Brain Regions Hypothalamus Research Articles

Gap Junctions between Neuronal Inputs But Not Gonadotropin-Releasing Hormone Neurons Control Estrous Cycles in the Mouse

The role of gap junctions in the neural control of fertility remains poorly understood. Using acute brain slices from adult GnRH-green fluorescent protein transgenic mice, individual GnRH neurons were filled with a mixture of fluorescent dextran and neurobiotin. No dye transfer was found between any GnRH neurons, although approximately 30% of GnRH neurons exchanged neurobiotin with closely apposed cells. Dual electrophysiological recordings from pairs of GnRH neurons revealed an absence of electrical coupling. Using adult connexin 36 (Cx36)-cyan fluorescent protein transgenic mice, Cx36 was identified in cells within several hypothalamic brain regions, including 64% of preoptic area kisspeptin neurons but not in GnRH neurons. To assess the potential role of Cx36 in non-GnRH neurons within the GnRH neuronal network (i.e. neurons providing afferent inputs to GnRH neurons), a calmodulin kinase IIα-Cre (CKC)-LoxP strategy was used to generate mice with a neuron-specific deletion of Cx36 beginning in the first postnatal week. Mutant female mice exhibited normal puberty onset but disordered estrous cyclicity, although their fecundity was normal as was their estrogen-negative and -positive feedback mechanisms. The effects of adult deletion of Cx36 from neurons were assessed using a tamoxifen-dependent inducible CKC-Cx36 transgenic strategy. Mutant mice exhibited the same reproductive phenotype as the CKC-Cx36 animals. Together these observations demonstrate that there is no gap junctional coupling between GnRH neurons. However, it is apparent that other neurons within the GnRH neuronal network, potentially the preoptic kisspeptin neurons, are dependent on Cx36 gap junctions and that this is critical for normal estrous cyclicity.

Novel galanin receptor subtype specific ligands in feeding regulation

Galanin a 29/30-residue neuropeptide has been implicated in several functions in the central nervous system, including the regulation of food consumption. Galanin and its analogues administered intraventricularly or into the hypothalamic region of brain have been shown to reliably and robustly stimulate the consumption of food in sated rodents. Three galanin receptor subtypes have been isolated, all present in the hypothalamus, but little is known about their specific role in mediating this acute feeding response. Presently, we introduce several novel GalR2 selective agonists and then compare the most selective of these novel GalR2 subtype selective agonists to known GalR1 selective agonist M617 for their ability to stimulate acute consumption of several foods shown to be stimulated by central administration of galanin. GalR1 selective agonist M617 markedly stimulated acute consumption of high-fat milk, but neither GalR2 selective agonist affected either high-fat milk or cookie mash intake. The present results are consistent with the involvement of GalR1 in mediating the acute feeding consumption by galanin and suggest an approach applicable to exploring galanin receptor specificity in normal and abnormal behavior and physiology.

Adolescent exposure to anabolic/androgenic steroids and the neurobiology of offensive aggression: A hypothalamic neural model based on findings in pubertal Syrian hamsters

Considerable public attention has been focused on the issue of youth violence, particularly that associated with drug use. It is documented that anabolic steroid use by teenagers is associated with a higher incidence of aggressive behavior and serious violence, yet little is known about how these drugs produce the aggressive phenotype. Here we discuss work from our laboratory on the relationship between the development and activity of select neurotransmitter systems in the anterior hypothalamus and anabolic steroid-induced offensive aggression using pubertal male Syrian hamsters ( Mesocricetus auratus) as an adolescent animal model, with the express goal of synthesizing these data into an cogent neural model of the developmental adaptations that may underlie anabolic steroid-induced aggressive behavior. Notably, alterations in each of the neural systems identified as important components of the anabolic steroid-induced aggressive response occurred in a sub-division of the anterior hypothalamic brain region we identified as the hamster equivalent of the latero-anterior hypothalamus, indicating that this sub-region of the hypothalamus is an important site of convergence for anabolic steroid-induced neural adaptations that precipitate offensive aggression. Based on these findings we present in this review a neural model to explain the neurochemical regulation of anabolic steroid-induced offensive aggression showing the hypothetical interaction between the arginine vasopressin, serotonin, dopamine, γ-aminobutyric acid, and glutamate neural systems in the anterior hypothalamic brain region.

Activation of extracellular signal-regulated kinases in social behavior circuits during resident-intruder aggression tests

Using a variety of experimental methods, a network of brain areas regulating aggressive behaviors has been identified in several groups of vertebrates. However, aggressive behavior expressed in different contexts is associated with different patterns of activity across hypothalamic and limbic brain regions. Previous studies in rodents demonstrated that short day photoperiods reliably increase both male and female aggression versus long day photoperiods. Here we used immunohistochemistry and western blots to examine the effect of photoperiod on phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK) in male California mice ( Peromyscus californicus) during resident-intruder tests. Phosphorylated ERK (pERK) can alter neuronal activity in the short term and in the long term acts as a transcription factor. In the posterior bed nucleus of the stria terminalis (BNST) males tested in aggression tests had more pERK positive cells when housed in short days but not long days. This result was replicated in western blot analyses from microdissected BNST samples. In the medial amygdala (MEA), immunostaining and western analyses showed that pERK expression also was generally increased in short days. Immunostaining was also used to examine phosphorylation of cyclic AMP response element binding protein (CREB). CREB can be phosphorylated by pERK as well as other kinases and functions primarily as a transcription factor. Intriguingly, aggressive interactions reduced the number of cells stained positive for phosphorylated CREB in the infralimbic cortex, ventral lateral septum and MEA. This effect was observed in mice housed in long days but not short days. Overall, these data suggest that different (but overlapping) networks of aggressive behavior operate under different environmental conditions.

Distribution of Kisspeptin Neurones in the Adult Female Mouse Brain

Kisspeptin-GPR54 signalling is essential for normal reproductive functioning. However, the distribution of kisspeptin neuronal cell bodies and their projections is not well established. The present study aimed to provide a detailed account of kisspeptin neuroanatomy in the mouse brain. Using a polyclonal rabbit antibody AC566, directed towards the final ten C-terminal amino acids of murine kisspeptin, three populations of kisspeptin-expressing cell bodies were identified in the adult female mouse brain. One exists as a dense periventricular continuum of cells within the rostral part of the third ventricle, another is found within the arcuate nucleus, and another is identified as a low-density group of scattered cells within the dorsomedial nucleus and posterior hypothalamus. Kisspeptin-immunoreactive fibres were abundant within the ventral aspect of the lateral septum and within the hypothalamus running in periventricular and ventral retrochiasmatic pathways. Notable exclusions from the kisspeptin fibre innervation were the suprachiasmatic and ventromedial nuclei. Outside of the hypothalamus, a small number of kisspeptin fibres were identified in the bed nucleus of the stria terminalis, subfornical organ, medial amygdala, paraventricular thalamus, periaqueductal grey and locus coerulus. All kisspeptin cell body and fibre immunoreactivity was absent in brain tissue from Kiss1 knockout mice. These observations provide a map of kisspeptin neurones in the mouse brain and indicate that a limited number of mostly medial hypothalamic and lateral septal brain regions are innervated by the three hypothalamic kisspeptin cell populations; the functions of these projections remain to be established.

A Tale of Two Hormones: Role of Leptin and Insulin in Hippocampal Synaptic Function

It is well documented that the endocrine hormones, leptin and insulin provide signals to specific hypothalamic brain regions to regulate energy balance. However, the past decade of research has not only revealed the widespread ex- pression of insulin and leptin receptors in the CNS, but has also identified numerous additional functions of these hor- mones in the brain. In particular, there is growing evidence that these hormones markedly influence hippocampal excita- tory synaptic transmission as well as hippocampal synaptic plasticity. More recent studies have also identified links be- tween dysregulation of leptin and insulin systems and the development of neurodegenerative disorders such as Alz- heimer's disease. Here we review the recent evidence supporting a role for these hormones in modulating hippocampal synaptic function in health and disease.

Altered emotional behaviors in the diabetes mellitus OLETF type 1 congenic rat

GPR10 is a G-protein-coupled receptor expressed in thalamic and hypothalamic brain regions, including the reticular thalamic nucleus (RTN) and periventricular nucleus (Pev), and the endogenous ligand for this receptor, prolactin-releasing peptide (PrRP), has demonstrated regulatory effects on the stress response. We produced a congenic rat by introducing the Dmo1 allele from the OLETF rat which encodes the amino acid sequences of GPR10 with a truncated NH 2-terminus, into the Brown–Norway background. Using receptor autoradiography, we determined a lack of specific [ 125I]PrRP binding in the RTN and Pev of these mutant rats compared to the control rats. Furthermore, intracerebroventricular injection of PrRP did not induce a significant increase of c- fos-like immunoreactivity in the paraventricular nucleus of the mutant rats compared to the control rats. The mutant rats also displayed a less anxious-like phenotype in three behavioral-based models of anxiety-like behavior (open field, elevated plus maze and defensive withdrawal test). These data show the mutant congenic rat, of which GPR10 neither binds nor responds to PrRP, expresses less anxious-like phenotypes. On the basis of these observations, the GPR10 might be a novel target for the developing new drugs against anxiety and/or other stress-related diseases.

Corticotropin releasing hormone receptor alterations elicited by acute and chronic unpredictable stressor challenges in stressor-susceptible and resilient strains of mice

Stressors increase corticotropin releasing hormone (CRH) functioning in hypothalamic and frontal cortical brain regions, and thus may contribute to the provocation of anxiety and depressive disorder. As the effects of stressors on these behavioral changes are more pronounced in some strains of mice (e.g., BALB/cByJ) than in others (e.g., C57BL/6ByJ), the present investigation assessed whether acute and chronic stressors would differentially influence CRH receptor immunoreactivity (ir-CRHr) and CRH receptor mRNA expression (CRH 1 and CRH 2) in the orbital frontal cortex (OFC) of these strains. An acute noise stressor, and to a greater extent a chronic, variable stressor regimen reduced ir-CRHr in BALB/cByJ mice. In contrast, in the hardier C57BL/6ByJ mice the acute stressor increased ir-CRHr in portions of the OFC, whereas a chronic stressor tended to reduce ir-CRHr. However, whereas the acute stressor did not influence CRH 1 mRNA expression, the chronic stressor increased CRH 1 mRNA expression in both mouse strains. The CRH 2 expression appeared in low abundance in both strains and was unaltered by the stressor. In addition to the OFC variations, quantitative immunohistochemistry indicated that the chronic stressor treatment increased CRH immunoreactivity in the median eminence of C57BL/6ByJ mice, but co-expression of CRH and arginine vasopressin (AVP) immunoreactivity was not provoked by the stressors. The data support the view that stressors provoke marked variations of ir-CRHr in the OFC that might contribute to the differential anxiety/depression-like profiles ordinarily apparent in the stressor-vulnerable and -resilient mouse strains.

Hormonal regulation of the arcuate nucleus melanocortin system

Over the past century, the hypothalamus has emerged as one of the critical sites involved in energy homeostasis. Degeneration studies in rats performed some six decades ago, first led to identifying hypothalamic subregions controlling food intake and body weight. The idea that the central nervous system (CNS), and the hypothalamus in particular, are key in metabolism regulation was reinforced by the discovery of leptin in 1994. Since the identification of leptin, enormous progress has been made in the understanding of the regulation of hypothalamic and extrahypothalamic brain regions that control food intake and energy expenditure by peripheral signals such as hormones. An important challenge is to decipher these complicated interactions between peripheral signals and neuronal circuits to better understand the etiology of metabolic disorders and to identify opportunities to intervene with pharmacological treatment. In this review, we focus on the hormonal regulation of the neuronal circuits of the arcuate nucleus of the hypothalamus: the melanocortin system.

Alzheimer Disease: What Role for Leptin and Insulin?

Among neurodegenerative diseases, Alzheimer disease (AD) is a leading cause of death in elderly individuals. AD is characterized, among other clinical findings, by unexplained weight loss, cachexia and altered immune function. Alteration in energy balance and nutritional status are relayed to the feeding-related hypothalamic nuclei by neuronal pathways and/or via alterations in the levels of eating-controlling hormones. The adipocyte-derived hormone, leptin, and the pancreatic-derived peptide, insulin, function as hormonal signaling mechanisms for fat deposition and play a key role in regulating food intake, body weight and energy homeostasis via their actions on specific hypothalamic nuclei. Moreover, leptin, insulin and their receptors are widely expressed in many hypothalamic and extra-hypothalamic brain regions indicating that these hormones may have other neuronal functions. Although emerging evidence supports the role of insulin resistance in the development of AD, the potential involvement of leptin in the pathogenic process of AD has been proposed only recently. Here we review recent reports and progress concerning the molecular mechanism and the potential role of leptin and insulin in AD.

Profiles of novel diurnally regulated genes in mouse hypothalamus: Expression analysis of the cysteine and histidine-rich domain-containing, zinc-binding protein 1, the fatty acid-binding protein 7 and the GTPase, ras-like family member 11b

Gene expression profiling of suprachiasmatic nucleus, ventrolateral preoptic area and the lateral hypothalamus was used to identify genes regulated diurnally in the hypothalamus of Mus musculus. The putative transcription regulator, cysteine and histidine-rich domain-containing, zinc binding protein 1, which had not been previously described in brain, was found to cycle diurnally in hypothalamus and forebrain with peak levels of mRNA expression during the dark phase. mRNA for the brain-type fatty acid binding protein 7 was found to change rhythmically in hypothalamic and extra-hypothalamic brain regions reaching peak levels early in the light phase suggesting that lipid metabolism is under circadian regulation in astrocytes. Rhythmically expressed genes in suprachiasmatic nucleus identified here were compared with previous reports in a meta-analysis. Genes held in common included fabp7, and the period gene, Per2. Also identified were genes implicated in guanosine-mediated signaling pathways that included dexamethasone-induced ras-related protein one ( dexras1), regulator of G-protein signaling ( rgs) 16, and ras-like family member 11b. Northern blotting confirmed diurnal changes in mRNA expression in the hypothalamus for these genes. Ras-like family member 11b was examined in more detail using in situ hybridization and antiphase diurnal changes in expression in suprachiasmatic nucleus and arcuate nucleus were identified implicating the gene in circadian-related, guanosine-mediated signaling. The transcription transactivator protein, CBP/p300-interacting transactivators with glutamic acid/aspartic acid-rich carboxyl-terminal domain, which had not been previously identified in brain, was enriched in suprachiasmatic nucleus and discrete regions of the hypothalamus and forebrain. The potential regulatory role of CBP/p300-interacting transactivators with glutamic acid/aspartic acid-rich carboxyl-terminal domain in the transcription of genes like TGF-α implicates the protein in diurnal activity rhythms. These results demonstrate the ability of gene expression profiling to identify potential candidates important in circadian or homeostatic processes.

Comparative anatomy of α2 and β adrenoceptors in the adult and developing brain of the marine teleost the red porgy (Pagrus pagrus, Sparidae): [3H]clonidine and [3H]dihydroalprenolol quantitative autoradiography and receptor subtypes immunohistochemistry

The present study aimed to determine the anatomic distribution and developmental profile of alpha(2) and beta adrenoceptors (AR) in marine teleost brain. Alpha 2 and beta adrenoceptors were studied at different developmental stages by using [(3)H]clonidine and [(3)H]dihydroalprenolol, respectively, by means of in vitro quantitative autoradiography. Furthermore, immunohistochemical localization of the receptor subtypes was performed to determine their cellular distribution. Saturation studies determined a high-affinity component of [(3)H]clonidine and [(3)H]dihydroalprenolol binding sites. High levels of both receptors were found in preglomerular complex, ventral hypothalamus, and lateral torus. Dorsal hypothalamus and isthmus included high levels of alpha(2) AR, whereas pretectum and molecular and proliferative zone of cerebellum were specifically characterized by high densities of beta AR. From the first year of life, adult levels of both AR were found in most medial telencephalic, hypothalamic, and posterior tegmental areas. Decreases in both receptors densities with age were prominent in ventral and posterior telencephalic, pretectal, ventral thalamic, hypothalamic, and tegmental brain regions. Immunohistochemical data were well correlated with autoradiography and demonstrated the presence of alpha(2A), alpha(2C), beta(1), and beta(2) AR subtype-like immunoreactivity. Both the neuronal (perikaryal or dendritic) and the glial localization of receptors was revealed. The localization and age-dependent alterations in alpha(2) and beta AR were parallel to plasticity mechanisms, such as cell proliferation in periventricular thalamus, hypothalamus, and cerebellum. In addition, the biochemical characteristics, distribution pattern, and neuronal or glial specificity of the receptors in teleost brain support a similar profile of noradrenergic transmission in vertebrate brain evolution.

D-amphetamine responses in catechol-O-methyltransferase (COMT) disrupted mice.

We have earlier found that 1). COMT inhibitors did not enhance amphetamine-induced dopamine efflux into striatal extracellular, that 2). they did not increase dopamine levels in striatal tissue and that 3). they did not potentiate amphetamine-induced turning behavior of hemiparkinsonian rats. Further, when COMT knockout mice were challenged with l-dopa or a dopamine transporter (DAT) inhibitor, an accumulation of dopamine occurred and the neurochemical and locomotor effects of l-dopa and GBR 12909 were modified accordingly. Since DAT inhibitors and amphetamine apparently have different mechanisms of action, we were interested to see how COMT knockout mice would react to d-amphetamine treatment. We measured the effects of d-amphetamine on locomotor activity and on the levels of catecholamines and their metabolites in striatal microdialysis fluid and in striatal, hypothalamic and cortical brain regions of COMT gene disrupted mice. Striatal dopamine receptor binding was also determined. RESULTS. After d-amphetamine administration, the DOPAC content in homozygous mice was 3-fold in the striatum, 17- to 18-fold in the cortex and 7- to 8-fold in the hypothalamus higher than in wild-type control mice, and there were no indications of genotypexsex interactions. However, the lack of COMT did not potentiate d-amphetamine-induced dopamine levels in brain tissue or in striatal extracellular fluid. D-amphetamine-induced (10 mg/kg) hyperlocomotion was less suppressed in male COMT knockout mice than in their wild-type counterparts. Striatal dopamine D(1) and D(2) receptor levels in male mice were not altered by COMT gene disruption. Changes in COMT activity modulates dopamine metabolism but the behavioral effects of d-amphetamine in male mice only to a small extent, and this action does not seem to depend on the actual extracellular dopamine concentration. Nor is it mediated through compensatory changes in dopamine D(1) and D(2) receptor levels. In dopaminergic neurons, the contribution of intracellular COMT remains secondary in conditions when dopamine is released by d-amphetamine.

Differential colocalization of Islet-1 and estrogen receptor alpha in the murine preoptic area and hypothalamus during development.

Estrogen receptor (ER) expression and regulation is vital to the correct functioning of the neuroendocrine brain. Islet-1 (Isl-1) is a LIM homeodomain-containing transcription factor that has been implicated in neuronal differentiation, is located in the hypothalamus, and can alter ER function in vitro. We have determined that Isl-1 is localized in several regions of the hypothalamus, including the ER rich areas of the ventromedial nucleus (VMH), the preoptic area, and the anterior hypothalamus. Using double-label immunocytochemistry, we examined the overlap between immunoreactive ERalpha and Isl-1 in these different hypothalamic brain regions. In the developing brain, almost 100% of VMH cells that contain immunoreactive ERalpha also contain Isl-1. However, in older animals, the percentage of double-label cells decreased below 70%. This change is due to a decrease in the number of cells containing Isl-1, because there was no difference in the number of ERalpha-containing cells. By contrast, in more anterior regions of the hypothalamus, cells containing both Isl-1 and ERalpha were less common, with the two populations adjacent to each other, rather than overlapping. These data suggest that, although Isl-1 and ERalpha can interact, they are not always found in the same cells and that regulation of ERalpha function is not under the same control in the VMH, preoptic area, and the anterior hypothalamus.

Sex differences in estrogen receptor α and β expression in vasopressin neurons of the supraoptic nucleus in elderly and Alzheimer’s disease patients: no relationship with cytoskeletal alterations

In various hypothalamic and adjacent brain regions we have previously found a remarkable increase in nuclear estrogen receptor staining in Alzheimer’s disease (AD). In order to see whether this was a general phenomenon or rather specific for those areas that are affected by the AD process we investigated ERα and ERβ expression in the arginine–vasopressin (AVP) neurons of the human dorsolateral suparoptic nucleus (dl-SON), that is the major source of plasma AVP. These neurons remain exceptionally intact in AD. Changes in ER expression were studied in relation to early Alzheimer changes (i.e. hyperphosphorylated tau) and neuronal metabolism in AD as determined by the size of the Golgi apparatus (GA) or cell size. No difference in neuronal metabolism (i.e. GA size or cell size) of AVP neurons was observed between AD and control patients and no early cytoskeletal AD alterations were found confirming the resistance of the dl-SON to AD. While no differences between AD and control patients were present for ERα and ERβ staining except for a lower proportion of nuclear ERβ AVP-positive neurons in AD subjects, complex sex differences not directly related to AD were observed within each group. The main finding of the present study is that in the dl-SON, that remains active and spared of AD changes, the increase in nuclear ERs seen in adjacent affected areas in AD patients does not occur. This indicates that a rise of nuclear ERs is not a generally occurring phenomenon but rather related to the pathogenetic alterations of the AD process.

Enhanced fos expression in the zebra finch (Taeniopygia guttata) brain following first courtship.

Young zebra finch males that court a female for the first time develop a stable preference for the females of that species. On the neuronal level, consolidation of the imprinted information takes place. Here we demonstrate that first courtship or being chased around in the cage leads to enhanced fos expression in forebrain areas implicated in learning and imprinting in zebra finch males compared with birds reared in isolation or in the aviary. Two of the forebrain areas highly active during first courtship (as demonstrated by the 14C-2-deoxyglucose technique), the imprinting locus latral neo/hyperstriatum ventrale (LNH) and the secondary visual area hyperstriatum accessorium/dorsale (HAD), demonstrate enhanced fos expression. Two other imprinting-related areas, the medial neo/hyperstriatum ventrale (MNH) and archistriatum/neostriatum caudale (ANC), do show c-fos induction; however, the areas are not congruous with those demarcated by the 2-DG autoradiographic studies. Additional telencephalic areas include the olfactory lobe, the information storage site lobus parolfactorius (LPO), the memory site hippocampus, the auditory caudomedial neostriatum implicated in the strength of song learning, and the caudolateral neostriatum, which is comparable to the mammalian prefrontal cortex. In addition, c-fos is induced by first courtship and chasing in neurosecretory cell groups of the preoptic area and hypothalamus associated with the repertoire of sexual behavior and stress or enhanced arousal. Enhanced fos expression is also observed in brainstem sources of specific (noradrenergic, catecholaminergic) and nonspecific (reticular formation) activating pathways with inputs to higher brain areas implicated in the imprinting process. Birds reared in isolation or alternatively in the aviary with social and sexual contact to conspecifics showed attenuated or no fos expression in most of the above-mentioned areas. First courtship and chasing both lead to enhanced uptake of 2-DG in the four imprinting areas, as well as subsequent changes in spine density-an anatomical manifestation of the imprinting process. fos expression in the imprinting and other telencephalic, preoptic, hypothalamic, and mesencephalic brain regions indicates processing of stimuli originating from exposure (like chasing) and the analysis of stimuli in a behaviorally relevant, sexually explicit context (like first courtship). c-fos induction in these brain areas indicates its involvement in the triggering of neural changes that accompany the learning process of imprinting, leading eventually to alterations in dendritic spine density in the zebra finch.

Acute central ghrelin and GH secretagogues induce feeding and activate brain appetite centers.

Ghrelin was recently identified as the endogenous ligand for the GH secretagogue (GHS) receptor. Like the synthetic GHSs [e.g. GH-releasing peptide-6 (GHRP-6)], ghrelin stimulates feeding and increases body weight in rats. The aim of this study was to identify brain regions that are activated by GHSs and determine whether the responses observed were secondary to food intake. In addition, possible mediators of GHS actions were examined. Intracerebroventricular (icv) injection of ghrelin or GHRP-6 into rats significantly stimulated food intake and transiently reduced core body temperature. The effect of both ghrelin and GHRP-6 on food intake was blocked by preadministration of a Y1 NPY receptor antagonist (BIBO3304). Using c-Fos immunohistochemistry, we demonstrated that icv ghrelin or GHRP-6 activated several hypothalamic brain regions, including the arcuate nucleus, paraventricular nucleus, dorsomedial nucleus, lateral hypothalamus, and two regions of the brainstem, the nucleus of the tractus solitarius and the area postrema. The cell activation induced by GHRP-6 was independent of food intake, as the same pattern and extent of c-Fos expression were observed in animals that were denied access to food following treatment. Finally, double immunohistochemistry indicated that orexin-containing, but not melanin-concentrating hormone-containing, neurons in the lateral hypothalamus were activated significantly by central administration of GHRP-6.

Developmental sex differences in glutamic acid decarboxylase (GAD 65) and the housekeeping gene, GAPDH

Previous work has demonstrated that the GABAergic system is involved in sexual differentiation of the rodent hypothalamus. The present study was designed to further examine this involvement by investigating developmental sex differences in GAD 65 protein levels in hypothalamic and extrahypothalamic brain regions known to be sexually dimorphic in adulthood. Brain nuclei were micro-dissected and GAD 65 protein levels were quantified using western immunoblotting. Sex differences in levels of GAD 65 were found in the dorsomedial nucleus and preoptic area of the hypothalamus and also the medial amygdaloid nucleus and CA1 subfield of the hippocampus. Unexpectedly, there were sex differences in protein levels of the housekeeping gene, GAPDH, cautioning against the use of GAPDH for standardizing protein samples during western immunoblotting.

Chronic exercise increases GAD gene expression in the caudal hypothalamus of spontaneously hypertensive rats

Previous studies have suggested that a γ-amino-butyric acid (GABA) deficit in the caudal hypothalamus (CH) of the spontaneously hypertensive rat (SHR) contributes to elevated levels of arterial pressure. The purpose of this study was to examine if SHR that underwent exercise training demonstrated a blunted development of hypertension and greater levels of glutamic acid decarboxylase (GAD) mRNA transcripts in the caudal hypothalamus. SHR were randomly paired and assigned to either a trained group (T; n=9) or a non-trained control group (NT; n=9). Trained animals were exercised for 10 weeks on a motorized treadmill while NT animals concurrently rested on a mock-treadmill. Following the 10-week training period, Northern blot analyses of mRNA for both the 65-kDa (GAD 65) and 67-kDa (GAD 67) isoforms of GAD were performed on tissue from caudal hypothalamic and cerebellar control brain regions. Exercise training simultaneously blunted the developmental rise in blood pressure in SHR (Δ59±9 mmHg in trained versus Δ77±9 mmHg in non-trained; P<0.03) and increased both GAD 65 (147±44%) and GAD 67 (162±77%) mRNA transcript levels in the CH ( P<0.05). In contrast, no difference was detected in GAD mRNA levels in the cerebellum between T and NT SHR. These findings are consistent with our previous functional studies and demonstrate that exercise can significantly and specifically upregulate GAD gene transcript levels in the caudal hypothalamus of hypertensive rats.

Distribution of estrogen receptor β immunoreactivity in the rat central nervous system

The discovery of estrogen receptor beta (ER beta) and subsequent localization of its mRNA in the rat central nervous system (CNS) has provided new insights about estrogen action in brain. A critical step in understanding the role of ER beta is demonstrating that the mRNA is translated into functional protein. The present study used a new ER beta-specific polyclonal antiserum (Z8P) and immunocytochemistry (ICC) to investigate the distribution of ER beta in the rat CNS. Ovariectomized female rats were perfusion fixed, and free-floating sections were incubated with Z8P. After visualization with a standard ABC method, nuclear immunoreactivity was seen in neurons throughout the brain, including the olfactory nuclei, laminae IV-VI of the cerebral cortex, medial septum, preoptic area, bed nucleus of the stria terminalis, supraoptic nucleus, paraventricular nucleus, zona incerta, medial and cortical amygdaloid nuclei, cerebellum, nucleus of the solitary tract, ventral tegmental area, and spinal trigeminal nucleus. Moreover, the results of a double-label ICC/ in situ hybridization study revealed that ER beta mRNA and immunoreactivity were colocalized in neurons of the brain, thus confirming the specificity of the antiserum. Through the use of Western blot analysis, Z8P was shown to recognize in vitro translated ER beta, but not ER alpha, as well as a 60-kDa protein from rat granulosa cells and ovary extracts. The results of these studies have demonstrated that (1) ER beta mRNA is translated into immunoreactive protein throughout the rat brain, and (2) ER beta resides in the cell nucleus. Together, these data provide an anatomic foundation for future studies and advance our understanding of estrogen action in hypothalamic and extrahypothalamic brain regions.