, and brain stem to the region of the GnRH neurons in the rostral preoptic area of the ram and to compare these imputs with those in the ewe. Sex differences were found in the number of retrogradely labeled cells in the dorsomedial and ventromedial nuclei. Retrogradely labeled cells were also observed in the lateral septum, preoptic area, organum vasculosum of the lamina terminalis, bed nucleus of the stria terminalis, stria terminalis, subfornical organ, periventricular nucleus, anterior hypothalamic area, lateral hypothalamus, arcuate nucleus, and posterior hypothalamus. These sex differences may partially explain sex differences in how GnRH secretion is regulated. Fluorescence immunohistochemistry was used to determine the neurochemical identity of some of these cells in the ram. Very few tyrosine hydroxylase-containing neurons in the A14 group (<1%), ACTH-containing neurons (<1%), and neuropeptide Y-containing neurons (1–5%) in the arcuate nucleus contained FluoroGold. The ventrolateral medulla and parabrachial nucleus contained the main populations of FluoroGold-containing neurons in the brain stem. Retrogradely labeled neurons were also observed in the nucleus of the solitary tract, dorsal raphe nucleus, and periaqueductal gray matter. Virtually all FluoroGold-containing cells in the ventrolateral medulla and about half of these cells in the nucleus of the solitary tract also stained for dopamine ß-hydroxylase. No other retrogradely labeled cells in the brain stem were noradrenergic. Although dopamine, ß-endorphin, and neuropeptide Y have been implicated in the regulation of GnRH secretion in males, it is unlikely that these neurotransmitters regulate GnRH secretion via direct inputs to GnRH neurons.

catecholamines, gonadotropin-releasing hormone, hypothalamus, neuropeptides, neurotransmitters


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reproduction is controlled centrally by GnRH. In the ram, this hormone is secreted into the hypophyseal portal blood in a pulsatile manner to regulate the secretion of the gonadotropins [1]. The characteristics of this pulsatile secretion are modified by a number of factors, including testicular steroids, season/photoperiod, nutrition, social cues, and more generally stress [2–4]. Information regarding these factors is relayed to the GnRH neurons by a complex network of neuronal pathways. The location of the neurons involved in these processes in the male remains largely unknown. The majority of the GnRH neurons in most mammalian species are located in the medial preoptic area, predominantly in the most rostral region [5]. The origin of the neuronal inputs to the medial preoptic area has been investigated in male rats using horseradish peroxidase as a retrograde tracer [6–8]. In these studies, the nondiscrete injections covered a large portion of the preoptic area, much of which contains very few GnRH neurons. Furthermore, the injection sites did not include the most rostral region of the preoptic area, where most GnRH neurons are found [5]. The information gained, therefore, is of limited use in the determination of potential inputs to the GnRH neurons in males.

Tillet et al. [9] used the retrograde tracer FluoroGold to map the location of cells that project to the rostral preoptic area in the ewe. Retrogradely labeled cells were observed throughout the hypothalamus and in a number of extrahypothalamic sites, including the amygdala, hippocampus, and a number of regions of the brain stem [9]. In the ewe, some orexin-containing neurons in the lateral hypothalamus provide direct input to GnRH neurons [10]. Noradrenergic neurons of the ventrolateral medulla in the caudal brain stem also project to the medial preoptic area in the ewe [11]. These neurons contain estrogen receptors [12] and thus may be involved in the feedback regulation of GnRH secretion by this steroid. Similar data are not available for the ram. The regulation of GnRH secretion in the sheep is sexually dimorphic, i.e., ewes demonstrate a positive feedback response to estrogen but rams do not. The brain area in which this positive feedback response appears to be generated is the region of the ventromedial/arcuate nuclei [13]. In addition, rams and ewes differ in the way stress influences LH secretion [14]. Furthermore, GnRH neurons in ram lambs receive only about half the number of synaptic inputs as do those neurons in ewe lambs [15], suggesting that the inputs to the GnRH neurons differ in males and females. Accordingly, the first aim of the present study was to map the distribution of cells in the hypothalamus and brain stem that project to the rostral part of the medial preoptic area in the ram and compare this information with the distribution and number of inputs to the rostral preoptic area in the ewe. This map will not identify the cells that provide direct input to the GnRH neurons but will identify the origin of cells that have the potential for such input. A second aim of the present study was to determine the neurochemical identity of some of the cells that project to the rostral part of the preoptic area. We identified the hypothalamic dopamine and pro-opiomelanocortin (POMC)-containing neurons because these have been implicated in the regulation of LH secretion in rams [16]. In addition, we investigated the inputs to the preoptic area of noradrenaline- and neuropeptide Y (NPY)-containing neurons because noradrenergic and NPY-containing fibers have been demonstrated in close proximity to or in synapse with GnRH neurons in the ewe [17, 18].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
This work was approved by the Ethics Committees of the Department of Physiology, Monash University, and of the Victorian Institute of Animal Science and was conducted in accordance with the Prevention of Cruelty to Animals Act, Victorian Government 1986, and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Retrograde Tracer Injection

Adult intact Romney Marsh rams (n = 7) and ewes (n = 6) were used for this study. Rams were injected at regular intervals throughout the whole year, and the ewes were injected during anestrus. Romney Marsh rams do not show a seasonal variation in sensitivity of GnRH/LH to testosterone negative feedback [19]. For tracer injection, the sheep were anesthetized by an i.v. injection of Thiopentone (May & Baker, Cheltenham, Australia), and anesthesia was maintained with halothane (3–5%) in oxygen. The animal's head was held in a stereotaxic frame, and a guide tube was inserted into the preoptic area using x-ray guidance, as described previously [20]. The target injection site was the rostral preoptic area, immediately dorsal to the organum vasculosum of the lamina terminalis, on the midline. Injections were made 3 mm below the tip of the guide tube. The retrograde tracer, FluoroGold (Fluorochrome, Inc., Denver, CO), was pressure injected as a 4% solution in 0.9% saline in a volume of 50 nl. Injections were made using a 1-µl syringe (SGE, Ringwood, Vic, Australia) with a 23-ga needle over 5 min. The needle was left in place for a further 15 min before withdrawal. To prevent hemorrhage of the perforated sagittal sinus, the guide tube was raised above the preoptic area and then fixed in place in the cerebrum with dental acrylic.

Tissue Collection

Three weeks after injection of tracer, the sheep were killed by an overdose of sodium pentobarbital (Valabarb; May & Baker), and the brain tissue was collected. The head was removed and perfused through both carotid arteries with 2 L normal saline containing 25 000 U heparin followed by 1.5 L of Zamboni fixative, the final 0.5 L containing 20% sucrose. The hypothalamus and brain stem were removed and placed in phosphate buffer containing 30% sucrose for 5 days. The tissues were then rinsed in buffer, frozen on dry ice, wrapped in parafilm, and stored in a container at -20°C until they were sectioned in a cryostat (40 µm). Sections were collected into cryoprotectant [21] and stored at -20°C.

Immunohistochemistry

All immunohistochemistry procedures were conducted on free-floating sections except as noted. Incubations were carried out on an orbital mixer at room temperature except for incubations with primary antibody, which were performed at 4°C. Sections were washed (three or four times for 10 min each time) in 0.05 M neutral PBS between changes of solution. The sections were first incubated for 20 min in 0.1% sodium borohydride to remove excess aldehydes and then in blocking solution containing 0.3% Triton X-100 and 5% normal goat serum (1 h). The sections were next incubated for 3 days in primary antibody plus 2% normal goat serum, 0.3% Triton X-100, and 0.1% sodium azide. Details of the primary antibodies used are given in Table 1.


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  		TABLE 1. Details of the primary antibodies used in this study

Following incubation in primary antibody, the sections were treated with Alexa 594-conjugated goat anti-rabbit serum (1:400) (Molecular Probes, Eugene, OR). The sections were then washed, mounted on gelatin-coated slides, and coverslipped using an antifade mountant (DAKO Australia, Botany, Australia). All sections were stored at 4°C and protected from light until analysis. Controls included omission of primary antibody, replacement of primary antibody with nonimmune serum, and incubation of primary antibody with inappropriate secondary antibody. No immunoreactivity was visible in sections following any of these control procedures. Sections stained for ACTH required pretreatment with 0.01% proteinase K (Roche Diagnostics Australia, Castle Hill, Australia) at 37°C. To minimize tissue damage from this treatment, sections were first mounted on positively charged slides (Superfrost Plus; Biolab Scientific, Clayton, Australia) and air-dried overnight. All washes and incubations for these sections were without agitation.

Analysis

Sections were examined for FluoroGold labeling using a Nikon epifluorescence microscope (excitation filter 330–380 nm, barrier filter 420 nm). The specificity of fluorescence was determined by substituting light at different wavelengths. Sections were analyzed at 480-µm intervals through the septum and hypothalamus from the diagonal band of Broca to the region of the mamillary bodies and through the brain stem from the most caudal region of the medulla through to the caudal part of the midbrain central gray. The distribution of labeled cells was mapped with an x,y plotting system (MD Plot; Minnesota Datametrics, St. Paul, MN). The number of retrogradely labeled cells per section in each nucleus/brain region was counted from these maps, and these numbers were compared in ewes and rams using the Mann-Whitney test. Where nuclei were paired, the total for each section is the sum of both nuclei.

Sections at 240-µm intervals through the preoptic region were stained for GnRH to determine the relationship of the injection site to the main GnRH neuron-containing region. To determine the neurochemical identity of some of the FluoroGold-containing cells in the hypothalamus, sections at 480-µm intervals were stained for tyrosine hydroxylase (TH) (throughout the hypothalamus), NPY, and ACTH (sections containing the arcuate nucleus only). Brain stem sections at 480- to 960-µm intervals were stained for dopamine ß hydroxylase (DBH). GnRH immunoreactive (GnRH-ir), TH-ir, NPY-ir, ACTH-ir, and DBH-ir cells were observed using a rhodamine filter (excitation 510–560 nm, barrier 590 nm). Cells were regarded as double labeled when they showed labeling under an ultraviolet filter (FluoroGold) and a rhodamine filter (immunostaining) but not at other wavelengths. Small differences in the placement of FluoroGold injection can result in differences in the distribution and numbers of retrogradely labeled cells; thus, colocalization data are reported for individual animals.

Abbreviations

The following abbreviations are used in Figures 1, 3, 4, 7, 8, 9, and 10: AC, anterior commissure; AHA, anterior hypothalamic area; AP, area postrema; Aq, cerebral aqueduct; ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; dBNST and vBNST, dorsal and ventral regions of the BNST, respectively; bp, brachium pontis; C, cuneate nucleus; cc, central canal; DBB, diagonal band of Broca; DMH, dorsomedial nucleus of the hypothalamus; DRN, dorsal raphe nucleus; DTg, dorsal tegmental nucleus; EC, external cuneate nucleus; Fx, fornix; GP, globus pallidus; IC, inferior colliculus; IO inferior olive; LC, locus ceruleus; LHA, lateral hypothalamic area; LPB, lateral parabrachial nucleus; LR, lateral reticular nucleus; LS, lateral septum; LV, lateral ventricle; ME, median eminence; Me5, mesencephalic nucleus of the trigeminal nerve; mlf, medial longitudinal fasciculus; MS, medial septum; MT, mamillothalamic tract; NT, needle track from FluoroGold injection needle; NTS, nucleus of the solitary tract; OC, optic chiasm; OT, optic tract; OVLT, organum vasculosum of the lamina terminalis; PAG, periaqueductal gray; Pe, periventricular area; cPe and rPe, caudal and rostral regions of the Pe, respectively; PeVN, periventricular nucleus; PH, posterior hypothalamus; POA, preoptic area; POC, preolivary complex; PT, pars tuberalis; PVN, paraventricular nucleus of the hypothalamus; PVT, paraventricular nucleus of the thalamus; SC, superior colliculus; SCP, superior cerebellar peduncle; SFO, subfornical organ; SMR, supramamillary recess of the third ventricle; sol, solitary tract; SP5, spinal nucleus of the trigeminal nerve; ST, stria terminalis; VLM, ventrolateral medulla; VMH, ventromedial nucleus of the hypothalamus; vPMN, ventral premamillary nucleus; 3V, Third cerebral ventricle; 4V, Fourth cerebral ventricle; dorsal motor nucleus of the vagus; 12, hypoglossal nucleus.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Injection Sites

The location of the injection sites for all seven rams is shown in Figure 1, which also shows the relationship of the injection site to the GnRH neurons. The boundaries of the injection sites were delineated based on the presence/absence of FluoroGold labeling within glial cells. The injection sites were approximately 1.5 mm wide and long and 2–2.5 mm high. Six of seven injections were into the rostral part of the medial preoptic area at or adjacent to the midline and covering the dorsal two thirds of the preoptic area. This region contains numerous GnRH neurons (Figs. 1 and 2). The injection in the remaining animal (no. 13) was slightly lateral to the midline, just impinging on the population of GnRH neurons (Fig. 1). The location of the injection sites for all of the ewes are shown in Figure 3. All of the ewes received an injection into the GnRH neurone-containing part of the rostral preoptic area. There was no difference in the distance of the injection site from the base of the brain (ram 2.72 ± 0.10 mm, ewe 2.49 ± 0.06 mm; P > 0.05).



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  		FIG. 1. Drawings of coronal sections through the preoptic area of the seven rams injected with retrograde tracer, showing injection sites (shaded area). Filled circles show individual GnRH-ir cells. Bar = 5 mm. Inset: An injection site (m, medial; d, dorsal; v, ventral). Bar = 250 µm. Abbreviations are listed in the text on page 1121



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  		FIG. 2. Injection site in the preoptic area of a ram. A) FluoroGold-labeled cells. B) GnRH-ir neurons (arrowheads). Note the blood vessels in both figures (highlighted by arrows) for comparison. Bar = 50 µm



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  		FIG. 3. Drawings of coronal sections through the preoptic area of the six ewes injected with retrograde tracer, showing injection sites (shaded area). Filled circles show individual GnRH-ir cells. In #5 and #9, 3V indicates third cerebral ventricle; other abbreviations defined in text on page 1121. Bar = 5 mm

Distribution of FluoroGold-Labeled Cells in the Hypothalamus of the Ram

FluoroGold labeling was observed within cytoplasmic granules in both cell bodies and dendrites. The intensity of labeling was variable, from highly intense where the labeling appears homogeneous to faint where the individual granules were readily apparent. FluoroGold injections that were made on the midline (rams 869 and 17) resulted in roughly equal labeling on both the left and right sides of the section. Injections that were lateral to the midline produced labeling that was predominantly on the ipsilateral side. The distribution of FluoroGold-labeled cells in the hypothalamus of ram 869 is shown in Figure 4. The majority of rams had a distribution of FluoroGold-labeled cells that was broadly similar to that in this animal, except in the relative labeling on the ipsilateral and contralateral sides.



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  		FIG. 4. Drawings of coronal sections through the hypothalamus of a ram from rostral (A) to caudal (I), showing the distribution of FluoroGold-labeled cells following an injection of FluoroGold down the midline in the rostral portion of the medial preoptic area (ram 869). Each dot illustrates approximately 10 FluoroGold-labeled cells. The shaded area indicates the injection site. Abbreviations defined in text on page 1121. Bar = 5 mm

The lateral septum contained a relatively high concentration of FluoroGold-containing cells (Table 2 and Figs. 4, A–C, and 5A), many of which were strongly labeled. Several retrogradely labeled cells were observed in the diagonal band of Broca (Fig. 4A). Few labeled cells were observed in the medial septum. FluoroGold-containing cells were observed throughout the preoptic area (Fig. 4, B and C). The organum vasculosum of the lamina terminalis (OVLT, Fig. 4B) and the ventral part of the bed nucleus of the stria terminalis (BNST, Fig. 4, B and C), also contained a relatively large number of FluoroGold-labeled cells. It was not possible, however, to determine the degree to which the labeling in the preoptic area, OVLT or ventral BNST was due to retrograde transport or diffusion of FluoroGold from the injection site. The intensity of labeled cells varied considerably in these regions, with both intensely labeled cells and very faint cells scattered throughout. The dorsal part of the BNST contained a moderate number of cells (Table 2 and Fig. 4, B and C). The stria terminalis itself contained a small number of cells clustered tightly together (Table 2 and Fig. 5B). The numbers of FluoroGold-labeled cells in the subfornical organ (Figs. 4C and 5C) varied considerably between animals (Table 2); rams that received more medial FluoroGold injections had higher numbers of labeled cells than did rams that received more lateral injections. Labeled cells were observed throughout the whole of the lateral preoptic area and into the anterior hypothalamic area (Table 2 and Fig. 4, C and D). No FluoroGold-labeled cells were detected in the suprachiasmatic or supraoptic nuclei. The rostral periventricular area, which included the periventricular nucleus and medial regions through the anterior hypothalamic area, contained a high density of FluoroGold-labeled cells (Table 2 and Figs. 4, C and D, and 5D). The caudal periventricular area, which incorporated the regions adjacent to the third ventricle above the median eminence, contained a medium density of FluoroGold-labeled cells (Table 2 and Fig. 4, E–H). In both parts of the periventricular area, the labeled cells were variable in intensity, with some cells being very strongly labeled but the majority being relatively weakly labeled (Fig. 5D). A high concentration of strongly labeled cells was observed in the paraventricular nucleus of the thalamus immediately adjacent to the lateral ventricle (Table 2 and Figs. 4, D–G, and 5E). No labeled cells were observed in the lateral retrochiasmatic area or the zona incerta. The dorsomedial nucleus contained a moderate number of retrogradely labeled cells, particularly in the medial half of the nucleus (Table 2 and Figs. 4, F–H, and 6A). The ventromedial nucleus only contained a small number of generally weakly labeled cells (Table 2 and Figs. 4, F–H, and 6C). The lateral hypothalamus contained a variable number of retrogradely labeled cells in the ventral regions, with most of them around the area lateral to the fornix (Table 2 and Fig. 4, F–H). Retrogradely labeled cells were also observed in the arcuate nucleus (Table 2 and Figs. 4, E–I, and 5F), mainly in the areas lateral to the third ventricle. Fewer FluoroGold-labeled cells were detected in the ventral part of the arcuate nucleus, which is the area below the third ventricle, adjacent to the median eminence. Strongly labeled cells were also observed around and below the supramamillary recess of the third ventricle (Fig. 4I). Relatively high numbers of retrogradely labeled cells were observed in the posterior hypothalamus, especially around the third ventricle and in the ventral premamillary nucleus (Table 2 and Fig. 4, H and I).


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  		TABLE 2. Mean number of retrogradely labeled cells counted per section* within hypothalamic and surrounding nuclei of all seven rams following FluoroGold injection in the rostral preoptic area


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  		TABLE 2. Extended



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  		FIG. 5. FluoroGold-labeled cells in a ram brain. A) Lateral septum. The lateral ventricle is immediately to the right of the image. B) Stria terminalis. C) Subfornical organ. D) Rostral periventricular nucleus. E) Paraventricular nucleus of the thalamus. F) Arcuate nucleus. Bar = 100 µm



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  		FIG. 6. FluoroGold-labeled cells in the dorsomedial nucleus of a ram (A) and a ewe (B) and in the ventromedial nucleus of a ram (C) and ewe (D). Bar = 100 µm

Sex Comparison in the Number of Retrogradely Labeled Cells in the Septopreoptic and Hypothalamic Regions

The number of retrogradely labeled cells within each nucleus was counted in sections through the septopreoptic region and hypothalamus, and the mean number of cells per section for each nucleus in each ram is shown in Table 2. The data for ewes injected with FluoroGold in the same region of the preoptic area are shown in Table 3 . There was no sex difference (P > 0.05) in the mean number of retrogradely labeled cells per section within any nucleus except in the dorsomedial and ventromedial hypothalamic nuclei. There was a significantly higher (P < 0.05) number of retrogradely labeled cells in the dorsomedial nucleus of the rams than the ewes (Tables 2 and 3 and Figs. 4, F–H, and 6, A and B). Conversely, there were significantly fewer (P < 0.05) retrogradely labeled cells in the ventromedial nucleus of the rams than the ewes (Tables 2 and 3 and Figs. 4, F–H, and 6, C and D).


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  		TABLE 3. Mean number of retrogradely labeled cells counted per section* within hypothalamic and surrounding nuclei of all six ewes following FluoroGold injection in the rostral preoptic area


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  		TABLE 3. Extended

Colocalization of FluoroGold with TH Immunoreactivity in the Hypothalamus of the Ram

In most rams, FluoroGold labeling was not detected in TH-immunoreactive (TH-ir) cells in any part of the hypothalamus. In one ram (no. 17), 4 double-labeled cells were counted out of 146 TH-ir cells in the A14 region. Fifteen double-labeled cells were observed in the A12 cell group, where there were >1500 TH-ir cells. Two other rams (nos. 35 and 16) had three double-labeled cells in each of the A14 and A12 regions. An example of a TH-ir cell that contained FluoroGold labeling is shown in Figure 7 (A and B). There were no FluoroGold-containing TH-ir cells in the A13 and A15 dopaminergic groups of any of the rams.



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  		FIG. 7. Colocalization of TH-ir (A), NPY-ir (C), and ACTH-ir (E) with FluoroGold labeling (B, D, and F, respectively) in the arcuate nucleus of a ram. Arrows show double-labeled cells. Abbreviations defined in text on page 1121. Bar = 20 µm

Colocalization of FluoroGold in NPY-ir Cells in the Hypothalamus of the Ram

The distribution of NPY-ir cells and FluoroGold-labeled cells in the arcuate nucleus and surrounding tissue is shown for ram 525 in Figure 8. An example of an NPY-ir cell with FluoroGold labeling is shown in Figure 7 (C and D). Such labeling was infrequently observed in the arcuate nucleus, occurring in about 2–3% of NPY-ir cells (Table 4). The degree of colocalization varied among sheep (Table 4). Most of the NPY-ir cells that were labeled with FluoroGold were in the caudal half of the arcuate nucleus (89 of 110 double-labeled cells counted). These double-labeled cells were predominantly in the ventral region of the arcuate nucleus, below the third ventricle (Fig. 8), where FluoroGold-labeled cells were rare.



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  		FIG. 8. Drawings of coronal sections through the medial basal hypothalamus of a ram (525), from rostral (A) to caudal (F), showing the distribution of FluoroGold-labeled cells in the arcuate nucleus and surrounding tissue (triangles), NPY-ir cells (filled circles), and double-labeled cells (stars). Each symbol represents approximately two labeled cells. Abbreviations defined in text on page 1121


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  		TABLE 4. Total number of FluoroGold (FG)-labeled cells, NPY-ir cells, and double-labeled cells in the arcuate nucleus of all seven rams injected with FluoroGold in the rostral preoptic area (11–14 sections/ram)

Colocalization of FluoroGold in ACTH-ir Cells in the Hypothalamus of the Ram

The distribution of ACTH-ir cells and FluoroGold-labeled cells in the arcuate nucleus and surrounding tissue is shown for ram 869 in Figure 9. An example of an ACTH-ir cell that contained FluoroGold labeling is shown in Figure 7 (E and F). FluoroGold-labeling in ACTH-ir cells in the arcuate nucleus was extremely rare, being observed in only 24 of 9771 ACTH-ir cells counted (Table 5).



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  		FIG. 9. Drawings of coronal sections through the medial basal hypothalamus of a ram (no. 869) from rostral (A) to caudal (D), showing the distribution of FluoroGold-labeled cells in the arcuate nucleus and surrounding tissue (triangles), ACTH-ir cells (filled circles), and double-labeled cells (stars). Each symbol represents approximately two labeled cells. Abbreviations defined in text on page 1121


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  		TABLE 5. Total number of FluoroGold (FG)-labeled cells, ACTH-ir cells, and double-labeled cells in the arcuate nucleus of six rams* injected with FluoroGold in the rostral preoptic area (9–12 sections/ram)

Distribution of FluoroGold-Labeled Cells in the Brain Stem of the Ram

The distribution of FluoroGold-labeled cells in the brain stem of the ram is shown in Figure 10. Examples of labeled cells are shown in Figure 11. Retrograde labeling of brain stem neurons was limited to a few discrete regions. In the medulla, labeled cells were observed in the ventrolateral medulla and, to a lesser extent, the nucleus of the solitary tract (Table 6 and Figs. 10, A–E, and 11, A, B, and E). These areas correspond to the regions of the A1 and A2 noradrenergic cell groups, and within the A1 region virtually all FluoroGold-labeled cells were DBH-ir (Table 7 and Fig. 11, C and D). By contrast, only 8–58% of FluoroGold-labeled cells in the nucleus of the solitary tract were DBH-ir. No FluoroGold-labeled cells were detected in any other parts of the medulla. Within the pons, the greatest number of FluoroGold-labeled cells was within the parabrachial nucleus (Table 6 and Figs. 10, F–H, and 11F). These cells were weakly labeled and were mainly concentrated within the lateral part of the nucleus, close to the edge of the tissue. One or two labeled cells per section were also observed in the dorsal raphe nucleus (Table 6). No cells were detected in the locus coeruleus. Several cells per section were observed in the caudal region of the midbrain central gray matter (Table 6 and Fig. 10, H and I). The rostral part of the midbrain was not examined. No FluoroGold-labeled cells in the pons were colabeled with DBH.



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  		FIG. 10. Drawings of coronal sections through the brain of a ram. A–E) Caudal brain stem, from caudal to rostral, showing the distribution of FluoroGold-labeled cells (triangles), DBH-ir cells (filled circles), and double-labeled cells (stars). F–I) Rostral brain stem, from caudal to rostral, showing the distribution of FluoroGold-labeled cells only (triangles). Each symbol represents approximately one labeled cell. Abbreviations defined in text on page 1121



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  		FIG. 11. FluoroGold-labeled cells in a ram. A and B) Labeled cells in the ventrolateral medulla. C and D) DBH-ir cells colocalized with a labeled cell in the ventrolateral medulla. E) Labeled cells in the nucleus of the solitary tract. F) Labeled cell in the lateral parabrachial nucleus. Bar = 100 µm


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  		TABLE 6. Mean number of retrogradely labeled cells counted per section within brain stem nuclei of all seven rams.*


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  		TABLE 7. Total number of FluoroGold (FG)-labeled cells, DBH-ir cells, and double-labeled cells in the ventrolateral medulla (A1 region) of five rams injected with FluoroGold in the rostral preoptic area (7–17 sections/ram)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We defined the inputs from the hypothalamus and brain stem to the rostral portion of the preoptic area in the ram. Because this region contains the majority of the GnRH neurons, these inputs are likely to include some of those that regulate GnRH secretion. Inputs are from various parts of the hypothalamus, but the major contribution is from the medial or periventricular region. In addition, inputs are received from discrete regions of the brain stem. We have quantified the inputs to the preoptic area from the hypothalamus and surrounding areas and used this information to compare the ram and the ewe. Sex differences in the number of cells that project to the rostral preoptic area were observed in two hypothalamic nuclei, the dorsomedial and ventromedial nuclei. Other sexual dimorphisms have been observed previously for the ventromedial nucleus in the sheep. Estrogen implants into the ventromedial nucleus of ovariectomized ewes have an initial negative feedback action that suppresses LH secretion, followed by the generation of an LH surge [13, 22]. By contrast, estrogen implants into the ventromedial nucleus of castrated rams have an inhibitory action on LH secretion [23, 24]. In addition, there are significantly more estrogen receptor-containing cells in the ventromedial nucleus in ewes than in rams [25]. Taken together, these data suggest that the ventromedial nucleus may be an important site for steroid feedback on GnRH/LH secretion in both ewes and rams, particularly in the sexual differentiation of the gonadotropin response to steroid feedback. The presence in the ventromedial nucleus of the ewe of greater numbers of cells that project to the preoptic area (present study) and greater number of estrogen receptor-containing cells [25] than in the ram raises the possibility that there is a group of estrogen receptor-containing neurons in this nucleus that project to the preoptic area and are unique to the female. If this is the case, then these neurons might be important for the relay of the positive feedback actions of estrogen to the GnRH neurons. This feedback action probably would not be via a direct projection to GnRH neurons because anterograde tracing studies have not shown any direct projections from the ventromedial nucleus to GnRH neurons in the preoptic area [26]. In any case, very few of the estrogen receptor-containing cells in the ventromedial nucleus of the ewe project to the preoptic area [27].

Neuronal tracing studies generally show a relatively high degree of interanimal variability, especially in the nonrodent species, where there is variation in the shape of the cerebral ventricles and associated brain structures. This study was no exception; we found variability in the number of retrogradely labeled cells in each nucleus among animals in the same group. This variability could be due to a number of factors, including slight variations in the placement of the tracer injection and in the area of diffusion of the tracer following injection. Conclusions regarding any sex differences in the numbers of cells that project from a given region to the preoptic area must be made with due regard to this variability.

Greater numbers of cells were retrogradely labeled in the dorsomedial nucleus in rams than in ewes. Little is known about the physiological role of this brain region in the sheep, but studies in rats [28, 29] suggest that it is important for the activation of the hypothalamus-pituitary-adrenal axis and the response to stress. Rams and ewes differ in the way that stress influences LH secretion [14], and the sex difference we observed may be related to this sexually dimorphic phenomenon. There is also a sex difference in the appetite/feeding response to leptin administration in sheep [30]. The dorsomedial nucleus may be associated with this sex difference because it has been implicated in the regulation of appetite and feeding in ewes [31]. Thus, the sex difference in the projections from the dorsomedial nucleus to the preoptic area may relate to this dimorphism.

Among the largest populations of FluoroGold-labeled cells observed were those in the arcuate nucleus, bed nucleus of the stria terminalis, and periventricular nucleus as well as throughout the preoptic area and to a lesser extent the ventromedial nucleus. These nuclei contain numerous androgen receptor-containing cells in the ram [32]. They also have many estrogen receptor {alpha}-containing cells, as do the lateral septum, organum vasculosum of the lamina terminalis, and anterior hypothalamic area [25], all regions that also received strong FluoroGold labeling. Some of the retrogradely labeled cells in these regions in the ram would likely contain androgen and/or estrogen receptors and therefore would be well placed to relay the negative feedback actions of testosterone/estrogen on GnRH secretion. Estrogen receptor-containing cells that have been retrogradely labeled from the rostral preoptic area have been localized in a number of regions in the ewe hypothalamus [27]. Further work is required to determine whether such associations also exist in the hypothalamus of the ram.

The low number of double-labeled cells detected in the hypothalamus might raise concern that our immunohistochemical procedures were causing fading of the FluoroGold fluorescence and, consequently, an underestimation of the number of double-labeled cells. We compared the number of FluoroGold-labeled cells in sections used for immunohistochemistry and in adjacent sections; the number of labeled cells was similar (data not shown). Thus, the low numbers of double-labeled cells in this part of the brain is unlikely to be due to fading of the FluoroGold-labeled cells.

Almost no TH-ir cells in the hypothalamus project to the rostral preoptic area in the ram, and the same is true in the ewe [9]. Thus, although there is evidence that dopamine is involved in the regulation of GnRH secretion in the ram [4], apparently there is no direct input of hypothalamic dopamine neurons onto GnRH neurons. Thus, dopaminergic regulation of GnRH secretion must be indirect, via interneurons. TH-ir fibers have been observed in close association with GnRH neurons in the ewe [17], but this input most likely is noradrenergic. Retrograde tracing studies [11] have shown that numerous DBH-ir (presumably noradrenergic) neurons in the ventrolateral medulla of the brain stem (A1 cell group) project to the rostral preoptic area in the ewe. The present study indicates that a similar pathway exists in the ram, although the physiological significance of this pathway remains unknown. In the ewe, neurons in the A1 cell group contain estrogen receptor {alpha} [12] and appear to play a role in the feedback actions of estrogen on GnRH/LH secretion [20, 33]. Intracerebroventricular injection of noradrenaline decreased plasma LH levels in wethers [34], suggesting that noradrenaline may have an inhibitory influence on GnRH secretion in the ram, but no other studies have investigated the role of noradrenaline in the regulation of GnRH secretion in the ram. Testosterone appears to regulate hypothalamic noradrenaline levels in male rats [35], but the little data concerning noradrenergic regulation of LH secretion in that species are conflicting [36, 37].

A low percentage of NPY-ir neurons in the arcuate nucleus of the ram projected to the rostral preoptic area. Most NPY cells are localized in the ventral arcuate nucleus, below the third ventricle, but this area contained few FluoroGold-labeled cells, which suggests that the NPY neurons in the arcuate nucleus have virtually no direct input onto GnRH neurons in the ram. The role of NPY in the regulation of GnRH secretion in the ram has not been investigated, although there is some evidence that NPY is involved in the regulation of GnRH in both the ewe [38, 39] and the male rat [40]. We investigated the inputs of arcuate NPY neurons to the rostral preoptic area because this is one of the few neuromodulators for which there are confirmed synapses onto GnRH neurons in the ewe [18]. In the ewe, the degree to which NPY neurons project from the arcuate nucleus to the preoptic area is unknown, and the NPY neurons that synapse onto GnRH neurons may not have originated in the arcuate nucleus but in the brain stem. NPY and noradrenaline are colocalized in some brain stem A1 and A2 neurons in the rat [41]. Whether NPY and noradrenaline are colocalized in the sheep brain stem is unknown, but it seems likely that some of the DBH-ir neurons in the A1 and A2 groups that we have shown to project to the preoptic area may also release NPY.

ACTH is a product of the POMC gene and as such can be considered a marker for ß-endorphinergic neurons in the arcuate nucleus. Less than 1% of ACTH-ir neurons project to the rostral preoptic area. The evidence is now very strong that ß-endorphin regulates GnRH secretion in the ram, exerting an inhibitory influence [42–44]. Anterograde tracing studies in the ewe suggest that there is virtually no direct input onto GnRH neurons from the regions of the arcuate nucleus, where ß-endorphin-containing cells are found [26]. Although ß-endorphin-containing terminals have been identified forming synapses onto GnRH neurons in male rats [45], the current data suggest that ß-endorphin must regulate GnRH secretion in the ram through an indirect manner.

The regulation of GnRH secretion is complex, involving the coordination of neurons scattered over a wide area of the forebrain in most mammalian species [5] to produce phasic activity that results in pulsatile secretion into the hypophyseal portal blood. In addition, a range of factors such as sex steroids, photoperiod, nutrition, and stress modify this pulse generator activity. This information is relayed to the GnRH neurons by a diverse network of neurons. The complexity of the system is highlighted in the present study by the observation that neurons that produce neurotransmitters known to be important in the regulation of GnRH secretion (vide supra) do not input to GnRH neurons directly. These data, combined with the results of our previous anterograde tracing studies in the ewe [26] that show few inputs to GnRH neurons from the arcuate-ventromedial region of the hypothalamus, suggest that most information from the periphery/external environment is relayed to the GnRH neurons via serial (polysynaptic) inputs. Many of these inputs to the GnRH neurons converge within integrating centers such as the BNST, preoptic area, and periventricular nucleus [26]. All of this information is processed and reduced such that the GnRH neurons themselves receive a relatively small amount of synaptic input [46].

The preoptic area is involved in a wide range of functions other than the control of GnRH secretion, including control of thermoregulation, sleep, blood pressure, respiration, behavior, and other neuroendocrine systems [47–50]. Therefore, some of the inputs must subserve these functions. Nevertheless, we focused our retrograde tracer injection sites in the rostral preoptic area, where most GnRH neurons are found, and our data provide an anatomical map of which parts of the brain are likely to directly regulate these cells. For at least some of the neuronal systems that regulate GnRH secretion, it appears that there are interneuronal pathways (vide supra), and because FluoroGold does not cross synapses [51], we assume that serial inputs to GnRH neurons are within the region of the injections. The preoptic area contains high numbers of cells that produce gamma amino butyric acid or glutamate [52] [53], and these cells are candidates for interneuronal transmission to GnRH cells.

We defined the source of inputs from the hypothalamus and brain stem to the rostral preoptic area in the ram. The distribution of these inputs is the same as in the ewe, but in two nuclei, the dorsomedial and ventromedial nuclei, the number of cells that project to the rostral preoptic area was different from that found in the ewe. Our results also indicate that regulation of GnRH secretion by dopamine, NPY, and ß-endorphin in the ram is unlikely to be via direct inputs to GnRH neurons, suggesting that there are serial and converging inputs to GnRH neurons.


   ACKNOWLEDGMENTS
 
We thank Bruce Doughton, Karen Briscoe, Linda Morrish, and Adam Link for animal care and assistance with surgery and tissue collection.


   FOOTNOTES
 
1 This work was supported by grants from the National Health and Medical Research Council of Australia (124352) and Monash University. Back

2 Correspondence and current address: Chris Scott, School of Biomedical Sciences, Charles Stuart University, Wagga Wagga, NSW 2650, Australia. FAX: 61 2 69332587; chscott{at}csu.edu.au Back

Received: 3 September 2002.

First decision: 26 September 2002.

Accepted: 7 October 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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