Hypothalamic Neurosecretory Neurons Research Articles

A versatile transcription factor: Multiple roles of orthopedia a (otpa) beyond its restricted localization in dopaminergic systems of developing and adult zebrafish (Danio rerio) brains.

Many transcription factors boost neural development and differentiation in specific directions and serve for identifying similar or homologous structures across species. The expression of Orthopedia (Otp) is critical for the development of certain cell groups along the vertebrate neuraxis, for example, the medial amygdala or hypothalamic neurosecretory neurons. Therefore, the primary focus of the present study is the distribution of Orthopedia a (Otpa) in the larval and adult zebrafish (Danio rerio) brain. Since Otpa is also critical for the development of zebrafish basal diencephalic dopaminergic cells, colocalization of Otpa with the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) is studied. Cellular colocalization of Otpa and dopamine is only seen in magnocellular neurons of the periventricular posterior tubercular nucleus and in the posterior tuberal nucleus. Otpa-positive cells occur in many additional structures along the zebrafish neuraxis, from the secondary prosencephalon down to the hindbrain. Furthermore, Otpa expression is studied in shh-GFP and islet1-GFP transgenic zebrafish. Otpa-positive cells only express shh in dopaminergic magnocellular periventricular posterior tubercular cells, and only colocalize with islet1-GFP in the ventral zone and prerecesscaudal periventricular hypothalamic zone and the perilemniscal nucleus. The scarcity of cellular colocalization of Otpa in islet1-GFP cells indicates that the Shh-islet1 neurogenetic pathway is not active in most Otpa-expressing domains. Our analysis reveals detailed correspondences between mouse and zebrafish forebrain territories including the zebrafish intermediate nucleus of the ventral telencephalon and the mouse medial amygdala. The zebrafish preoptic Otpa-positive domain represents the neuropeptidergic supraopto-paraventricular region of all tetrapods. Otpa domains in the zebrafish basal plate hypothalamus suggest that the ventral periventricular hypothalamic zone corresponds to the otp-expressing basal hypothalamic tuberal field in the mouse. Furthermore, the mouse otp domain in the mammillary hypothalamus compares partly to our Otpa-positive domain in the prerecess caudal periventricular hypothalamic zone (Hc-a).

An increased extrasynaptic NMDA tone inhibits A-type K+ current and increases excitability of hypothalamic neurosecretory neurons in hypertensive rats.

A functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A-type K+ current (IA ) influences homeostatic firing responses of magnocellular neurosecretory cells (MNCs) to a physiological challenge. However, whether an altered eNMDAR-IA coupling also contributes to exacerbated MNC activity and neurohumoral activation during disease states is unknown. We show that activation of eNMDARs by exogenously applied NMDA inhibited IA in MNCs obtained from sham, but not in MNCs from renovascular hypertensive (RVH) rats. Neither the magnitude of the exogenously evoked NMDA current nor the expression of NMDAR subunits were altered in RVH rats. Conversely, we found that a larger endogenous glutamate tone, which was not due to blunted glutamate transport activity, led to the sustained activation of eNMDARs that tonically inhibited IA , contributing in turn to higher firing activity in RVH rats. Our studies show that exacerbated activation of eNMDARs by endogenous glutamate contributes to tonic inhibition of IA and enhanced MNC excitability in RVH rats. We recently showed that a functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A-type K+ current (IA ) influences the firing activity of hypothalamic magnocellular neurosecretory neurons (MNCs), as well as homeostatic adaptive responses to a physiological challenge. Here, we aimed to determine whether changes in the eNMDAR-IA coupling also contributed to exacerbated MNC activity during disease states. We used a combination of patch-clamp electrophysiology and real-time PCR in MNCs in sham and renovascular hypertensive (RVH) rats. Activation of eNMDARs by exogenously applied NMDA inhibited IA in sham rats, but this effect was largely blunted in RVH rats. The blunted response was not due to changes in eNMDAR expression and/or function, since neither NMDA current magnitude or reversal potential, nor the levels of NR1-NR2A-D subunit expression were altered in RVH rats. Conversely, we found a larger endogenous glutamate tone, resulting in the sustained activation of eNMDARs that tonically inhibited IA and contributed also to higher ongoing firing activity in RVH rats. The enhanced endogenous glutamate tone in RVH rats was not due to blunted glutamate transporter activity. Rather, a higher transporter activity was observed, which possibly acted as a compensatory mechanism in the face of the elevated endogenous tone. In summary, our studies indicate that an elevated endogenous glutamate tone results in an exacerbated activation of eNMDARs, which in turn contributes to diminished IA magnitude and increased firing activity of MNCs from hypertensive rats.

Structural Reconstruction of the Perivascular Space in the Adult Mouse Neurohypophysis During an Osmotic Stimulation.

Oxytocin (OXT) and arginine vasopressin (AVP) neuropeptides in the neurohypophysis (NH) control lactation and body fluid homeostasis, respectively. Hypothalamic neurosecretory neurones project their axons from the supraoptic and paraventricular nuclei to the NH to make contact with the vascular surface and release OXT and AVP. The neurohypophysial vascular structure is unique because it has a wide perivascular space between the inner and outer basement membranes. However, the significance of this unique vascular structure remains unclear; therefore, we aimed to determine the functional significance of the perivascular space and its activity-dependent changes during salt loading in adult mice. The results obtained revealed that pericytes were the main resident cells and defined the profile of the perivascular space. Moreover, pericytes sometimes extended their cellular processes or 'perivascular protrusions' into neurohypophysial parenchyma between axonal terminals. The vascular permeability of low-molecular-weight (LMW) molecules was higher at perivascular protrusions than at the smooth vascular surface. Axonal terminals containing OXT and AVP were more likely to localise at perivascular protrusions than at the smooth vascular surface. Chronic salt loading with 2% NaCl significantly induced prominent changes in the shape of pericytes and also increased the number of perivascular protrusions and the surface area of the perivascular space together with elevations in the vascular permeability of LMW molecules. Collectively, these results indicate that the perivascular space of the NH acts as the main diffusion route for OXT and AVP and, in addition, changes in the shape of pericytes and perivascular reconstruction occur in response to an increased demand for neuropeptide release.

Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition.

Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus–pituitary–somatotropic hypoplasia. The unbiased smpd3−/− mouse mutant and derived smpd3−/− primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3−/− mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition.

Abstract 054: Prorenin Stimulates Firing Activity in Hypothalamic Neurons via Angiotensin II-Dependent and -Independent Mechanisms

The brain RAS plays a central role in cardiovascular homeostasis by modulating sympathetic nerve activity and hormone secretion. Still, the relevance of intrinsic brain RAS function is questioned because renin levels are too low to have an impact on angiotensin II (AngII) formation. The discovery of the prorenin receptor (PRR) brought new support to the brain RAS involvement in neurohumoral regulation. However, the mechanisms underlying PRR-mediated actions remain unknown. We used whole cell patch-clamp electrophysiology in hypothalamic slices to examine the effects of prorenin (PR) on the excitability of magnocellular neurosecretory neurons (MCNs) of the supraoptic nucleus and parvocellular RVLM-projecting presympathetic neurons of the paraventricular nucleus (PVN-RVLM), which play integral roles in cardiovascular control, and have been implicated in the pathophysiology of neurogenic hypertension. PR application (2.5nM) increased the firing activity of both MCNs (basal 1.3±0.4 Hz vs PR 3.3±0.7 Hz; p=0.0086) and parvocellular neurons (basal 0.5±0.1 Hz vs PR 2.1±0.4 Hz; p=0.0006), including a subset of PVN-RVLM neurons (basal 0.5±0.1 Hz vs PR 1.4±0.4 Hz; p=0.048). In MCNs, this effect was blocked by a PRR antagonist ( PRO20 ; 250nM; basal 1.9±0.6 Hz vs PRO20 1.3±0.5 Hz; p=0.017), but persisted in the presence of the AT1 receptor blocker, losartan (50μM; basal 0.8±0.2 Hz vs losartan 2.3±0.5 Hz; p=0.0018). Conversely, PR effects on PVN neurons persisted with PRO20 (basal 0.9±0.2 Hz vs PRO20 1.6±0.3 Hz; p=0.013), but were largely (~75%) blunted with losartan (basal 0.2±0.04 Hz vs losartan 0.6±0.1 Hz; p=0.0193; Δ basal 1.6±0.4 Hz vs Δ losartan 0.4±0.1 Hz; p=0.0175). These results suggest that while PR actions are primarily PRR-mediated in MCNs, they are predominantly AngII-dependent in PVN cells. Lastly, PR effects were abolished in both cell types by dialyzing neurons with the Ca 2+ chelator BAPTA (10mM; MCNs: basal 0.6 ± 0.1 Hz vs BAPTA 0.3 ± 0.1 Hz; p=0.0277; PVN: basal 0.4±0.05 Hz vs BAPTA 0.5±0.2 Hz; p=0.6468). Our results show for the first time that PR stimulates firing activity in both hypothalamic neurosecretory and presympathetic neurons, and support distinct AngII-independent and dependent signaling mechanisms in each neuronal population.

In vivo processing and release into the circulation of GFP fusion protein in arginine vasopressin enhanced GFP transgenic rats: response to osmotic stimulation.

Arginine vasopressin (AVP) is a neurohypophysial hormone synthesized as a part of a prepropeptide precursor containing the signal peptide, AVP hormone, AVP-associated neurophysin II and copeptin in the hypothalamic neurosecretory neurons. A transgenic (Tg) rat line expressing the AVP-eGFP fusion gene has been generated. To establish the AVP-eGFP Tg rat as a unique model for an analysis of AVP dynamics in vivo, we first examined the in vivo molecular dynamics of the AVP-eGFP fusion gene, and then the release of GFP in response to physiological stimuli. Double immunoelectron microscopy demonstrated that GFP was specifically localized in neurosecretory vesicles of AVP neurons in this Tg rat. After stimulation of the posterior pituitary with high potassium we demonstrated the exocytosis of AVP neurosecretory vesicles containing GFP at the ultrastructural level. Biochemical analyses indicated that the AVP-eGFP fusion gene is subjected to in vivo post-translational modifications like the native AVP gene, and is packaged into neurosecretory vesicles as a fusion protein: copeptin1-14 -GFP. Moreover, GFP release into the circulating blood appeared to be augmented after osmotic stimulation, like native AVP. Thus, here we show for the first time the in vivo molecular processing of the AVP-eGFP fusion gene and stimulated secretion after osmotic stimulation in rats. Because GFP behaved like native AVP in the hypothalamo-pituitary axis, and in particular was released into the circulation in response to a physiological stimulus, the AVP-eGFP Tg rat model appears to be a powerful tool for analyzing neuroendocrine systems at the organismal level.

A Novel Pathway Regulates Thyroid Hormone Availability in Rat and Human Hypothalamic Neurosecretory Neurons

Hypothalamic neurosecretory systems are fundamental regulatory circuits influenced by thyroid hormone. Monocarboxylate-transporter-8 (MCT8)-mediated uptake of thyroid hormone followed by type 3 deiodinase (D3)-catalyzed inactivation represent limiting regulatory factors of neuronal T3 availability. In the present study we addressed the localization and subcellular distribution of D3 and MCT8 in neurosecretory neurons and addressed D3 function in their axons. Intense D3-immunoreactivity was observed in axon varicosities in the external zone of the rat median eminence and the neurohaemal zone of the human infundibulum containing axon terminals of hypophysiotropic parvocellular neurons. Immuno-electronmicroscopy localized D3 to dense-core vesicles in hypophysiotropic axon varicosities. N-STORM-superresolution-microscopy detected the active center containing C-terminus of D3 at the outer surface of these organelles. Double-labeling immunofluorescent confocal microscopy revealed that D3 is present in the majority of GnRH, CRH and GHRH axons but only in a minority of TRH axons, while absent from somatostatin-containing neurons. Bimolecular-Fluorescence-Complementation identified D3 homodimers, a prerequisite for D3 activity, in processes of GT1-7 cells. Furthermore, T3-inducible D3 catalytic activity was detected in the rat median eminence. Triple-labeling immunofluorescence and immuno-electronmicroscopy revealed the presence of MCT8 on the surface of the vast majority of all types of hypophysiotropic terminals. The presence of MCT8 was also demonstrated on the axon terminals in the neurohaemal zone of the human infundibulum. The unexpected role of hypophysiotropic axons in fine-tuned regulation of T3 availability in these cells via MCT8-mediated transport and D3-catalyzed inactivation may represent a novel regulatory core mechanism for metabolism, growth, stress and reproduction in rodents and humans.

Altered astrocyte glutamate transporter regulation of hypothalamic neurosecretory neurons in heart failure rats

Neurohumoral activation, which includes augmented plasma levels of the neurohormone vasopressin (VP), is a common finding in heart failure (HF) that contributes to morbidity and mortality in this disease. While an increased activation of magnocellular neurosecretory cells (MNCs) and enhanced glutamate function in HF is well documented, the precise underlying mechanisms remain to be elucidated. Here, we combined electrophysiology and protein measurements to determine whether altered glial glutamate transporter function and/or expression occurs in the hypothalamic supraoptic nucleus (SON) during HF. Patch-clamp recordings obtained from MNCs in brain slices show that pharmacological blockade of astrocyte glutamate transporter 1 (GLT1) function [500 μM dihydrokainate (DHK)], resulted in a persistent N-methyl-D-aspartate receptor (NMDAR)-mediated inward current (tonic I(NMDA)) in sham rats, an effect that was significantly smaller in MNCs from HF rats. In addition, we found a diminished GLT1 protein content in plasma membrane (but not cytosolic) fractions of SON punches in HF rats. Conversely, astrocyte GLAST expression was significantly higher in the SON of HF rats, while nonselective blockade of glutamate transport activity (100 μM TBOA) evoked an enhanced tonic I(NMDA) activation in HF rats. Steady-state activation of NMDARs by extracellular glutamate levels was diminished during HF. Taken together, these results support a shift in the relative expression and function of two major glial glutamate transporters (from GLT1 to GLAST predominance) during HF. This shift may act as a compensatory mechanism to preserve an adequate basal glutamate uptake level in the face of an enhanced glutamatergic afferent activity in HF rats.

(Re-)activation of neurons in aging and dementia: Lessons from the hypothalamus

Our hypothesis is that there is ‘wear and tear’ in the brain, which is the basis of the process of aging, but that stimulation of brain function may slow down brain aging and diminish the risk for neurodegenerative diseases such as Alzheimer's disease (AD), possibly by activating repair mechanisms. Evidence supporting this hypothesis is presented in this review. During normal aging and in AD, cell loss is not as prominent a phenomenon as is often presumed. In fact, unaltered neuronal numbers have been reported in many brain areas in AD, e.g. in the nucleus basalis of Meynert (NBM) where the number of large neurons decreases while that of small neurons increases. Decreased neuronal activity is an essential characteristic of AD, and a substantial decrease of cerebral glucose metabolism may even precede cognitive impairments. Some hypothalamic neurons remain intact and active during the process of aging, others become even hyperactive, which may lead to disorders. Arginine vasopressin (AVP) levels were found to be higher in the elderly than in young subjects. There is an age-related, sex-dependent activation of the AVP neurons in the supraoptic nucleus (SON) and in the paraventricular nucleus (PVN), which may be the basis of analogous changes in the prevalence of hypertension and hyponatraemia in the elderly. No significant functional loss of magnocellular hypothalamic neurosecretory neurons were found in the SON or PVN in AD. The activity of the corticotrophin releasing hormone (CRH) neurons in the hypothalamic PVN is the basis for the activity of the hypothalamo-pituitary-adrenal (HPA) axis, which is activated during aging in a sex-dependent way, and even more activated in AD. The activated HPA axis is a risk for depression. Environmental stimulation increases brain reserve. An increase in time spent on intellectual activities was associated with a significant decrease in probability to get AD, and occupation has even a stronger indication of diminished risk for dementia. A series of observations showed that a dysfunctional clock may underlie the disordered rhythms in AD. Additional bright light improved the rest–activity rhythms, while giving bright light and/or melatonin to AD patients ameliorated the progression of cognitive and noncognitive symptoms. This implies that neurons affected by AD can still be reactivated if the right stimuli are applied. Unknown diffusible factors from the neural stem cells improve the survival of aged and degenerating neurons in postmortem human brain slice cultures. Gene therapy with nerve growth factor aimed at the NBM showed metabolic activation of various brain regions. A microarray study of the prefrontal cortex in the course of AD revealed an increased expression of genes related to synaptic activity and changes in plasticity during the very early pre-symptomatic stages, which is proposed to represent a coping mechanism against increased soluble β-amyloid levels. In brief, these examples of the ‘use it or lose it’ principle during the course of aging or AD now provide novel targets for the development of therapeutic strategies aiming at the prevention and treatment of AD.

Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction

In polyglutamine diseases including Huntington's disease (HD), mutant proteins containing expanded polyglutamine stretches form nuclear aggregates in neurons. Although analysis of their disease models suggested a significance of transcriptional dysregulation in these diseases, how it mediates the specific neuronal cell dysfunction remains obscure. Here we performed a comprehensive analysis of altered DNA binding of multiple transcription factors using R6/2 HD model mice brains that express an N-terminal fragment of mutant huntingtin (mutant Nhtt). We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons. We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides. In contrast to Brn-2, its functionally related protein, Brn-1, was not sequestered by mutant Nhtt but was upregulated in R6/2 brain, except in hypothalamus. Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

Dendritic synthesis and release of the neuropeptide galanin: Morphological evidence from studies on rat locus coeruleus neurons

Dendritic synthesis and release of neuropeptides have been demonstrated in hypothalamic neurosecretory neurons. Here we tested whether this mechanism may be applicable to galanin in rat locus coeruleus (LC) neurons. Light and electron microscopic immunocytochemistry demonstrated the occurrence of galanin peptide in secretory granules within dendritic processes. In situ hybridization at the light and electron microscopic level showed galanin mRNA in these LC processes. Moreover, the levels of the transcript and its extension into the dendrites were significantly increased by reserpine treatment. Finally, evidence for exocytotic release from large dense-core vesicles in LC dendrites was obtained by using tannic acid-treated slices. Taken together, these results indicate that galanin can be synthesized in, and possibly released from, LC. These data support a key function of the neuropeptide galanin as an auto-paracrine local modulator in a major, strongly divergent monoaminergic regulatory system.

Estrogen enhances expression of the complement C5a receptor and the C5a-agonist evoked calcium influx in hormone secreting neurons of the hypothalamus

In the present study we examined presence of the complement C5a receptor (C5aR) in hypothalamic neurosecretory neurons of the rodent brain and effect of estrogen on C5aR expression. Whole cell patch clamp measurements revealed that magnocellular neurons in the supraoptic and paraventricular nuclei of hypothalamic slices of the rats responded to the C5aR-agonist PL37-MAP peptide with calcium ion current pulses. Gonadotropin-releasing hormone (GnRH) producing neurons in slices of the preoptic area of the mice also reacted to the peptide treatment with inward calcium current. PL37-MAP was able to evoke the inward ion current of GnRH neurons in slices from ovariectomized animals. The amplitude of the inward pulses became higher in slices obtained from 17β-estradiol (E2) substituted mice. Calcium imaging experiments demonstrated that PL37-MAP increased the intracellular calcium content in the culture of the GnRH-producing GT1-7 cell line in a concentration-dependent manner. Calcium imaging also showed that E2 pretreatment elevated the PL37-MAP evoked increase of the intracellular calcium content in the GT1-7 cells. The estrogen receptor blocker Faslodex in the medium prevented the E2-evoked increase of the PL37-MAP-triggered elevation of the intracellular calcium content in the GT1-7 cells demonstrating that the effect of E2 might be related to the presence of estrogen receptor. Real-time PCR experiments revealed that E2 increased the expression of C5aR mRNA in GT1-7 neurons, suggesting that an increased C5aR synthesis could be involved in the estrogenic modulation of calcium response. These data indicate that hypothalamic neuroendocrine neurons can integrate immune and neuroendocrine functions. Our results may serve a better understanding of the inflammatory and neurodegeneratory diseases of the hypothalamus and the related neuroendocrine and autonomic compensatory responses.

Changes in vasotocin immunoreactivity of paraventricular nuclei and adrenal function of Japanese quail in relation to different phases of sexual development

In relevance to osmoregulatory and reproductive functions, activity of hypothalamic neurosecretory neurons may also vary seasonally. The current study was performed to determine annual changes in ir-AVT neurons of hypothalamus and adrenal gland function. We examined changes in ir-AVT neuron by immunohistochemical method and plasma testosterone was measured by enzyme immunoassay. The steroidogenic interrenal activity was studied by histological and biochemical methods. Birds were sampled in February (quiescent), April (recrudescent), June (breeding) and November (regressive). A significant and gradual increase in the number of ir-AVT neurons was observed from quiescent to breeding phase which decreased during regressive phase of annual gonadal cycle. The gradual increase in ir-AVT neurons along with annual gonadal activity of quail were accompanied by increase in plasma levels of testosterone. These results indicate a functional interaction between sex steroid and AVT synthesizing neurons. Adrenal activity (as judged by weight, ascorbic acid content, cortical cord width and cortico-medullary ratio) was also maximum during breeding phase. It is thus postulated that domesticated quail when exposed to natural day length (NDL), exhibits seasonal/annual cyclicity in vastocinergic activity and adrenal function which may be due to difference in sex steroid hormone.

Neutral Sphingomyelinase (SMPD3) Deficiency Causes a Novel Form of Chondrodysplasia and Dwarfism That Is Rescued by Col2A1-Driven smpd3 Transgene Expression

Neutral sphingomyelinase SMPD3 (nSMase2), a sphingomyelin phosphodiesterase, resides in the Golgi apparatus and is ubiquitously expressed. Gene ablation of smpd3 causes a generalized prolongation of the cell cycle that leads to late embryonic and juvenile hypoplasia because of the SMPD3 deficiency in hypothalamic neurosecretory neurons. We show here that this novel form of combined pituitary hormone deficiency is characterized by the perturbation of the hypothalamus-pituitary growth axis, associated with retarded chondrocyte development and enchondral ossification in the epiphyseal growth plate. To study the contribution by combined pituitary hormone deficiency and by the local SMPD3 deficiency in the epiphyseal growth plate to the skeletal phenotype, we introduced the full-length smpd3 cDNA transgene under the control of the chondrocyte-specific promoter Col2a1. A complete rescue of the smpd3(-/-) mouse from severe short-limbed skeletal dysplasia was achieved. The smpd3(-/-) mouse shares its dwarf and chondrodysplasia phenotype with the most common form of human achondrodysplasia, linked to the fibroblast-growth-factor receptor 3 locus, not linked to deficits in the hypothalamic-pituitary epiphyseal growth plate axis. The rescue of smpd3 in vivo has implications for future research into dwarfism and, particularly, growth and development of the skeletal system and for current screening and future treatment of combined dwarfism and chondrodysplasia.

Glutamatergic innervation of the hypothalamic median eminence and posterior pituitary of the rat

Recent studies have localized the glutamatergic cell marker type-2 vesicular glutamate transporter (VGLUT2) to distinct peptidergic neurosecretory systems that regulate hypophysial functions in rats. The present studies were aimed to map the neuronal sources of VGLUT2 in the median eminence and the posterior pituitary, the main terminal fields of hypothalamic neurosecretory neurons. Neurons innervating these regions were identified by the uptake of the retrograde tract-tracer Fluoro-Gold (FG) from the systemic circulation, whereas glutamatergic perikarya of the hypothalamus were visualized via the radioisotopic in situ hybridization detection of VGLUT2 mRNA. The results of dual-labeling studies established that the majority of neurons accumulating FG and also expressing VGLUT2 mRNA were located within the paraventricular, periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area. In contrast, only few FG-accumulating cells exhibited VGLUT2 mRNA signal in the arcuate nucleus. Dual-label immunofluorescent studies of the median eminence and posterior pituitary to determine the subcellular location of VGLUT2, revealed the association of VGLUT2 immunoreactivity with SV2 protein, a marker for small clear vesicles in neurosecretory endings. Electron microscopic studies using pre-embedding colloidal gold labeling confirmed the localization of VGLUT2 in small clear synaptic vesicles. These data suggest that neurosecretory neurons located mainly within the paraventricular, anterior periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area secrete glutamate into the fenestrated vessels of the median eminence and posterior pituitary. The functional aspects of the putative neuropeptide/glutamate co-release from neuroendocrine terminals remain to be elucidated.

Neutral sphingomyelinase 2 ( smpd3 ) in the control of postnatal growth and development

Neutral sphingomyelinases sphingomyelin phosphodiesterase (SMPD)2 and -3 hydrolyze sphingomyelin to phosphocholine and ceramide. smpd2 is expressed ubiquitously, and smpd3 is expressed predominantly in neurons of the CNS. Their activation and the functions of the released ceramides have been associated with signaling pathways in cell growth, differentiation, and apoptosis. However, these cellular responses remain poorly understood. Here we describe the generation and characterization of the smpd3(-/-) and smpd2(-/-)smpd3(-/-) double mutant mouse, which proved to be devoid of neutral sphingomyelinase activity. SMPD3 plays a pivotal role in the control of late embryonic and postnatal development: the smpd3-null mouse develops a novel form of dwarfism and delayed puberty as part of a hypothalamus-induced combined pituitary hormone deficiency. Our studies suggest that SMPD3 is segregated into detergent-resistant subdomains of Golgi membranes of hypothalamic neurosecretory neurons, where its transient activation modifies the lipid bilayer, an essential step in the Golgi secretory pathway. The smpd3(-/-) mouse might mimic a form of human combined pituitary hormone deficiency.

Ecology meets endocrinology: environmental sex determination in fishes.

Van Valen (1973) characterized evolution as the control of development by ecology. Sex determination in fishes provides some clear examples of this “control” in operation. Teleost fishes show a remarkable variety of sex determination and differentiation patterns. These range from systems in which sex is determined by sex chromosomes, as in birds and mammals, to simultaneous hermaphrodites that alternate spawning as a female and male on a second to second basis. This extraordinary flexibility may result from a combined lack of developmental constraint on reproductive structures in many lineages and selection for sexual lability in the face of environmental unpredictability. This review addresses environmental influences on sex determination and differentiation in fishes. There is a variety of documented environmental influences on sex determination (ESD) in fishes. We focus here on two classes of examples where the key environmental cues are of clear ecological relevance, the effects appear especially likely to be important as a normal part of the life history, and where there is evidence suggesting the sexual patterns observed represent adaptations that increase individual fitness. These classes are sex determination that is controlled by social interactions (behavioral sex determination [BSD]) (Crews 1993) and temperaturedependent sex determination (TSD). Sex determination controlled by social influences can occur before or after sexual maturation but appears to maximize the expected reproductive success of individuals in both cases. Here we first address BSD and then TSD in fishes. For each pattern of sex determination, we discuss selection pressures that appear to favor these patterns, examples of each, and what is known regarding the underlying physiological mechanisms. For more comprehensive and general reviews of patterns and mechanisms of sex determination in fishes, the reader is referred to several excellent reviews (Nakamura et al. 1998; Baroiller et al. 1999; Baroiller and D’Cotta 2001; Piferrer 2001). The major focus in studies of physiological mediation of teleost sex determination is what is referred to by endocrinologists as the hypothalamo-pituitary-gonadal (HPG) axis (Fig. 1). This axis consists primarily of hypothalamic neurosecretory neurons producing gonadotropin-releasing hormone (GnRH), gonadotropins produced in and released from the pituitary gland (GtH I and GtH II), and the gonad as the major site of steroid biosynthesis with its steroid metabolizing enzymes, steroid hormone receptors, and a variety of other proteins that mediate steroid hormone action. One steroid biosynthetic enzyme that has been a particularly fruitful focus in correlative and manipulative studies of vertebrate sex determination is cytochrome P-450 aromatase. This enzyme catalyzes the conversion of androgens to estrogens (primarily testosterone to estradiol-17 ). Aromatase expression correlates with female determination in a variety of vertebrates, and aromatasespecific antagonists can block female development in fishes, amphibians, reptiles, and birds (Elbrecht and Smith 1992; Lance and Bogart 1992; Crews et al. 1994; Wennstrom and Crews 1995; Kitano et al. 1999; D’Cotta et al. 2001). Estradiol-17 plays a central role in female reproductive physiology in fishes, whereas the androgen 11-ketotestosterone (11-KT) is crucial to gamete maturation and the expression of secondary sexual characteristics in males (Borg 1994; Brantley et al. 1993). Importantly, testosterone levels often do not differ between male and female fishes or are higher in females (Borg 1994). Because of the central role of aromatase in the biosynthesis of estrogens, it will be a focus in consideration of mechanisms by which environmental information leads to sex determination responses. More generally, our understanding of vertebrate sexual function indicates the HPG axis plays the key role in transducing environmental information into gonadal determination, differentiation, and maturation events. A general theme of this review is where and how this transduction may occur in the HPG axis.

Space flight affects magnocellular supraoptic neurons of young prepuberal rats: transient and permanent effects

Effects of microgravity on postural control and volume of extracellular fluids as well as stress associated with space flight may affect the function of hypothalamic neurosecretory neurons. Since environmental modifications in young animals may result in permanent alterations in neuroendocrine function, the present study was designed to determine the effect of a space flight on oxytocinergic and vasopressinergic magnocellular hypothalamic neurons of prepuberal rats. Fifteen-day-old Sprague–Dawley female rats were flown aboard the Space Shuttle Columbia (STS-90, Neurolab mission, experiment 150) for 16 days. Age-matched litters remained on the ground in cages similar to those of the flight animals. Six animals from each group were killed on the day of landing and eight animals from each group were maintained under standard vivarium conditions and killed 18 weeks after landing. Several signs of enhanced transcriptional and biosynthetic activity were observed in magnocellular supraoptic neurons of flight animals on the day of landing compared to control animals. These include increased c-Fos expression, larger nucleoli and cytoplasm, and higher volume occupied in the neuronal perikaryon by mitochondriae, endoplasmic reticulum, Golgi apparatus, lysosomes and cytoplasmic inclusions known as nematosomes. In contrast, the volume occupied by neurosecretory vesicles in the supraoptic neuronal perikarya was significantly decreased in flight rats. This decrease was associated with a significant decrease in oxytocin and vasopressin immunoreactive levels, suggestive of an increased hormonal release. Vasopressin levels, cytoplasmic volume and c-Fos expression returned to control levels by 18 weeks after landing. These reversible effects were probably associated to osmotic stimuli resulting from modifications in the volume and distribution of extracellular fluids and plasma during flight and landing. However, oxytocin levels were still reduced at 18 weeks after landing in flight animals compared to controls. This indicates that space flight during prepuberal age may induce irreversible modifications in the regulation of oxytocinergic neurons, which in turn may result in permanent endocrine and behavioral impairments.

Activation of translation in pituitary gonadotrope cells by gonadotropin-releasing hormone.

The neuropeptide GnRH is a central regulator of mammalian reproductive function produced by a dispersed population of hypothalamic neurosecretory neurons. The principal action of GnRH is to regulate release of the gonadotropins, LH and FSH, by the gonadotrope cells of the anterior pituitary. Using a cultured cell model of mouse pituitary gonadotrope cells, alphaT3-1 cells, we present evidence that GnRH stimulation of alphaT3-1 cells results in an increase in cap-dependent mRNA translation. GnRH receptor activation results in increased protein synthesis through a regulator of mRNA translation initiation, eukaryotic translation initiation factor 4E-binding protein, known as 4EBP or PHAS (protein, heat, and acid stable). Although the GnRH receptor is a member of the rhodopsin-like family of G protein-linked receptors, we show that activation of translation proceeds through a signaling pathway previously described for receptor tyrosine kinases. Stimulation of translation by GnRH is protein kinase C and Ras dependent and sensitive to rapamycin. Furthermore, GnRH may also regulate the cell cycle in alphaT3-1 cells. The activation of a signaling pathway that regulates both protein synthesis and cell cycle suggests that GnRH may have a significant role in the maintenance of the pituitary gonadotrope population in addition to directing the release of gonadotropins.

Chapter 1.2 Functions of the perikaryon and dendrites in magnocellular vasopressin-secreting neurons: New insights from ultrastructural studies

Magnocellular hypothalamic neurosecretory neurons secreting vasopressin or oxytocin provide a robust model system for the investigation and understanding of many aspects of peptidergic neuronal function. Many of their functions and the cellular organelles involved are well understood. However, recent ultrastructural studies have thrown new light on various aspects of magnocellular neurosecretory function which have not previously received much attention. This review concerns two of these: the effects of mutations in the vasopressin gene on the handling of the translated peptide by the rough endoplasmic reticulum; and the role of the magnocellular dendrites in the production, secretion and localisation of peptides. Investigation of the synthesis of proteins derived from vasopressin genes which have undergone various mutations has at the moment provided more answers than questions: Why do some abnormal products accumulate as masses of peptide in the rough endoplasmic reticulum while others do not? Why do accumulations in humans appear to be damaging to the neurons while those in the rat do not? Investigations of the role of dendrites in the production and release of peptides show that the dendrites have all the machinery needed for protein translation and appear to synthesize locally proteins required for dendritic function. Of particular interest is the possibility that various transmitter receptor proteins could be synthesized in the dendrites close to the synapses in which they become localized. Precisely how such membrane proteins are inserted into the synaptic complex is, however, unclear, because the most part of the dendrites lack any form of the Golgi packaging organelle that can be recognised as such either by immunocytochemistry or electron microscopy. Better established is the ability of magnocellular dendrites to secrete either vasopressin or oxytocin in response to a variety of stimuli including sex steroids. This local release of peptide into the magnocellular nuclei has important but as yet incompletely defined effects on the functioning of the neurons.