Expression of estrogen and progesterone receptors in glutamate and GABA neurons of the pubertal female monkey hypothalamus.

We have previously reported direct glutamate (Glu) synapses upon GnRH-containing neurons in the primate hypothalamus, and extensive interactions between Glu and aminobutyric acid (GABA) neurons in areas associated with reproductive function. Both Glu and GABA are known to affect peripubertal GnRH neurohormone release, but their relative roles remain unclear. In a developmental survey, estrogen receptors (ER) and progesterone receptors (PR) were virtually undetectable after immunostaining the hypothalamus of prepubertal monkeys, but were clearly evident in neurons of adults. We hypothesized, therefore, that Glu and GABA neurons which develop ER or PR expression during puberty may participate in reactivation of the hypothalamic-pituitary-gonadal axis. To identify those neurons in midpubertal female cynomolgus monkeys, we performed immunofluorescence staining for ER or for PR in separate sets of hypothalamic sections, and then immunostained for Glu or for glutamate decarboxylase (GAD, to identify GABA neurons) using a contrasting fluorophore. ER and PR were localized in the cytoplasm and nuclei of Glu and GAD neurons in nine hypothalamic and related brain regions. Quantitation revealed intranuclear ER in an average of 80% of the Glu neurons in all regions analyzed, and an average of 84% of the GAD neurons in all regions except the supraoptic nucleus (28%). Intranuclear PR expression was more variable, occurring in an average of 93% of the Glu neurons in seven regions, but in only 41% in the medial preoptic area, and 0% in the arcuate-periventicular zone. In addition, while intranuclear PR was seen in 96% of the GAD neurons in the septum, it appeared in 67% of the GAD neurons in the paraventricular nucleus, 47% in the medial preoptic area, 40% in the periventricular zone, and was absent from neurons in the supraoptic nucleus and mammillary bodies. In summary, certain subpopulations of Glu and GABA neurons in principal hypothalamic regions of the female monkey express ER and PR at midpuberty. Taken together with previous findings, these results suggest that Glu and GABA neurons which become sensitive to steroid hormones may help regulate GnRH neurohormone release and promote the onset of puberty. Since neuronal expression of ER or PR connotes sensitivity to gonadal feedback, and intranuclear translocation signals transcriptional activation, these results provide insights into the specific neuronal events involved in the peripubertal transition in primates.

Estrogen and progesterone receptor expression in neuroendocrine and related neurons of the pubertal female monkey hypothalamus.

Expression of hypothalamic estrogen receptors (ER) and progesterone receptors (PR) is barely evident in prepubertal monkeys but is prominent in adults. To investigate whether adult patterns of ER and PR expression are established in mid-pubertal female cynomolgus monkeys, we labeled neuroendocrine (NEU) neurons by microinjection of retrograde tracer into the median eminence, and then identified ER and PR by specific immunostaining in separate sets of hypothalamic sections. ER and PR appeared in the cytoplasm and nuclei of cells identified exclusively as neurons, and retrograde tracer remained clearly visible in the cytoplasm of NEU neurons after immunostaining. Numbers of NEU and related neurons expressing ER or PR were quantified in principal hypothalamic regions. In the supraoptic nucleus, almost all neurons analyzed (n = 580) contained ER (94%) with many also NEU (73% ER + NEU), while lesser amounts of the neurons examined (n = 214) expressed PR (75%) and were NEU (53% PR + NEU). In the paraventricular nucleus, most of the neurons analyzed (n = 302) contained ER (90% ER; 54% ER + NEU), but few of the neurons studied (n = 269) contained PR (34% PR; 19% PR + NEU). In the periventricular zone, nearly all neurons examined (n = 795) contained ER (95% ER; 48% ER + NEU), but fewer of those studied (n = 298) exhibited PR (79% PR; 47% PR + NEU). In the arcuate-periventricular zone, all neurons examined (n = 542) contained ER (100%) but few were NEU (4% ER + NEU), while nearly all neurons studied (n = 418) contained PR (95%), some of which were NEU (21% PR + NEU). Neurons expressing ER were also prevalent in areas without NEU labeling, including the diagonal band of Broca, medial preoptic area, and mammillary bodies, but were less common in the septum and dorsomedial hypothalamus. Likewise, neuronal PR expression was seen frequently in the mammillary bodies, but occurred less often in the diagonal band of Broca, medial preoptic area, and dorsomedial hypothalamus. Neurons in the suprachiasmatic nucleus and lateral hypothalamic area lacked retrograde labeling. These results identify the principal sites and subsets of NEU and related neurons which express ER and PR in the mid-pubertal female monkey hypothalamus. They appear to correlate well with known populations of steroid-sensitive NEU neurons present in these areas in adults. The data also suggest that functional patterns of ER and PR expression arise upon reactivation of the hypothalamic-pituitary-gonadal axis at puberty. The degrees of receptor expression and of nuclear translocation most likely reflect peripubertal changes in the levels of gonadal steroids. Taken together, these results provide important insights into the mechanisms and development of neuroendocrine control during the pubertal period in primates.

Glutamate and GABAergic neurointeractions in the monkey hypothalamus: a quantitative immunomorphological study.

Glutamate (Glu) and gamma-aminobutyric acid (GABA) are the most abundant excitatory and inhibitory neurotransmitters in the mammalian hypothalamus. Glu and GABA-containing neurons have both been shown to synapse with gonadotropin-releasing hormone (GnRH) and other neuroendocrine systems in the hypothalamus of several species. Since their direct interactions could play a pivotal role in governing neuroendocrine function, we performed double-label immunostaining for Glu and for glutamic acid decarboxylase (GAD) as a marker for GABAergic neurons in hypothalamic sections from adult female cynomolgus monkeys. Ultrastructural analysis of 785 Glu-immunoreactive (-ir) and GAD-ir elements in the medial septum (MS), arcuate nucleus-ventral hypothalamic tract (VHT1), supraoptic nucleus (SON), paraventricular nucleus (PVN), and median eminence (ME) revealed that 63% were Glu-ir, 28% were GAD-ir, and 9% were Glu + GAD-ir. In addition, we observed surprisingly consistent labeling of 2-4% somata (SOM), 65-80% dendrites (DEN), and 15-30% axons and terminals (AXO) in all of these areas. Characterization of 177 interactions (36% synapses, 64% contacts) by pre/post-transmitter content indicated that 29% contained Glu/GAD, 15% Glu/Glu, and 15% Glu/Glu + GAD, while 16% were unlabeled/Glu, 9% were unlabeled/GAD, and 16% expressed other transmitter combinations. Regional analysis of these interactions showed that 43% occurred in the MS, 22% in VHT1, 14% in SON, 9% in PVN, and 12% in the ME. AXO/DEN interactions made up 51% of all labeled interactions characterized, and were comprised 29% of Glu/GAD, 22% of Glu/Glu, and 18% of the Glu/Glu, and 18% of the Glu/Glu + GAD type. AXO/DEN synapses were more prevalent than contacts in all areas except the PVN and of course the ME, where anatomical synapses do not occur. AXO/SOM interactions represented approximately 15% of all those identified, and were predominantly unlabeled/Glu (71%) and unlabeled/GAD (18%) synapses. Almost all (95%) AXO/SOM synapses and 75% of the contacts occurred in the MS. DEN/DEN interactions, 28% of the total, were composed 50% of Glu/GAD, 12% of Glu/Glu, and 18% of the Glu/Glu+GAD type. The relatively few DEN/DEN synapses all appeared in the MS, whereas much more abundant DEN/DEN contacts were more widely distributed. DEN/SOM interactions, 6% of the total, appeared only as contacts, with the majority (60%) again located in the MS. In addition, the MS contained 48% of all asymmetrical synapses (vs. 35% in VHT1 and 17% in SON), 62% of all symmetrical synapses (vs. 19% in VHT1 and 14% in SON), and 35% of all contacts (vs. 21% in VHT1 and 12% in SON) identified.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutamate-immunoreactive neurons and their gonadotropin-releasing hormone-neuronal interactions in the monkey hypothalamus.

Glutamate (Glu) is the most prevalent excitatory neurotransmitter in the brain and has been implicated in the regulation of GnRH secretion in several mammalian species, including the monkey. To investigate the neuroanatomical basis for Glu-GnRH interactions, we performed an immunocytochemical study at both the light and electron microscopic levels on the brains of four female and five male macaques. Initially, we determined the location of Glu-immunoreactive (-ir) elements using a monoclonal antibody specific for glutaraldehyde-fixed Glu (Glu-2) and 3,3'-diaminobenzidine-4-HCl (DAB). Glu-ir was observed in the cytoplasm and to a variable degree in the nuclei of neurons in the diencephalon. Cytoplasmic staining was particularly intense in numerous neurons in the arcuate nucleus, supraoptic nucleus, and many paraventricular nucleus neurons. Short Glu-ir processes were evident in these and other hypothalamic regions and were extremely dense in the infundibular stalk and median eminence. Prior absorption of the Glu-2 antibody with a Glu-glutaraldehyde-BSA conjugate completely abolished all immunostaining in both neuronal nuclei and cytoplasm. Double label Glu-GnRH immunostaining for light microscopy was performed using Glu-2 and DAB without enhancement, and a polyclonal antibody (LR1 or LR2) with silver-enhanced DAB for Glu and GnRH, respectively. Glu-ir interactions with GnRH-ir cell bodies were not apparent, but a few Glu-ir axons seemed to contact GnRH-ir dendrites in the organum vasculosum of the lamina terminalis, medial septum, and arcuate nucleus regions. Reciprocal interactions occurred more frequently, however, in which GnRH-ir axons and dendritic fibers engaged Glu-ir cell bodies en passant, particularly toward the medial and posterior hypothalamus. For ultrastructural analyses, Glu-ir elements were stained with the Glu-2 antibody and 15 nm immunogold or DAB. Electron microscopy demonstrated that Glu-ir was associated with clear microvesicles within the neuronal cytoplasm. Glu-ir processes made classical asymmetrical synapses with one another and received asymmetrical synapses from unlabeled afferents. In sections double labeled for Glu with immunogold and for GnRH with DAB, axo-somatic interactions were not observed. However, axo-dendritic Glu-GnRH synapses were seen, which usually exhibited Glu-ir labeling of terminal vesicles and inconsistent postsynaptic densities, with GnRH-ir neurosecretory granules sometimes congregated in the apposing dendrite or spine. Surprisingly, reverse GnRH-Glu interactions were observed more frequently.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide Y system of the female monkey hypothalamus: retrograde tracing and immunostaining.

Neuropeptide Y (NPY) stimulates the release of hypothalamic gonadotropin-releasing hormone (GnRH) as well as pituitary gonadotropins in the presence of ovarian steroids, but inhibits release in their absence. In primates, however, the effects of NPY depend largely upon the site and method of administration. In ovariectomized monkeys, NPY infusion into the stalk-median eminence reportedly causes a dose-response increase in GnRH secretion in the absence of gonadal steroids. To help elucidate these findings, we investigated the NPY system and its neuroendocrine (NEU) component in the primate brain by retrograde tracing and immunostaining. One adult female and 1 juvenile female cynomolgus monkey were given microinjections of retrograde tracer into the median eminence (ME). Two weeks later, they were perfused with fixative, and series of 40-microns frontal vibratome sections were collected at 500-microns intervals through 4 mm of the forebrain. Injection sites were not visible in the juvenile female monkey ME, so this animal served as a neurosurgical and injection control. Sections were immunostained using a polyclonal NPY antiserum and the peroxidase antiperoxidase (PAP) technique. NPY immunostaining in another adult female cynomolgus monkey and in a late fetal female and a neonatally castrated adult male rhesus monkey gave essentially similar results. NPY-immunoreactive (NPY-IR) neurons were widely distributed throughout the caudate nucleus, but appeared concentrated within specific hypothalamic areas. Their number, as well as the number of NEU neurons, was nearly equal in bilaterally paired areas and on both sides of the hypothalamus overall. Ratios of retrogradely labeled NPY-IR neurons to the number of NPY-IR somata were expressed as percentages of NEU NPY-IR neurons for each side and in each area. These averaged 65% in the supraoptic nucleus (SON), 41% in the paraventricular nucleus (PVN), 32% in the medial preoptic area (MPOA), which has only one quarter of their number of NPY-IR cells, and 11% in the medial basal hypothalamus (MBH). NPY-IR fiber densities were highest in the area olfactoria, medial septal and ventromedial nuclei. They were high in the tuberculum olfactorium, lateral septum, nucleus accumbens, MPOA, PVN, dorsomedial nucleus and regions of the MBH including the arcuate nucleus, tuber cinereum and ventral hypothalamic tract (VHT). NPY fiber densities were moderate in the vertical portion of the diagonal band of Broca, the ventral part of the caudate nucleus, the anterior commissural nucleus and the lateral preoptic area, as well as the anterior and lateral hypothalamic areas, the anterior ventral periventricular area, the suprachiasmatic nucleus and the dorsolateral SON.(ABSTRACT TRUNCATED AT 400 WORDS)

Interactions between vasopressin- and gonadotropin-releasing-hormone-containing neuroendocrine neurons in the monkey supraoptic nucleus.

Vasopressin (VP) is a hypophysiotropic hormone which is also implicated in the control of gonadotropin-releasing hormone (GnRH) secretion. We examined whether VP- and GnRH-immunoreactive (-IR) elements interact directly in the supraoptic nucleus (SON) of cynomolgus monkeys. Neuroendocrine (NEU) neurons in 4 juveniles were retrogradely labeled from the median eminence with wheat germ agglutinin apohorseradish peroxidase conjugated to gold before aldehyde perfusion. Frontal vibratome sections were immunostained for GnRH with peroxidase-antiperoxidase (PAP) and for VP with 5- or 15-nm gold. Many of the GnRH-IR and more than half the VP-IR cell bodies in the SON were NEU. VP-IR elements formed axodendritic and axosomatic symmetrical synapses with one another. In addition, VP-IR boutons also synapsed with NEU GnRH-IR neurons. Although GnRH axon terminals and dendrites contacted VP-IR dendrites and NEU cell bodies, we were unable to find convincing examples of GnRH/VP synapses through serial sections, perhaps due to the use of PAP-diaminobenzidine as the GnRH (afferent) immunolabel. In summary, our study demonstrates anatomical synapses between VP-IR and other VP and GnRH-IR neurons in the SON, in which postsynaptic VP or GnRH cell bodies were NEU. On the other hand, reciprocal GnRH/VP contacts but no true synapses were seen. However, the results suggest coordinated roles for VP and GnRH in NEU control of gonadotropin secretion. Whether VP itself and/or coexistent neuroeffectors act directly on NEU GnRH secretion remains to be determined. As such, VP neurons could help coordinate suppression of gonadotropins and augmentation of glucocorticoids during the stress response in primates.

Opioid synapses on vasopressin neurons in the paraventricular and supraoptic nuclei of juvenile monkeys.

Opioid peptide- as well as vasopressin-containing neurons synapse on gonadotropin releasing hormone neurons in juvenile macaques. In this study we performed double-label immunostaining for opioid and vasopressin neurons in the paraventricular and supraoptic nuclei in order to assess their interrelationships. Neuroendocrine neurons in the hypothalamus were prelabeled by microinjection of electron-dense retrograde tracer into the median eminence, and were easily identified in frontal Vibratome sections. Sections through the paraventricular and supraoptic nuclei were immunostained for vasopressin with the peroxidase-antiperoxidase technique, and for opioids using the indirect immunogold method. By light microscopy, opioid-immunoreactive inputs appeared to innervate an average of 39% of the vasopressin neurons in the paraventricular nucleus and 33% in the supraoptic nucleus, and were more prevalent anteriorly. Clusters of opioid afferents formed cup-like calices around major processes of many vasopressin neurons, especially in the paraventricular nucleus. Electron microscopy revealed that these groups of opioid axon terminals made frequent symmetrical and fewer asymmetrical synapses on both neuroendocrine and non-neuroendocrine vasopressinergic cell bodies and dendrites. Our study did not reveal vasopressin-opioid synapses in these hypothalamic nuclei, but this does not preclude the possibility of their existence elsewhere. These results indicate that opioid afferents modulate vasopressin neuronal activity in the monkey paraventricular and supraoptic nuclei. Previous results have suggested that corticotropin releasing hormone acts via vasopressinergic neurons to stimulate opioid neuronal activity and to inhibit gonadotropin releasing hormone release. Taken together, the data suggest that stressful stimuli could initiate a series of neuropeptidergic interactions which ultimately alter pulsatile gonadotropin releasing hormone secretion and thus gonadotropin secretion in primates.

Location of the neuroendocrine gonadotropin-releasing hormone neurons in the monkey hypothalamus by retrograde tracing and immunostaining*,**.

Abstract In order to localize neuroendocrine gonadotropin-releasing hormone (GnRH) neurons in the monkey hypothalamus, four juvenile cynomolgus macaques (one female, three males) were each given two or three microinjections (0.2 to 0.3 mul per site) of the retrograde tracer wheat germ agglutinin-apoHorseradish peroxidase-10 nm colloidal gold into the superficial, median eminence region of the infundibular stalk. Five to 15 days following surgery, the brains were fixed by perfusion and vibratomed at 40 mum in the frontal plane. Every 12th section was immunostained with rabbit anti-GnRH using the peroxidase anti-peroxidase technique with diaminobenzidine as the chromogen. Neuroendocrine GnRH neurons were easily identified in tissue sections as brown, immunostained cell bodies containing more than three distinct, dark blue, tracer-filled lysosomes. Neuronal counts from each complete series of sections were compiled by anatomical region, and the percentages of GnRH and neuroendocrine GnRH neurons determined. The highest proportion of neuroendocrine GnRH neurons (with projections to the median eminence) occurred in the ventral hypothalamic tract, especially in its medial third (71%), and in the supraoptic decussation just anterior to it. Proportions decreased moving laterally into the middle third (58%) and lateral third (25%) of the ventral hypothalamic tract. Further anterior and lateral, progressively smaller but significant neuroendocrine GnRH contributions were found in the supraoptic nucleus (57%) and lateral hypothalamus (33%), and in the medial preoptic area (26%). Although the medial preoptic area contained a greater percentage of the total GnRH-immunoreactive cell bodies (36%) than the ventral hypothalamic tract (27%), as a whole, the ventral hypothalamic tract contained 60% of the neuroendocrine GnRH neurons compared to only 25% from the medial preoptic area. Large numbers of GnRH cell bodies found in the diagonal band of Broca near the organum vasculosum of the lamina terminalis were not retrogradely labeled. GnRH neurons were not observed in the arcuate nucleus, the few in the paraventricular nucleus were not neuroendocrine, and the contribution from the periventricular zone was negligible. Our results here are the first to identify the neurons giving rise to the neuroendocrine GnRH system in juvenile monkeys. The data indicate that more GnRH neurons close to the infundibulum serve a neuroendocrine (perhaps hypophysiotropic) role than do those in more anterior areas. Furthermore, they suggest that the ventral hypothalamic tract is the most important, and perhaps most influential, neuroendocrine GnRH cell group in primates. The data substantiate the observed autonomy of the medial basal hypothalamus in controlling gonadotropin secretion and menstrual cyclicity in these animals. However, they also infer that perhaps 60% of the GnRH neurons do not project to the primate median eminence, and thus may serve other non-neuroendocrine functions.

Location of the neuroendocrine dopamine neurons in the monkey hypothalamus by retrograde tracing and immunostaining.

Abstract In order to localize neuroendocrine dopamine neurons in the monkey hypothalamus, one female and three male juvenile cynomolgus macaques were each given two or three microinjections (0.2 to 0.3 mul per site) of the retrograde tracer wheat germ agglutinin-apoHorseradish peroxidase-10 nm colloidal gold into the superficial, median eminence region of the infundibular stalk. Five to 15 days following surgery, the brains were fixed by perfusion, and vibratomed at 40 pm in the frontal plane. Every 12th section was immunostained with rabbit anti-tyrosine hydroxylase using the peroxidase anti-peroxidase technique with diaminobenzidine as the chromogen. Neuroendocrine, immunoreactive neurons were easily recognized as brown, immunopositive cell bodies containing more than three distinct dark blue granules, confirmed by electron microscopy to be tracer-filled lysosomes. Neuronal counts from each complete series of sections were compiled by anatomical region, and the percentages of tyrosine hydroxylase-immunoreactive and neuroendocrine, immunoreactive neurons determined. Although regional and interanimal variations were observed, we estimated that 5,400 of the total 19,000 tyrosine hydroxylaseimmunoreactive neurons in the juvenile macaque hypothalamus were neuroendocrine. When averaged by anatomical region, the suprachiasmatic nuclear groups contained 7% of all immunoreactive neurons (50% were neuroendocrine) and 15% of all neuroendocrine, immunoreactive neurons in these animals. The combined periventricular zones contained 20% of all immunoreactive neurons (more than 50% of ventral and 38% of dorsal were neuroendocrine) and 58% of all neuroendocrine, immunoreactive neurons. The paraventricular nucleus included 50% of all immunoreactive neurons, more than any other nucleus, (3% were neuroendocrine) and 11% of the total neuroendocrine, immunoreactive neurons. The ventral paraventricular nucleus contained only 2% of all immunoreactive neurons (13% were neuroendocrine) and 3% of the total neuroendocrine group. The zona incerta contained 15% of all immunoreactive neurons (0% were retrogradely labeled) but 0% of the neuroendocrine cells. The arcuate nucleus subdivisions contained about 5% of all immunoreactive neurons (more than 60% were neuroendocrine) and 8% of the neuroendocrine population. The ventral hypothalamic tract contained about 1% of all immunoreactive neurons (medially, 63%, and further laterally, 25% were neuroendocrine) and 5% of all neuroendocrine, immunoreactive neurons in these animals. The presence of the retrograde tracer from the median eminence in tyrosine hydroxylase-immunoreactive neurons, combined with knowledge of the location of dopaminergic cell groups, permitted assessment of the A11-A14 dopaminergic neurons which project to the primate infundibulum. Neuroendocrine dopamine neurons occurred predominantly in the All periventricular zones (65% of the total), being greatest around the ventral aspect of the entire third ventricle. They were less numerous in more dorsal regions of All extending up to the level of the paraventricular nucleus. The A12 arcuate (tuberoinfundibular) projection (15% of the total) was not nearly as prominent as All in primates, in contrast to rodents. None of the A13 incertohypothalamic dopamine neurons (0%) projected to the median eminence. The A14 anterior-ventral periventricular region, including the suprachiasmatic nuclear groups, provided the substantial remainder (20%) of all neuroendocrine dopamine neurons. In summary, our results suggest the involvement of a regionally specific dopaminergic system in the hypothalamic control of anterior pituitary hormone secretion in primates. The data also indicate that 75% of all tyrosine hydroxylase-immunopositive neurons do not project to the median eminence, and probably serve other functions. Although the retrograde tracer may not have labeled all neuroendocrine dopamine neurons, it may have identified some dopamine neurons which only interact with other median eminence nerve terminals, or other types of tyrosine hydroxylase-containing, neuropeptidergic neurons which project to the infundibulum. However, considering the known locations of dopaminergic neurons and the large numbers of labeled cells, the results here are a reliable indication of the diverse origins of median eminence-directed dopamine neurons in the juvenile primate.

Corticotropin-releasing factor neurons innervate dopamine neurons in the periventricular hypothalamus of juvenile macaques. Synaptic evidence for a possible companion neurotransmitter.

Corticotropin-releasing factor (CRF) and dopamine (DA) are important integrators of the endocrine and autonomic response to stress. CRF neurons in the anterior portions of the periventricular nucleus (PV) and parvocellular paraventricular nucleus (pvPVN) occur close to A14 DA neurons in these same locations. Since CRF has been shown to act as an excitatory neurotransmitter, possible CRF interactions with the DA system were investigated using double-label immunocytochemistry. Coronal vibratome sections through the PV and pvPVN were obtained from colchicine-treated and nontreated juvenile female cynomolgus macaques. They were sequentially immunostained for tyrosine hydroxylase (TH) (to identify DA neurons) with PAP and DAB, and for CRF using 15 nm colloidal gold. By light microscopy, areas of coincidence of TH- and CRF-immunoreactive cell bodies in the PV and pvPVN were obvious, but double-stained elements were not observed. By electron microscopy, asymmetrical synapses frequently occurred between CRF axons and TH dendrites or somata. Symmetrical axosomatic synapses sometimes appeared adjacent to these CRF/TH synapses, while symmetrical axoaxonic synapses were rare. We conclude that CRF neuronal efferents synaptically activate A14 DA neurons in the primate PV and pvPVN. Parallel CRF/DA symmetrical synapses also suggest coexistence of a companion transmitter within some of these same CRF neurons. Our own previous work and recent independent studies indicate that this transmitter is probably GABA. Thus the CRF neuronal system, which is known to alter secretion of several pituitary hormones, may also act through hypothalamic periventricular DA neurons to mediate other responses to stress.

Erythrocyte enzyme polymorphisms in the Kamboh caste of Patiala, Punjab.

Data are presented on six erythrocyte enzyme polymorphisms (ADA, GLO I, EsD, AK, AP, GPI) in the Kamboh, a scheduled caste inhabiting the Patiala district of the Indian state of Punjab. With their high ADA2 (18.5%), Pa (42.5%) and GPI3 (5.3%) and a rather low GLO1 (18.8%) frequencies, the Kamboh differ from all other populations of Punjab.

The distribution of AB0 and LH blood groups in some north Indian populations.

The distribution of AB0 and LH blood groups among five Punjabi populations from North India (Jat Sikh, Bania, Brahmin, Sikh Khatri and Hindu Khatri) is reported. Significant differences have been found in many cases regarding the distribution of AB0 and LH systems, especially between Sikh Khatris and Hindu Khatris, who are usually pooled for population genetic studies.

Infundibular gonadotropin-releasing hormone neurons are inhibited by direct opioid and autoregulatory synapses in juvenile monkeys.

A consistent group of gonadotropin-releasing hormone (GnRH) cell bodies occurs in the ventral hypothalamic tract at the infundibular lip (IL), just below the arcuate nucleus (ARC), at the site of the so-called GnRH 'pulse generator'. Immunocytochemical studies were performed to examine contacts between these GnRH neurons and nearby opioid peptide (OP) neurons in the ARC. Vibratome sections of the medial basal hypothalamus were obtained from colchicine-treated, perfusion-fixed juvenile female rhesus macaques. They were sequentially immunostained for GnRH using the peroxidase antiperoxidase (PAP) technique and for adrenocorticotropic hormone (to identify OP neurons) using colloidal gold. The PAP and colloidal gold markers could be clearly differentiated at both the light and electron microscopic levels. OP+ and GnRH+ neuronal cell bodies occurred close together in the ARC-IL region, sometimes within the same electron microscope grid square. At the electron microscopic level, OP+ axons formed symmetrical synapses with GnRH+ somata and proximal axons, suggesting a pronounced inhibitory influence on GnRH neuronal activity. Examples of OP+/GnRH+ axodendritic and dendrodendritic contacts were also observed. Furthermore, symmetrical synapses between GnRH+ axons and GnRH+ perikarya or dendrites were occasionally present. The data obtained here clearly indicate that direct OP inhibition of GnRH 'pulse generator' neurons occurs at the ARC-IL in juvenile primates. It is suggested that these OP neurons help mediate steroid-negative feedback at the hypothalamic level. Furthermore, it is suggested that OP/GnRH and GnRH/GnRH inhibitory contacts may play a role in maturation and control of reproductive function.

Immunostaining reveals accumulation of serotonin and coexistence with tyrosine hydroxylase in hypothalamic neurons of acutely stalk-sectioned baboons.

The distribution of serotonin (5-HT) and tyrosine hydroxylase (TH) was examined in the hypothalamus of juvenile baboons, 24 h after infundibular stalk section. Simultaneous immunostaining for 5-HT with peroxidase-antiperoxidase (PAP) and TH with 15 nm colloidal gold (IGS) was performed on Vibratome sections from 3 operated and 1 control female. Light microscopy revealed fine 5-HT immunopositive (5-HT+) fibers, presumably axons, in the suprachiasmatic nuclei and ventromedial hypothalamus (VMH) after stalk section. In addition, focal accumulations of swollen and heavily stained 5-HT+ fibers occurred on the side of the surgical approach. Enlarged fibers were densest in the medial preoptic area, lateral and VMH areas, and the median eminence. TH immunoreactivity (TH+) in VMH cell bodies and axons was only slightly increased over that in controls. Electron microscopy of areas of 5-HT+ and TH+ overlap (medial VMH and adjacent periventricular zone) showed that 5-HT+ profiles were mostly unmyelinated axons and irregular varicosities. A few myelinated 5-HT+ axons were also observed. TH+ perikarya, dendrites, axons and terminals showed gold labeling characteristic for this enzyme. However, colocalization of 5-HT (PAP) and TH (IGS) was present in a number of fiber varicosities in experimental animals only. Both single- and double-labeled profiles occurred in individual thin sections, thus arguing against antibody cross-reactivity. These results indicate that: hypothalamic 5-HT+ fibers project to the median eminence in primates; 5-HT fibers become more obvious after stalk section due to accumulation of transmitter; focal 5-HT+ immunoreactivity in the hypothalamus can increase dramatically after distant and mild surgical trauma, and coexistence of 5-HT and TH in single neurons can appear after acute stalk section and/or trauma in experimental animals. These findings might represent uptake of exogenous 5-HT or amplified expression of endogenous neurotransmitter, suggesting that plasticity of transmitter phenotype might follow acute surgical and/or endocrine intervention in mature primate brain. Neuroendocrine studies employing the stalk-sectioned primate might thus be radically affected.

GABAergic and catecholaminergic synaptic interactions in the macaque hypothalamus: double label immunostaining with peroxidase-antiperoxidase and colloidal gold.

Immunogold staining (IGS) for glutamic acid decarboxylase (GAD) was combined with the peroxidase-antiperoxidase (PAP) technique for tyrosine hydroxylase (TH) to analyze gamma-aminobutyric acid-catecholaminergic neuronal interactions in the rhesus hypothalamus. At the light-microscopic level, TH-immunoreactive (-IR) perikarya and their fibers (brown) were observed in the anterior ventral periventricular area (AVPV), the arcuate nucleus (ARC) and the adjacent periventricular zone (ARC-PVZ). GAD-IR processes (light red) were also present throughout the hypothalamus and appeared to contact some TH-IR neurons. At the electron-microscopic level, PAP was present in perikarya, dendrites, axons and axon terminals of TH-IR neurons. Colloidal gold particles (15 nm) were found only in dendrites and axon terminals of GAD-IR neurons. Labeled GAD terminals typically contained small, clear synaptic vesicles, while TH terminals contained these and sometimes one or two dense-core vesicles. In the ARC and ARC-PVZ, asymmetrical (Gray I) axodendritic synapses occurred between GAD and TH-IR profiles, with TH/GAD directionality more prevalent. Symmetrical (Gray II) synapses were less common, with either TH or GAD presynaptic in axodendritic and dendrodendritic contacts. GAD/GAD interactions were not observed, but TH/TH contacts appeared to be mostly dendrodendritic. In the AVPV, only symmetrical synapses were encountered, and their directionality was difficult to determine. GAD- and TH-IR dendrites frequently established dendrodendritic synapses, but GAD/TH dendrosomatic synapses were seldom seen. These results illustrate the complex interactions of GAD- and TH-containing elements in the neuroendocrine hypothalamus.

Ultrastructural analysis of synapses involving tyrosine hydroxylase-containing neurons in the ventral periventricular hypothalamus of the macaque.

Immunocytochemical staining for tyrosine hydroxylase (TH) in the adult macaque brain revealed a network of catecholaminergic (CA) cell bodies and fibers in the arcuate (ARC), anterior ventral periventricular (APV) and lateral suprachiasmatic nuclei (SCN). Coronal Vibratome sections immunostained with PAP or colloidal gold (15 nm) were thin sectioned and examined by electron microscopy. We examined 280 TH-immunopositive processes in individual or in serial thin sections. Of these, 190 engaged in a total of 270 synapses identified as Gray Type I asymmetrical synapses (AS) with distinct postsynaptic densities or Gray Type II symmetrical synapses (SS) without such specializations. The majority (80%) of all synapses were axodendritic, 63% of which exhibited SS and 37% AS, representing almost all of the AS observed. In nearly every case, unlabeled axon terminals containing round, 45 nm, clear vesicles and occasional small dense core vesicles contacted TH-labeled dendrites. About 15% of the synapses were dendrodendritic, all of which were symmetrical. Rare contacts involving other elements (axosomatic, dendrosomatic) constituted only 5% of the total, and occurred predominantly as SS. The predominance of AS and the prevalence of SS almost exclusively on TH-containing dendrites indicates that these CA neurons receive extensive afferent input from other neurotransmitters. TH-labeling of both neural elements in most dendrodendritic, and in some axodendritic SS, also suggests that they modulate one another within the ARC, APV and SCN. The results suggest that these CA neurons perform an important role in local integration, and may act elsewhere to affect the common final pathway of the neuroendocrine system in primates.