Khushdev K. Thind

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

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 μl 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 μm 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.

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)

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.

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.

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.

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)

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.

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.

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.

Location of the Neuroendocrine Dopamine Neurons in the Monkey Hypothalamus by Retrograde Tracing and Immunostaining

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 μl 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.