Maternally involved galanin neurons in the preoptic area of the rat.

Recent selective stimulation and ablation of galanin neurons in the preoptic area of the hypothalamus established their critical role in control of maternal behaviors. Here, we identified a group of galanin neurons in the anterior commissural nucleus (ACN), and a distinct group in the medial preoptic area (MPA). Galanin neurons in ACN but not the MPA co-expressed oxytocin. We used immunodetection of phosphorylated STAT5 (pSTAT5), involved in prolactin receptor signal transduction, to evaluate the effects of suckling-induced prolactin release and found that 76 % of galanin cells in ACN, but only 12 % in MPA were prolactin responsive. Nerve terminals containing tuberoinfundibular peptide 39 (TIP39), a neuropeptide that mediates effects of suckling on maternal motivation, were abundant around galanin neurons in both preoptic regions. In the ACN and MPA, 89 and 82 % of galanin neurons received close somatic appositions, with an average of 2.9 and 2.6 per cell, respectively. We observed perisomatic innervation of galanin neurons using correlated light and electron microscopy. The connection was excitatory based on the glutamate content of TIP39 terminals demonstrated by post-embedding immunogold electron microscopy. Injection of the anterograde tracer biotinylated dextran amine into the TIP39-expressing posterior intralaminar complex of the thalamus (PIL) demonstrated that preoptic TIP39 fibers originate in the PIL, which is activated by suckling. Thus, galanin neurons in the preoptic area of mother rats are innervated by an excitatory neuronal pathway that conveys suckling-related information. In turn, they can be topographically and neurochemically divided into two distinct cell groups, of which only one is affected by prolactin.

Neural interaction between galanin-like peptide (GALP)- and luteinizing hormone-releasing hormone (LHRH)-containing neurons.

Galanin-like peptide (GALP), commonly known as an appetite-regulating peptide, has been shown to increase plasma luteinizing hormone (LH) through luteinizing hormone-releasing hormone (LHRH). This led us to investigate, using both light and electron microscopy, whether GALP-containing neurons in the rat brain make direct inputs to LHRH-containing neurons. As LHRH-containing neurons are very difficult to demonstrate immunohistochemically with LHRH antiserum without colchicine treatment, we used a transgenic rat in which LHRH tagged with enhanced green fluorescence protein facilitated the precise detection of LHRH-producing neuronal cell bodies and processes. This is the first study to report on synaptic inputs to LHRH-containing neurons at the ultrastructural level using this transgenic model. We also used immunohistochemistry to investigate the neuronal interaction between GALP- and LHRH-containing neurons. The experiments revealed that GALP-containing nerve terminals lie in close apposition with LHRH-containing cell bodies and processes in the medial preoptic area and the bed nucleus of the stria terminalis. At the ultrastructural level, the GALP-positive nerve terminals were found to make axo-somatic and axo-dendritic synaptic contacts with the EGFP-positive neurons in these areas. These results strongly suggest that GALP-containing neurons provide direct input to LHRH-containing neurons and that GALP plays a crucial role in the regulation of LH secretion via LHRH.

Peptidergic and catecholaminergic synaptic contacts onto nucleus preopticus medianus neurons projecting to the subfornical organ in the rat.

The nucleus preopticus medianus (POMe) is known to be a key site in regulation of cardiovascular and body fluid homeostasis. To clarify the regulation mechanism to the POMe, the innervation pattern of synapses made by axon terminals immunoreactive to beta-endorphin, neuropeptide Y and tyrosine hydroxylase onto POMe neurons projecting to the subfornical organ (SFO) was investigated in the rat. After injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex, into the SFO, many neurons were retrogradely labeled in the POMe, more frequently in its dorsal part. Electron microscopy of the POMe revealed that beta-endorphin- and tyrosine hydroxylase-immunoreactive axon terminals formed predominantly axo-somatic synapses, and neuropeptide Y-immunoreactive axon terminals formed more axo-dendritic than axo-somatic synapses with retrogradely labeled neurons. The present localization patterns of POMe neurons retrogradely labeled from the SFO and the type of synapses of axon terminals immunoreactive to three neurochemical markers on these neurons were compared to those of POMe neurons retrogradely labeled from the paraventricular hypothalamic nucleus demonstrated in our previous report.

Sexually dimorphic hormonal regulation of the gap junction protein, CX43, in rats and altered female reproductive function in CX43+/- mice.

Astrocytic gap junctional communication is important in steroid hormone regulation of reproductive processes at the level of the hypothalamus, including estrous cyclicity and sexual behavior. We examined the effects of estradiol and progesterone on the abundance of the gap junctional protein, connexin 43 (CX43), which is highly expressed in astrocytes. Gonadectomized rats received hormone treatments that induce maximal sexual behavior and gonadotropin surges in females (estrogen for 48 h followed by progesterone, estrogen alone or progesterone alone). Control animals received vehicle (oil) injections. In the female rat preoptic area (POA), containing the gonadotropin-releasing hormone (GnRH) cell bodies, treatment with estrogen, progesterone or estrogen + progesterone significantly increased CX43 protein levels in immunoblots. In contrast, estrogen + progesterone significantly decreased CX43 levels in the male rat POA. This sexually dimorphic hormonal regulation of CX43 was not evident in the hypothalamus, which contains primarily GnRH nerve terminals. Treatment with estrogen + progesterone significantly decreased CX43 levels in both the male and female hypothalamus. To examine the role of CX43 in female reproductive function, we studied heterozygous female CX43 (CX43+/-) mice. Most mutant mice did not show normal estrous cycles. In addition, when compared to wild type females, CX43+/- mice had reduced lordosis behavior. These data suggest that hypothalamic CX43 expression is regulated by steroid hormones in a brain-region-specific and sexually dimorphic manner. Therefore, gap junctional communication in the POA and hypothalamus may be a factor regulating the estrous cycle and sexual behavior in female rodents.

Ultrastructural evidence for luteinizing hormone-releasing hormone neuronal control of estrogen responsive neurons in the preoptic area.

Both estrogen receptor (ER) immunoreactive (ir) and LHRH-ir neurons and processes are present in the preoptic area of the guinea pig. This experiment was conducted to determine if LHRH-ir terminals interact synaptically with ER-ir cells. A light microscopic dual chromogen immunocytochemical technique employing diaminobenzidine (DAB) and nickel-enhanced DAB for LHRH and ER localization, respectively, revealed that many varicose LHRH-ir fibers coursed in close proximity to ER-ir cells in the anterior part of the preoptic area at the preventricular periventricular nucleus (Pep), suggesting the likelihood of synaptic interactions. Ultrastructural analysis was performed using DAB and 3,3',5,5'-tetramethylbenzidine (TMB) for LHRH and ER localization, respectively. DAB labeling in LHRH-ir neurons appeared as a dense flocculent product dispersed throughout the cytoplasm. TMB stained ER-ir neurons contained electron dense crystalline spicules located predominantly in their nuclei. Numerous TMB labeled ER-ir neurons were present in the Pep, and occasionally occurred in clusters, closely apposed to one another. Many LHRH-ir terminals made synaptic contact or were apposed to unlabeled dendrites, while fewer contacted perikarya. Most significantly, ER-ir neurons showing clear evidence of intranuclear TMB crystals received synaptic input from LHRH-ir terminals. In addition, LHRH-ir terminals lacking synaptic specializations were also in direct apposition to ER-ir perikarya. These results provide morphological evidence that LHRH-ir neurons can regulate ER-ir neurons in the preoptic area. Since LHRH-ir cells are thought to be regulated by estrogen responsive neurons, interaction of LHRH terminals with ER-ir cells may represent a regulatory feedback circuit between the two systems.

Studies on the role of the preoptic area in the control of reproductive function in the rat.

The present study was designed to determine the effects of bilateral lesions restricted to the medial preoptic area on reproductive function in rats. Adult female rats were placed in a stereotaxic instrument, and bilateral lesions were made in the medial preoptic area of the diencephalon using an anodic current and platinum or stainless steel electrodes. Both types of lesions produced identical effects. Most animals with lesions restricted to the medial preoptic area showed repeated periods of pseudopregnancy for the duration of their lives. Ova were detected in the oviducts on the day of estrus between pseudopregnancies, and deciduomata could be produced by uterine traumatization during pseudopregnancy. Daily administration of lergotrile mesylate, a dopamine agonist, for about two weeks, to the pseudopregnant rats with lesions resulted in the appearance of normal 4 or 5-day estrous cycles. These results suggest that the medial preoptic area may not be necessary for ovulation in the rat, and that the normal function of the preoptic area in the control of reproductive cyclicity may be mediated via dopaminergic neurons. In agreement with earlier studies, we found that lesions that extended into the anterior hypothalamus resulted in constant estrus.

Estrogen receptor-immunoreactive glia, endothelia, and ependyma in guinea pig preoptic area and median eminence: electron microscopy.

The presence of estrogen receptors (ERs) in nonneural cells in brain, including glia, ependyma, and endothelia, has not previously been documented with electron microscopy. This study employed immunocytochemistry to investigate whether ER immunoreactivity (ER-ir) is present in glial, ependymal, or endothelial cells in the medial preoptic area (POA) and median eminence (ME) in the brain of gonadally intact female guinea pigs. Tissue sections through these regions were immunostained with monoclonal antibody H222 for ER localization using 3,3',5,5'-tetramethylbenzidine (TMB) as the chromogen. ER-ir cells were identified ultrastructurally by the presence of distinct spicule-like TMB crystals in nuclei. While neurons constituted the clear majority of ER-immunopositive cells, labeled astrocytes, ependyma, and endothelia were also present. Distinct intranuclear TMB crystals were present in astrocytes at the anterior pole of the POA within the preventricular periventricular nucleus, anterior compact subnucleus of the medial preoptic nucleus (MPNa), and organum vasculosum of the lamina terminalis, indicating ER-ir. In the MPNa, cell counts performed at the ultrastructural level revealed that 9.6% (15 of 156) of the astrocytes were ER-ir. To further explore the relationship of ERs with astrocytes, ER/glial fibrillary acidic protein (GFAP) double labeling experiments were performed using TMB and diaminobenzidine tetrahydrochloride for ER and GFAP localization, respectively. These studies verified the presence of ERs in astrocytes at the anterior pole of the POA and demonstrated the presence of ERs in GFAP-ir cells in the ME. Cell counts at the ME showed that 23 of 50 (46%) GFAP-ir cells were ER-ir. ER-ir was also present in scattered ependymal cells lining the third ventricle at the POA and overlying the ME. Typically, approximately four to eight ER-ir ependymal cells were present around the perimeter of the third ventricle, although occasionally small aggregations of greater numbers of labeled cells were observed. Both common ependyma and cells morphologically identified as tanycytes were ER-ir. Some endothelial cells and vascular smooth muscle cells also contained ERs. While approximately 11% of the vessels were lined by ER-ir cells in sections through the MPNa and preventricular periventricular nucleus, approximately 15% of the vessels were labeled in the organum vasculosum of the lamina terminalis. In the ME a greater percentage (59%) of the vessels contained ER-ir endothelial cells. Collectively, these results indicate that in addition to regulating the activity of neurons, estrogen may affect brain function through effects exerted on astrocytes, ependymal cells, and endothelial cells.

Androgen receptor immunoreactivity in forebrain axons and dendrites in the rat.

As members of the steroid receptor superfamily, androgen receptors (ARs) have been traditionally identified as transcription factors. In the presence of ligand, ARs reside in the nucleus, where, upon ligand binding, the receptors dimerize and bind to specific response elements in the promoter region of hormone-responsive genes. However, in this report, we describe the discovery that ARs are also present in axons and dendrites within the mammalian central nervous system. AR expression in axons was identified in the rat brain at the light microscopic level using two different antibodies directed against the N terminus of the AR protein and nickel intensified 3'-3'-diaminobenzidine, and also using fluorescence methods and confocal microscopy. This distribution was confirmed at the ultrastructural level. In addition, AR immunoreactivity was identified in small dendrites at the ultrastructural level. AR-immunoreactive axons were observed primarily in the cerebral cortex and were rare in regions where nuclear AR expression is abundant. The observation that ARs are present in axons and dendrites highlights the possibility that androgens play an important and novel extra-nuclear role in neuronal function.

Effects of gonadal steroids on the ultrastructure of GnRH neurons in the rhesus monkey: synaptic input and glial apposition.

The secretion of the gonadotropins is modulated by the gonadal steroids, but the means by which these effects are mediated are not well understood. The present anatomical study was undertaken to investigate the possibility that the GnRH system responds to alterations in the gonadal steroid environment with reversible changes in synaptic input and glial wrapping such as have been observed in other neuroendocrine systems. The ultrastructure of GnRH neurons was studied in the preoptic area and medial basal hypothalamus of rhesus monkeys in various steroid conditions including five intact cycling, four long-term ovariectomized animals, two long-term ovariectomized animals with steroid replacement (LtOVX+), and two animals replaced with steroid at the time of ovariectomy (StOVX+). Electron micrographic montages of GnRH neuronal profiles were analyzed using computerized morphometrics, and the percentages of the length of perikaryal membrane immediately apposed by glial processes and that with postsynaptic modification were calculated. Ovariectomy resulted in a significant increase in the apposition of glial processes to GnRH perikaryal membranes and a significant decrease in their innervation in both brain regions. There was also a higher incidence of GnRH neurons with immunostaining confined to secretory granules and a decrease in the volume of nucleoli, both of which could be interpreted as indications that GnRH peptide synthesis was reduced in ovariectomized animals. After an ovarian steroid replacement regimen which mimicked two menstrual cycles, the innervation of GnRH neurons was increased and the glial ensheathment was partially reduced. This was true for both the LtOVX+ and StOVX+ steroid-replacement groups. GnRH neurons in the medial basal hypothalamus received more synaptic input than did those in the preoptic area, regardless of the steroid condition of the animal. The degree of glial ensheathment of GnRH neurons in the preoptic area became significantly greater than that in the medial basal hypothalamus after ovariectomy. These observations suggest there may be differences in the role of GnRH neurons in these two brain regions. These immunocytochemical ultrastructural studies provide strong evidence that alterations in the gonadal steroid milieu can produce morphological changes in the GnRH neuron and its immediate environment in the primate.

Progesterone modulation of androgen receptors in the brain and pituitary of male guinea pigs.

Androgen receptors (AR) were determined in cytosol and nuclear extracts of pituitary and neural tissue from intact male guinea pigs by a binding assay using [3H]dihydrotestosterone as ligand. Saturation analyses of cytosol from hypothalamus-preoptic area (POA)-amygdala regions and anterior pituitary revealed receptors (ARc) with apparent Kd values of 2.52 and 3.83 X 10(-10) M, respectively. Nuclear salt extracts from the same tissues contained receptors (ARn) with Kd values of 4.38 and 5.12 X 10(-10) M. Reproductive behavior of 10 males was observed with receptive females for 10 min once a week. After 4 weeks, half of the animals received 10 mg progesterone (P)/day for an additional 4 weeks. P treatment significantly (P less than 0.05) increased latency to first mount and decreased mounts per test period. After behavioral testing, analysis of the AR content of specific brain regions revealed that the highest concentrations of ARc and ARn were in the POA and medial basal hypothalamus, and the lowest were in the cerebral cortex. The ARn content was significantly suppressed in POA and medial basal hypothalamus (P less than 0.05) from P-treated males compared to the control value. These data show that AR content is highest in areas thought to control behavior and gonadotropin release within the brain of the male guinea pig. In addition, the antiandrogenic actions of P on the central nervous system, which in this experiment were expressed as a significant decline in reproductive behavior, may be explained by its interference with the retention of the AR in the nucleus.

Aging changes in the beta-endorphin neuronal system in the preoptic area of the C57BL/6J mouse: ultrastructural analysis.

In hypothalami of aging rodents, beta-endorphin (beta-EP) neuron number and content are reduced. The objectives of this study were: first, to analyze ultrastructurally the population of neuronal elements in a selected region of the preoptic area (POA) in young and old mice; second, to study the beta-EP neuronal system in the same region to determine whether or not this population remains stable with age. Vibratome sections from the most caudal POA through the diagonal band of Broca were examined by light microscopy and immunocytochemistry in mature, cycling (5-6 months old) and old, acyclic, disease-free (24-26 months old) mice. A subset of beta-EP-like perikarya and associated structures was observed in the periventricular POA. When this subregion was examined at the ultrastructural level, there was a significant decrease in the number of recipient dendrites [3.78 +/- 0.04 SEM/micron 2 young vs. 0.82 +/- 0.03/micron 2 old; p < 0.007, analysis of variance (ANOVA)], but a significant increase in the number of nonmyelinated axons (20.0 +/- 2.6/micron 2 young vs. 26.8 +/- 0.7/micron 2 old; p < 0.05). Immunolabeled terminals that contained a synapse comprised 2.56 +/- 0.08% of all terminals with synapses in young mice but only 0.34 +/- 0.04% in old ones when corrected for surface area examined (p < 0.03). A significant age-related loss was also observed in the nonmyelinated beta-EP-labeled axon population (1.50 +/- 0.10% young vs. 0.40 +/- 0.01% old; p < 0.009, ANOVA). We conclude that there are critical changes in the microenvironment of the POA in old, noncycling female mice that are likely to affect neuron function.

Increased synaptic input to gonadotropin-releasing hormone neurons in aged, virgin, male Sprague-Dawley rats.

Using double-label ultrastructural immunocytochemistry, we found the synaptic input to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area of aged (20 months old), virgin, male Sprague-Dawley rats to be denser than that in young adults (3 months old). These results confirmed earlier observations on F-344 virgin male rats. The aging F-344 rat, however, is prone to testicular tumor and so it was essential to see if the phenomenon was reproducible in another rat strain. In the first study, a portion of the increase in synaptic density was due to an increase in the proportion of synapses containing pleiomorphic vesicles, frequently associated with the neurotransmitter GABA. We tested the possibility directly using a double-label protocol for GnRH and glutamic acid decarboxylase (GAD). However, in the present study the density of input by GABA did not change with age. This inhibitory amino acid represented about 10% of the total innervation in young animals; but, in aged animals, because the total synaptic input was greater, GABA represented only about 4% of the innervation. Synaptic vesicles within GAD-immunoreactive terminals were uniformly clear and spherical, suggesting that pleiomorphic vesicle shape is not an appropriate criterion for GABAergic innervation.

Ovariectomy and age alter gonadotropin hormone releasing hormone-noradrenergic interactions.

The noradrenergic (NA) system influences gonadotropin hormone releasing hormone (GnRH) neurons resulting in the luteinizing hormone surge. Direct neuroanatomical interactions between preoptic area (POA) GnRH neuronal elements and NA [i.e., dopamine-beta-hydroxylase (DBH)] terminals; effects of short-term ovariectomy (S-OVX) on these interactions; and the stability of these interactions with age were studied in young (5-month-old) proestrous, 5-month-old S-OVX (6d), old (24-month-old) constant diestrous, and 24-month-old long-term (L)-OVX mice. Proestrous females demonstrated direct interactions between NA terminals and GnRH neuronal elements. The percentage of GnRH dendritic profiles contacted by DBH terminals at proestrous (6.96 +/- 1.07%) did not differ versus young S-OVX females (4.55 +/- 0.91%). No GnRH-NA interactions were observed in old mice. S-OVX resulted in a decrease in DBH terminals but an increase in dendrite and nonmyelinated axon number versus young proestrous females (p < or = 0.05 ANOVA). Findings show direct GnRH-DBH interactions, confirm S-OVX effects on neural ultrastructure, and demonstrate some S-OVX changes resembling those in older mice. L-OVX failed to prevent aging changes. Diminished capacity to produce normal estrous cycles in aging females could result in part from absence of direct GnRH-DBH interactions.

Electron microscopic analysis of synaptic inputs from the median preoptic nucleus and adjacent regions to the supraoptic nucleus in the rat.

The median preoptic nucleus (MnPo) is critical for normal fluid balance, mediating osmotically evoked drinking and neurohypophysial hormone secretion. The influence of the MnPo on vasopressin and oxytocin release is in part through direct connections to the supraoptic and paraventricular nucleus. In the present investigation the synaptic contacts between the MnPo and supraoptic neurons were investigated in rats by ultrastructural examination of terminals labeled anterogradely with the tracers Phaseolus vulgaris-leucoagglutinin or biotinylated dextran. At the light microscopic level, labeled fibers within the supraoptic nucleus branched frequently, were punctuated by varicosities, and were distributed throughout the nucleus without preference for the known distributions of oxytocin and vasopressin neurons. At the ultrastructural level, synapses were associated with many of these varicosities. The ratio of labeled axodendritic to axosomatic synapses encountered was roughly consistent with a uniform innervation of dendrites and somata. The great majority of synapses were characterized by symmetrical contacts. Similar results were found for a few injections made in the organum vasculosum of the lamina terminalis, just rostral to the MnPo, and in the immediately adjacent periventricular preoptic area. Coupled with other recent anatomical and electrophysiological evidence, these results suggest there is a strong monosynaptic pathway from structures along the ventral lamina terminalis to the supraoptic nucleus.

Ultrastructure and synaptic organization of luteinizing hormone-releasing hormone (LHRH) neurons in the anestrous ewe.

Electron microscopic immunocytochemistry was employed to examine the ultrastructure of luteinizing hormone-releasing hormone (LHRH) neurons and their projections to the median eminence in the sheep brain. LHRH perikarya in the preoptic area of anestrous ewes are less innervated than nonimmunoreactive cells in the same sections, but still receive numerous synaptic inputs, primarily onto distal dendrites and small somatic protuberances. Axon terminals synapsing upon LHRH cells contain a combination of clear spherical vesicles and larger dense-core vesicles. Interestingly, LHRH cell bodies and dendrites are almost entirely surrounded by glial processes. These processes intervene between immunoreactive elements that at a light microscopic level appear to be in contact with each other. Thus no evidence was obtained at the ultrastructural level for contacts among adjacent LHRH cells or dendrites in the preoptic area. Synaptic inputs onto LHRH cell bodies and dendrites appear to penetrate this glial sheath. In contrast to the absence of contacts among LHRH cells in the preoptic area, individual LHRH terminals in the median eminence are often clustered in direct plasma membrane contact. Comparisons between animals of differing reproductive status are needed to determine whether alterations in synaptic inputs, glial ensheathment, or LHRH-LHRH appositions, may underlie seasonal changes in the activity of LHRH neurons.

Luteinizing hormone releasing hormone containing synapses in the diagonal band and preoptic area of the guinea pig.

Light microscopic immunocytochemical processing of the region of the ventral diagonal band of Broca (vertical limb) and the medial preoptic area of the guinea pig for LHRH revealed complex axonal bundles. The axons were of several different shapes and widths and showed varicosities of varying form. When such tissue was embedded and processed for electron microscopic localization of immunoreaction product, numerous LHRH-positive synapses were seen. The synapses were made by large varicosities. They were all axodendritic and showed a well-defined synaptic cleft, as well as a clustering of immunoreactive large granules and immunonegative small, clear vesicles. Postsynaptic densities were either very small or absent. These data show that LHRH is present in direct synapses within the CNS and is, therefore, in a position to be released and act as a neurotransmitter/neuromodulator as well as a neurohormone.

Efferent connections from the lateral hypothalamic region and the lateral preoptic area to the hypothalamic paraventricular nucleus of the rat.

The lateral preoptic and lateral hypothalamic regions contain the majority of the cell groups embedded in the fibre trajectories of the medial forebrain bundle on its course through the hypothalamus. Recent studies have extended considerably the parcellation of the lateral hypothalamic region, and therefore, the need to emphasize new insights into the anatomical organisation of projections from the neurons of the lateral hypothalamic region. In the present study we describe the anatomical organisation of efferent projections from the lateral preoptic and lateral hypothalamic regions to the hypothalamic paraventricular nucleus (PVN) on the basis of retrograde- and anterograde-tracing techniques. Iontophoretic injections of the retrograde tracer, cholera toxin subunit B, into the PVN revealed that most hypothalamic nuclei project to the PVN. Within the lateral hypothalamic region, retrogradely labelled cells were concentrated in the intermediate hypothalamic area, the lateral hypothalamic area, and the perifornical nucleus, whereas fewer retrogradely labelled cells were found in the lateral preoptic area. To determine the distribution of terminating fibres in subnuclei of the heterogeneous PVN, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were delivered into distinct areas of the lateral hypothalamic region. Neurons of the intermediate hypothalamic area projected mainly to the PVN subnuclei, which contained parvicellular neuroendocrine cells. In contrast, neurons of the rostral and tuberal parts of the lateral hypothalamic area and the perifornical nucleus projected to the PVN subnuclei, which contained parvicellular neurons that send descending projections to preganglionic cell groups in the medulla and spinal cord. The perifornical nucleus was the only area within the lateral hypothalamic region that consistently innervated magnocellular perikarya of the PVN. Finally, all areas of the lateral hypothalamic region contributed substantially to fibres terminating in the perinuclear shell of the PVN. These results demonstrate that anatomically distinct areas of the lateral hypothalamic region have distinct projections to subnuclei of the PVN and further substantiate the view that the lateral hypothalamic region as well as the PVN constitute anatomically and functionally heterogeneous structures.

Effects of testosterone on a selected neuronal population within the preoptic sexually dimorphic nucleus of the Japanese quail.

The effects of testosterone on the volume and cytoarchitecture of the sexually dimorphic nucleus of the preoptic area (POM) were investigated in male and female Japanese quail. It was confirmed that castration decreases the POM volume in males and that, in gonadectomized birds of both sexes, testosterone increases this volume to values similar to those observed in intact sexually mature males. This suggests that the sex difference in POM volume results from a differential activation by T so that this brain morphological characteristic is not truly differentiated in the organizational sense. This conclusion was extended here by demonstrating that males exposed to a photoperiod simulating long days and that are known to have high plasma levels of testosterone have a larger POM than short-day males that have inactive testes. Detailed morphometric studies of POM neurons revealed a structural heterogeneity within the nucleus. A population of large neurons (cross-sectional area larger than 70-80 microns2) was well represented in the dorsolateral but was almost absent in the medial part of POM. This lateral population of neurons was sensitive to variations of testosterone levels in males but not in females. The cross-sectional area, diameter, and perimeter of the dorsolateral neurons were significantly increased in males exposed to high testosterone levels (intact birds exposed to long days or castrated birds treated with the steroid). These changes were not observed in the medial part of the nucleus. Interestingly, the size of the dorsolateral neurons was not affected by testosterone treatments in females. These results suggest that the swelling of neurons in the lateral POM of males might be responsible for the increase in total volume of the nucleus, which is observed in physiological situations associated with a high testosteronemia. In addition, the sensitivity to testosterone of the dorsolateral neurons in the POM appears to be sexually differentiated. This differential response to testosterone might represent a truly dimorphic feature in the organizational sense and additional studies manipulating the early steroid environment should be performed to test this possibility.

An aromatase-associated cytoplasmic inclusion, the "stigmoid body," in the rat brain: II. Ultrastructure (with a review of its history and nomenclature).

The ultrastructure of aromatase-associated "stigmoid (dot-like) structures," which were detected in a previous study using light-microscopic immunohistochemistry (Shinoda et al.: J. Comp. Neurol. 322:360-376, '92), were examined in the rat medial preoptic region, bed nucleus of the stria terminalis, medial amygdaloid nucleus, and arcuate nucleus by pre- and post-embedding marking with a polyclonal antibody against human placental antigen X-P2 (hPAX-P2) for immuno-electron microscopic analysis. The immunoreactive stigmoid structure was identified as a distinct, non-membrane-bounded cytoplasmic inclusion (approximately 1-3 microns in diameter), which has a granulo-fuzzy texture with moderate-to-low electron density in non-immunostained preparations. It consists of at least four distinct granular and three distinct fibrillo-tubular elements forming a granulo-fibrillar conglomerate. This type of inclusions was formally termed the "stigmoid body" under the electron microscope. The stigmoid body is composed of the outer granulo-fibrillar and inner hyaloplasmic compartments. The immunoreactivity for hPAX-P2 is mainly localized to the former, especially to the low density granulo-fuzzy materials associated with the fibrillo-tubular elements. Identification of the ultrastructure of stigmoid body clarified their prevalence not only in the limbic and hypothalamic regions, but also in sex-steroid-sensitive peripheral tissues (e.g., peripheral sensory ganglia, ovary, testis) by consulting earlier electron-microscopic studies. Reviewing the history and nomenclature of this inclusion body, we reorganized the terminology of related neuronal cytoplasmic inclusions, the terms of which have often been confused, and discussed its functional significance on the basis of the present and previously accumulated data. In conclusion, we emphasized the importance of the stigmoid bodies in the sex-steroid-sensitive neural system because of their large size, high frequency, specific distribution in brains and peripheral tissues, effects of sex-steroids, and immunological and histochemical characteristics of the antibody marking the inclusion. The stigmoid bodies may provide a subcellular site for sex-steroid metabolism in their target tissues and play a critical role in cytosolic modulation of their actions (e.g., by aromatization) prior to their receptor binding.

Effects of sex and androgen treatment on dendritic dimensions of neurons in the sexually dimorphic preoptic/anterior hypothalamic area of male and female ferrets.

A sexually dimorphic group of cells at the dorsal border of the preoptic/anterior hypothalamic area (POA/AH) of ferrets has been previously identified in Nissl-stained tissue. In this study, Golgi-stained tissue was examined in order 1) to determine whether sex differences exist in dendritic dimensions of neurons from this region, and 2) to assess the effects of adult androgen treatment on dendritic morphology in ferrets of both sexes. Brains from adult ferrets given daily injections of testosterone propionate (5 mg/kg body weight) or oil vehicle for 5 weeks after gonadectomy were impregnated by Golgi-Cox procedures. After sectioning at 120 microns, 78 multipolar neurons were selected from the sexually dimorphic POA/AH of 12 ferrets and reconstructed in three dimensions with the aid of a computer-assisted neuron tracing system. Large sex differences were observed in somal area and most aspects of dendritic morphology, including total length, number of branches, and total dendritic surface area. Androgen also appeared to accentuate dendritic arborization in both sexes, but this effect was weaker than the sex effect, more apparent in males than females, and restricted to fewer variables. The most statistically significant effects of adult androgen treatment in males were found for total dendritic surface area and percentage of fourth order dendrites, and in females, average dendritic thickness. These data show that strong sex differences exist in dendritic structure of neurons in the POA/AH, and suggest that alterations in levels of gonadal steroids in adulthood may promote synaptic remodeling in a region of the brain involved in the control of sexually dimorphic behaviors.