Vitamin D hormone confers neuroprotection in parallel with downregulation of L-type calcium channel expression in hippocampal neurons.

Although vitamin D hormone (VDH; 1,25-dihydroxyvitamin D(3)), the active metabolite of vitamin D, is the major Ca(2+)-regulatory steroid hormone in the periphery, it is not known whether it also modulates Ca(2+) homeostasis in brain neurons. Recently, chronic treatment with VDH was reported to protect brain neurons in both aging and animal models of stroke. However, it is unclear whether those actions were attributable to direct effects on brain cells or indirect effects mediated via peripheral pathways. VDH modulates L-type voltage-sensitive Ca(2+) channels (L-VSCCs) in peripheral tissues, and an increase in L-VSCCs appears linked to both brain aging and neuronal vulnerability. Therefore, we tested the hypothesis that VDH has direct neuroprotective actions and, in parallel, targets L-VSCCs in hippocampal neurons. Primary rat hippocampal cultures, treated for several days with VDH, exhibited a U-shaped concentration-response curve for neuroprotection against excitotoxic insults: lower concentrations of VDH (1-100 nm) were protective, but higher, nonphysiological concentrations (500-1000 nm) were not. Parallel studies using patch-clamp techniques found a similar U-shaped curve in which L-VSCC current was reduced at lower VDH concentrations and increased at higher (500 nm) concentrations. Real-time PCR studies demonstrated that VDH monotonically downregulated mRNA expression for the alpha(1C) and alpha(1D) pore-forming subunits of L-VSCCs. However, 500 nm VDH also nonspecifically reduced a range of other mRNA species. Thus, these studies provide the first evidence of (1) direct neuroprotective actions of VDH at relatively low concentrations, and (2) selective downregulation of L-VSCC expression in brain neurons at the same, lower concentrations.

Contrasting mammalian PTH promoters: identification of transcription factors controlling PTH gene expression.

Parathyroid hormone (PTH) is a key component in the maintenance of calcium and phosphate homeostasis. The steady-state expression of the PTH gene can be modeled as a balance between transcriptional activators and repressors. During renal failure, the gradual loss of kidney function is often accompanied by increased circulating concentrations of PTH and decreased synthesis of 1,25-di-hydroxyvitamin D3 (1,25(OH)2D3). The latter finding results in impaired calcium absorption and the removal of a known repressor of PTH gene transcription. Current regimens for treating secondary hyperparathyroidism associated with renal insufficiency are focused on boosting activities that repress PTH gene transcription or secretion of the hormone, and involve the use of vitamin D and its analogues or calcimimetic agents. However, in recent years, concerns have arisen over the use of the steroid hormone and alternative treatments are being sought. Here, we present new information regarding transcription factors controlling PTH gene expression, which include the specificity proteins (Sp) and the nuclear factor Y (NF-Y) complex. A highly conserved DNA response element for the Sp proteins has been identified in mammalian promoters, while an NF-Y binding site is uniquely positioned in the human promoter. Both of these factors are expressed in the parathyroid gland and their DNA elements appear to be functioning as activators of PTH gene expression. Further elucidation of such pathways may offer novel approaches for treating hyperparathyroidism associated with renal failure via suppression of transcriptional activators. That work is currently in progress.

Parathyroid hormone/parathyroid hormone-related peptide type 1 receptor in human bone.

The parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor (denoted as PTH-1R) is a key signaling factor through which calcium-regulating hormones PTH and PTHrP exert their effects on bone. There are contradictory reports regarding the capability of osteoclasts to express PTH-1R. To address this issue in humans, bone biopsy specimen samples from 9 normal controls and 16 patients with moderate to severe secondary renal hyperparathyroid bone disease (2 degrees HPT) with elevated PTH levels were studied to determine whether osteoclasts in the bone microenvironment express PTH-1R messenger RNA (mRNA) and protein. We report that osteoclasts express the PTH-1R mRNA but the protein is detected only in patients with 2 degrees HPT. The PTH-1R mRNA and protein also were found in osteoblasts, osteocytes, and bone marrow cells. Receptor expression was higher in osteoclasts and osteoblasts of patients with 2 degrees HPT than normal controls (98.0 +/- 1.1% vs. 65.7 +/- 14.3% and 65.8 +/- 3.4% vs. 39.1 +/- 6.2%; p < 0.01, respectively). Approximately half of osteoclasts found in bone of patients with 2 degrees HPT have the PTH-1R protein. In patients with 2 degrees HPT, a positive relationship exists between erosion depth, a parameter of osteoclastic activity, and the percentage of osteoclasts with PTH-1R protein (r = 0.58; p < 0.05). In normal controls, an inverse relationship exists between the percentage of osteoblasts with receptor mRNA, mRNA signals/cell, and serum PTH levels (r = -0.82 and p < 0.05 and r = -0.78 and p < 0.01, respectively). The results provide the novel evidence of PTH-1R in human osteoclasts and suggest a functional role for the receptors in 2 degrees HPT.

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.

Localization of C-type natriuretic peptide mRNA in rat hypothalamus.

Central or peripheral administration of C-type natriuretic peptide (CNP) affects numerous neuroendocrine systems, including the hypothalamo-pituitary-gonadal, hypothalamo-pituitary-adrenocortical and hypothalamo-neurohypophysial axes. The present report characterizes the distribution of CNP mRNA in hypothalamus, providing the first definition of CNP-containing neuroendocrine circuits. In situ hybridization histochemical analysis revealed high expression of CNP mRNA in the anteroventral periventricular nucleus (AVPv) and in hypothalamic arcuate nucleus (ARC). Hybridization signals of significantly lower intensity were seen in the medial, median and periventricular preoptic area, the supraoptic, dorsomedial, ventral premammillary and lateral mammillary nuclei and in the posterior hypothalamic area. A few scattered CNP mRNA containing cells were visualized in the medial parvocellular paraventricular nucleus, posterior magnocellular paraventricular nucleus and lateral hypothalamic area. In the AVPv and ARC the pattern of CNP mRNA distribution paralleled that of ANP mRNA. The results indicate a distribution of CNP mRNA associated with key neuroendocrine systems, and underscores the potential importance of this novel natriuretic peptide in neuroendocrine regulation.

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.

Targeted overexpression of insulin-like growth factor I to osteoblasts of transgenic mice: increased trabecular bone volume without increased osteoblast proliferation.

Insulin-like growth factor I (IGF-I) is an important growth factor for bone, yet the mechanisms that mediate its anabolic activity in the skeleton are poorly understood. To examine the effects of locally produced IGF-I in bone in vivo, we targeted expression IGF-I to osteoblasts of transgenic mice using a human osteocalcin promoter. The IGF-I transgene was expressed in bone osteoblasts in OC-IGF-I transgenic mice at high levels in the absence of any change in serum IGF-I levels, or of total body growth. Bone formation rate at the distal femur in 3-week-old OC-IGF-I transgenic mice was approximately twice that of controls. By 6 weeks, bone mineral density as measured by dual energy x-ray, and quantitative computed tomography was significantly greater in OC-IGF-I transgenic mice compared with controls. Histomorphometric measurements revealed a marked (30%) increase femoral cancellous bone volume in the OC-IGF-I transgenic mice, but no change in the total number of osteoblasts or osteoclasts. Transgenic mice also demonstrated an increase in the osteocyte lacunea occupancy, suggesting that IGF-I may extend the osteocyte life span. We conclude that IGF-I produced locally in bone osteoblasts exerts its anabolic effect primarily by increasing the activity of resident osteoblasts.

Distribution of natriuretic peptide precursor mRNAs in the rat brain.

Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) represent members of a recently discovered neuropeptide family involved in central regulation of endocrine and autonomic functions. The present study employed an in situ hybridization approach to provide the first detailed comparative mapping of ANP, BNP, and CNP mRNAs in brain. Results indicate that ANP mRNA is highly expressed in anterior olfactory nuclei, limbic cortices, dorsal endopiriform nucleus, hippocampal subfield CA1, cortical amygdaloid nuclei, medial habenula, anteroventral periventricular and arcuate nuclei, periventricular stratum, zona incerta, mammillary nuclei, inferior olive, nucleus ambiguus, and pontine paragigantocellular nuclei. CNP mRNA is expressed at highest levels in olfactory nuclei, limbic cortices, dorsal endopiriform nucleus, hippocampal subfields CA1-3, anteroventral periventricular and arcuate nuclei, and numerous brainstem regions (including the pontine, lateral reticular, solitary tract, prepositus hypoglossal, and spinal trigeminal nuclei). Positive labeling for BNP mRNA was not observed in brain. The presence of both ANP and CNP mRNA in the same regions of distinct nuclei (e.g., the anteroventral periventricular and arcuate nuclei) suggests the potential for coexpression. Overall, the present data are consistent with a prominent role for both ANP and CNP in neuroendocrine regulation and central cardiovascular integration. The extensive localization of ANP and/or CNP mRNA in olfactory nuclei, limbic cortex, hippocampus, amygdala and diencephalic limbic relays further indicate a putative role for ANP and CNP as neuromodulators of olfactory/limbic information processing.

Localization of natriuretic peptide-activated guanylate cyclase mRNAs in the rat brain.

Physiological actions of atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) are elaborated by membrane-bound natriuretic peptide receptors (NPRs). These receptors possess intracellular guanylate cyclase domains that mobilize cyclic guanosine monophosphate upon binding of peptide. Two distinct NPR subtypes have been described in brain: the NPR-A selectively binds ANP, whereas NPR-B exhibits high affinity for CNP. To define further the potential domains of ANP and CNP action in brain, the present study used in situ hybridization histochemistry to map NPR-A and NPR-B mRNA-expressing cell populations. Significant levels of neuronal NPR-A mRNA expression were observed only in the mitral cell layer of the olfactory bulb, medial habenula, subfornical organ, and area postrema. Expression of NPR-A mRNA was observed in forebrain white matter tracts, suggesting synthesis in glial cells. In contrast, NPR-B mRNA was widely expressed throughout the neuraxis. In the telencephalon, signal was abundant throughout limbic cortex and neocortex, olfactory bulb, hippocampus, and amygdala. Intense NPR-B mRNA hybridization was observed in preoptic-hypothalamic neuroendocrine circuits and in motor nuclei of cranial nerves. Intermediate expression of NPR-B mRNA was observed in brainstem nuclei controlling autonomic function. Labeling for NPR-B but not NPR-A mRNA was observed in pituicytes in the neural lobe of the pituitary and in scattered cells of the anterior pituitary. These results suggest that CNP is the primary biologically active natriuretic peptide in brain. In contrast with NPR-B, NPR-A appears to be expressed largely in restricted cell populations containing high levels of ANP and in circumventricular organs. These data implicate the NPR-A in autoregulation of ANP neurons and central registration of cardiac ANP release.

Characterization of vitamin D receptor immunoreactivity in human bone cells.

The present study examined the expression of the vitamin D receptor (VDR) in adult human bone by immunohistochemical analysis. Antiserum from goats immunized with an N-terminal rat VDR peptide was purified by affinity chromatography. The purified antiserum recognized both endogenous rat and recombinant human VDR in Western blots. The purified antiserum was also able to specifically supershift the recombinant human VDR when analyzed in mobility shift assays. Immunohistochemical analysis of MG-63 cells, a human osteoblastic cell line known to express the VDR, revealed prominent staining over the nuclei of these cells. Immunostaining was greatly attenuated in the presence of an excess of the immunizing peptide. Analysis of bone biopsy samples from 16 normal human subjects immunostained for VDR protein showed strong, immunopositive staining over bone cells, particularly osteoblasts, in keeping with prior studies. In addition, there was significant immunoreactivity observed in nuclei of osteoclasts, lining cells and scattered bone marrow stromal cells of the adult human bone. Results showed that 298 osteoblasts out of 808 (36.9%) examined were immunopositive. It was also observed that 29 osteoclasts out of 125 (23%) contained VDR immunoreactivity. The ability to detect VDR in osteoclasts and stromal cell populations suggests that in addition to regulating osteoblast function, these other cell types are also direct targets of the hormone's action. These results demonstrate the utility of this purified antiserum in detecting the VDR in a variety of molecular techniques and should prove useful in examining receptor expression in various pathological conditions.

Evidence of functional vitamin D receptors in rat hippocampus.

The steroid hormone vitamin D has important biological roles in calcium transport, cell growth, and cell differentiation. Its cellular activities are mediated by high affinity interaction with the vitamin D receptor. In brain, autoradiographic, immunohistologic, and messenger RNA expression studies implicate a number of neuronal systems, including the hippocampus, as potential targets of vitamin D. However, cellular distribution and protein expression, and binding of the receptor to vitamin D response elements have yet to be established in hippocampus. This investigation was undertaken to characterize the vitamin D receptor in rat hippocampus with western blot, immunocytochemistry, and gel shift analyses. The presence of the receptor protein in hippocampus extracts was revealed with western blotting using an anti-rat vitamin D receptor antiserum. In vivo and in vitro immunocytochemical results confirmed the presence of vitamin D receptor in neuronal and glial cells. In the hippocampus, the receptor was localized in pyramidal and granule cell layers, CA1, CA2, and CA3 subfields and in the dentate gyrus. Double labeling for the vitamin D receptor and glial fibrillary acidic protein revealed that glia also expressed the receptor protein. Gel shift analyses evaluated with the murine osteopontin vitamin D response element indicated a specific, bound receptor-containing complex from hippocampal extracts. Altogether, these findings clearly document the localization of vitamin D receptor in rat hippocampus and that hippocampus contains vitamin D receptors capable of specifically binding to DNA. In combination with reports of a neuroprotective role for vitamin D in hippocampal cell survival, these data suggest that the endogenous vitamin D receptor may mitigate processes related to cellular homeostasis, perhaps through a calcium buffering mechanism.

Further Evidence that Most Luteinizing Hormone-Releasing Hormone Neurons are not Directly Estrogen-Responsive: Simultaneous Localization of Luteinizing Hormone-Releasing Hormone and Estrogen Receptor Immunoreactivity in the Guinea-Pig Brain.

Gonadotropin secretion from the pituitary is regulated in large part by steroid action on the brain. An important question concerns whether luteinizing hormone-releasing hormone (LHRH) neurons themselves transduce steroid signals, or whether, alternatively, steroid-sensitive interneuronal populations regulate their activity. A previous study in the rat employing steroid autoradiography combined with LHRH immunocytochemistry revealed that only an exceedingly small percentage of LHRH-immunoreactive (ir) neurons was estrogen concentrating. This study has examined the relationship of estrogen receptive and LHRH-ir cells in the male and female guinea-pig brain with double label immunocytochemistry. Since estrogen receptor-ir, as demonstrated with antibody H222, is known to be confined predominantly to the cell nucleus, whereas LHRH-ir is localized mainly in the cytoplasm, single cells can be double-labeled. Diaminobenzidine tetrahydrochloride was used for localization of LHRH-ir while nickel-enhanced diaminobenzidine tetrahydrochloride was used for localization of estrogen receptor-ir. The results revealed that there were many brain nuclei that contained both LHRH and estrogen receptor-positive cells, including the preventricular periventricular nucleus, the anterior subcompact nucleus of the medial preoptic nucleus (MPNa), the remainder of the medial preoptic nucleus, the retrochiasmatic area, and the anterior, dorsomedial, ventrolateral and arcuate nuclei. However, of a total of 2,604 LHRH-ir cells that were examined, we observed only 5 double-labeled cells (<0.2%). The double-labeled cells were not restricted to a single nucleus; they were present in the MPNa, the retrochiasmatic area and the arcuate nucleus. Moreover, at the light microscopic level, LHRH cells quite frequently appeared to be apposed to estrogen receptor-positive cells (8.8% in the female), especially in the MPNa. These results lend further support to the notion that estrogen-mediated regulation of the LHRH system is achieved primarily through estrogen receptive interneurons. However, due to the existence of LHRH-LHRH synaptic interactions, the possibility also exists that a small population of estrogen-sensitive LHRH neurons could contribute to generalized activation of the LHRH system.

The C-type natriuretic peptide receptor is the predominant natriuretic peptide receptor mRNA expressed in rat hypothalamus.

The natriuretic peptide receptors (NPR) are membrane-bound guanylate cyclases with extracellular binding domains specific for particular members of the natriuretic peptide family. NPR-A binds atrial natriuretic peptide (ANP) with high affinity, whereas the NPR-B appears to be specific for C-type natriuretic peptide (CNP). Previous data indicating extensive overlap between localization of ANP and CNP in hypothalamic neuroendocrine circuits suggest the importance of determining whether specificity of natriuretic peptide action may be conferred via receptor type present on target cells. To address this issue, we used in situ hybridization histochemistry to localize NPR-A and NPR-B mRNA in the hypothalamus. NPR-A mRNA was not found in substantial abundance in any hypothalamic nucleus; however, detectable NPR-A signal was observed in other brain regions, including the subfornical organ and medial habenula. In contrast, NPR-B mRNA was expressed throughout the hypothalamus, including neurons of the magnocellular and parvocellular paraventricular, the arcuate, and the supraoptic nuclei. Expression was also seen in other nuclei essential to neuroendocrine control, including the median preoptic, anteroventral periventricular, tuberomammilary, ventromedial and suprachiasmatic nuclei. NPR-B mRNA was also observed in the neural lobe of the pituitary gland, suggesting expression by pituicytes. The results suggest that NPR-B is the primary natriuretic peptide receptor in hypothalamus, and by inference indicate that CNP is the primary active natriuretic peptide in neuroendocrine regulation.

Colocalization of natriuretic peptide and estrogen receptor immunoreactivities in preoptic nuclei in the female rat.

Estrogen is known to play an important role in regulating reproductive function in female rats through actions exerted at the preoptic area, a part of the brain that is markedly sexually dimorphic and which contains abundant estrogen receptors. A critical question to our understanding of estrogen's action on the brain is to identify the types of neurons that contain estrogen receptors (ER). Previous studies have shown that atrial natriuretic peptide (ANP) is in abundance in the preoptic area, and that ANP and other natriuretic peptides are capable of regulating gonadotropin secretion. In an effort to determine whether ERs are present in natriuretic peptide-immunoreactive (NP-ir) neurons in the preoptic area of the rat, double label immunocytochemistry was performed. Since ER-ir, as demonstrated with antibody H222 is known to be localized predominantly in cell nuclei, while NP-ir is present in the cytoplasm, single cells can be double labeled. Diaminobenzidine tetrahydrochloride was used for localization of NP-ir neurons, while nickel-enhanced diaminobenzidine tetrahydrochloride was used for localization of ER-ir. The results revealed that many nuclei throughout the preoptic area contained neurons that were ER-ir or NP-ir and that a substantial number were double labeled. Cell counts in selected preoptic nuclei and components, including the anteroventral periventricular nucleus, periventricular preoptic nucleus, medial part of the medial preoptic nucleus, and central part of the medial preoptic nucleus revealed that 13.6%, 11.1%, 13.5%, and 24.4%, respectively, of the NP-ir neurons in these nuclei also contained ER-ir. Collectively, a total of 14.9% of the NP-ir neurons in these nuclei also contained ER-ir.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and glucocorticoid regulation of natriuretic peptide clearance receptor (NPR-C) mRNA in rat brain and choroid plexus.

The natriuretic peptide clearance receptor (NPR-C) binds atrial natriuretic peptide, brain natriuretic peptide and C-type natriuretic peptide with high affinity. This receptor lacks an intracellular guanylate cyclase domain, and is believed to exert biological actions by sequestration of released natriuretic peptides and/or inhibition of adenylate cyclase. The present report summarizes the first detailed mapping of NPR-C mRNA in rat brain. In situ hybridization analysis revealed high levels of NPR-C mRNA expression in frontal and retrosplenial granular cortices, medial preoptic nucleus, ventral cochlear nucleus and choroid plexus. NPR-C mRNA expression was also observed in deep layers of neocortex and limbic cortex, posterior cortical amygdala, ventral subiculum, amygdalohippocampal area, and dentate gyrus. Positive hybridization signal was observed in both anterior and intermediate lobes of the pituitary gland. Regulatory studies indicated that expression of NPR-C mRNA was increased in the medial preoptic nucleus of adrenalectomized rats, suggesting negative glucocorticoid regulation. No changes in NPR-C mRNA expression were observed in frontal cortex or choroid plexus. These results suggest a role for the NPR-C in modulation of natriuretic peptide availability and/or adenylate cyclase activity in a subset of central natriuretic peptide circuits concerned with cortical, olfactory and neuroendocrine functions. Response of the NPR-C gene to changes in circulating hormones suggests the capacity for glucocorticoid modulation of natriuretic peptide action at the receptor level.

The role of bone biopsy in clinical practice and research.

Survival rates of patients on dialysis have increased with improved dialytic therapy. However, the resultant increased duration of dialysis has led to a rise in renal osteodystrophy (ROD). Because this metabolic bone disease can produce fractures, bone pain, and deformities late in the course of the disease, prevention and early treatment are essential. Types of ROD include predominant hyperparathyroid bone disease, low turnover bone disease (including osteomalacia and adynamic bone disease), and mixed uremic osteodystrophy. Serum PTH levels are commonly used to assess bone turnover in dialyzed patients. However, a recent study in our laboratory found that serum PTH levels between 65 and 450 pg/ml seen in the majority of dialysis patients are not predictive of the underlying bone disease. To date, bone biopsy is the most powerful and informative diagnostic tool to provide important information on precisely the type of renal osteodystrophy affecting patients, the degree of severity of the lesions, and the presence and amount of aluminum deposition in bone. Bone biopsy is not only useful in clinical settings but also in research to assess the effects of new therapies on bone. The methods of in situ hybridization histochemistry (ISHH) and immunohistochemistry (IHC) are providing the means to study local biomolecules that play a role in bone metabolism. As these research tools become more refined, they will become increasingly valuable in the study of bone. Alternatives to the bone biopsy continue to be pursued, but they have not been proven to have the same specificity or sensitivity to effectively determine the potential value of a specific therapeutic regimen.

Estrogen receptive neurons in the preoptic area of the rat are postsynaptic targets of a sexually dimorphic enkephalinergic fiber plexus.

The periventricular preoptic area (pePOA) is a sexually dimorphic component of the rat forebrain that contains a sexually dimorphic Met-enkephalin immunoreactive (ENK-ir) fiber plexus. This plexus is especially dense in the female while only scattered ENK-ir fibers are present in the pePOA of the male. Abundant estrogen receptive neurons are located in the pePOA of both the female and male. This experiment was conducted to determine if estrogen receptive neurons in the pePOA are postsynaptic targets of ENK-ir terminals. Double label ultrastructural localization of estrogen receptor (ER)-ir neurons and ENK-ir fibers was performed using the chromogens 3,3',5,5'-tetramethylbenzidine (TMB) and diaminobenzidine tetrahydrochloride (DAB), respectively. TMB-stained ER-ir neurons contained electron dense crystalline spicules located predominantly in their nuclei. Flocculent DAB reaction product was distributed over membraneous structures in ENK-ir fibers and terminals. Numerous ER-ir neurons were present in the pePOA of the male and female. In females, many ENK-ir terminals, both synaptic and non-synaptic, contacted the perikarya of ER-ir neurons. In contrast, many fewer ENK-ir terminals made contact on ER-ir neurons in the male. Thus, these results provide morphological evidence that ENK-ir neurons can regulate ER-ir neurons in the pePOA. Moreover, because expression of the ENK-ir pePOA fiber plexus is estrogen-sensitive in the female, these results suggest strongly that estrogen may regulate these neurons both pre- and postsynaptically. Finally, these results provide additional evidence for the involvement of the sexually dimorphic pePOA ENK-ir fibers plexus in the control of estrogen-mediated function in the female.

Estrous cycle-associated axosomatic synaptic plasticity upon estrogen receptive neurons in the rat preoptic area.

This study examined the hypothesis that synaptic relationships change on a daily basis in the anterior preoptic area of the intact, cycling female rat. Specifically, the anteroventral periventricular nucleus (AVPv) was chosen for analysis due to its abundant estrogen- and progesterone-receptive neurons and its critical role in the control of gonadotropin secretion. Ultrastructural analysis revealed that in the 24 h between proestrus and estrus, there was a 39% increase in axosomatic synapses upon AVPv neurons. In the subsequent 24 h to metestrus, the number of synapses decreased by 22%. Additional data showed that ovariectomy resulted in more axosomatic synapses in the AVPv relative to proestrus (46%) and metestrus (35%). Another component of the study investigated synaptic relationships on estrogen receptor-immunoreactive (ER-ir) and non-ER-ir neurons across the estrous cycle. ER-ir neurons received significantly more synaptic input at proestrus and estrus than did non-ER-ir neurons. At metestrus and following ovariectomy, no significant differences were present. The present study indicates that estrous cycle-associated synaptic plasticity occurs in the AVPv and lends further support to the critical role of this nucleus in regulation of gonadotropin secretion. Moreover, it provides the first evidence that ER- and non-ER-ir neurons are differentially innervated.

Estrogen-receptive neurons in the anteroventral periventricular nucleus are synaptic targets of the suprachiasmatic nucleus and peri-suprachiasmatic region.

The anteroventral periventricular nucleus (AVPv) in the rat preoptic area is a key site underlying control of the steroid dependent preovulatory gonadotropin surge. Estrogen and progesterone receptor-containing neurons in the preoptic/hypothalamic continuum, particularly those in the AVPv, are believed to transduce steroidal signals and, in turn convey this information to the LHRH system, which lacks steroid receptors. In addition to the influence of the gonadal steroids, the precise timing of the preovulatory gonadotropin surge is believed to be regulated by the hypothalamic suprachiasmatic nucleus (SCN). The SCN and peri-SCN neurons send efferent projections rostrally to the anterior preoptic area suggesting that circadian signals are communicated synaptically to steroid-responsive neurons in the AVPv. To test this hypothesis, ultrastructural double label immunocytochemistry was conducted to determine whether SCN efferents contact estrogen receptor-immunoreactive neurons in the AVPv. Brain sections with SCN injections of phaseolus vulgaris leucoagglutinin (PHA-L) were immunostained for estrogen receptors and PHA-L. Light and electron microscopic data show that the anterior preoptic area received robust PHA-L-immunoreactive efferents from SCN neurons and immediately adjacent subparaventricular zone. In particular, the AVPv contained abundant labeled fibers and terminal boutons. Ultrastructurally, SCN- and subparaventricular zone-derived terminals synaptically contacted the perikaryon of many estrogen receptor-immunoreactive neurons in the AVPv. The perikarya of unlabeled neurons were also contacted, but the majority of the labeled contacts were observed upon neuronal processes. These results demonstrate that estrogen responsive AVPv neurons are regulated by SCN efferents. Furthermore, the present data provide strong support to the idea of collective control of pituitary gonadotropin release by steroid sensitive and circadian signal neural pathways.

Molecular bone morphometry.

Quantitative histomorphometric assessment of bone biopsies represents a powerful and informative method for the study of metabolic bone diseases. It is the gold standard against which the noninvasive "diagnostic" markers of bone metabolism as well as newly available therapeutic modalities are tested. With the rapid progress in technology of molecular biology, identification of systemic and local biomolecules known to regulate bone metabolism can now be achieved. The study of localization, levels of expression, and synthesis of these factors in bone and its microenvironment is possible through applications of in situ hybridization histochemistry (ISHH) and immunohistochemistry (IHC). Application of ISHH allows study of specific mRNA expression. IHC determines the presence and distribution of target protein in cells. These two methodologies provide the link between the cellular processes of mRNA transcription and translation to the working protein. Combining the established bone histomorphometric techniques with ISHH and IHC elevates the study of bone to new heights, i.e., cellular and molecular mechanistic issues can now be studied.