Audrey F. Seasholtz

Leptin action via neurotensin neurons controls orexin, the mesolimbic dopamine system and energy balance

Leptin acts on leptin receptor (LepRb)-expressing neurons throughout the brain, but the roles for many populations of LepRb neurons in modulating energy balance and behavior remain unclear. We found that the majority of LepRb neurons in the lateral hypothalamic area (LHA) contain neurotensin (Nts). To investigate the physiologic role for leptin action via these LepRb(Nts) neurons, we generated mice null for LepRb specifically in Nts neurons (Nts-LepRbKO mice). Nts-LepRbKO mice demonstrate early-onset obesity, modestly increased feeding, and decreased locomotor activity. Furthermore, consistent with the connection of LepRb(Nts) neurons with local orexin (OX) neurons and the ventral tegmental area (VTA), Nts-LepRbKO mice exhibit altered regulation of OX neurons and the mesolimbic DA system. Thus, LHA LepRb(Nts) neurons mediate physiologic leptin action on OX neurons and the mesolimbic DA system, and contribute importantly to the control of energy balance.

Mineralocorticoid receptor overexpression in forebrain decreases anxiety-like behavior and alters the stress response in mice.

Although numerous stress-related molecules have been implicated in vulnerability to psychiatric illness, especially major depression and anxiety disorders, the role of the brain mineralocorticoid receptor (MR) in stress, depression, and affective function is not well defined. MR is a steroid hormone receptor that detects circulating glucocorticoids with high affinity and has been primarily implicated in controlling their basal level and circadian rhythm. To specifically address the role of MR in hypothalamic-pituitary-adrenal axis activity and anxiety-related behaviors, we generated transgenic mice with increased levels of MR in the forebrain (MRov mice) by using the forebrain-specific calcium/calmodulin-dependent protein kinase II α promoter to direct expression of MR cDNA. A mild but chronic elevation in forebrain MR results in decreased anxiety-like behavior in both male and female transgenic mice. Female MRov mice also exhibit a moderate suppression of the corticosterone response to restraint stress. Increased forebrain MR expression alters the expression of two genes associated with stress and anxiety, leading to a decrease in the hippocampal glucocorticoid receptor (GR) and an increase in serotonin receptor 5HT-1a, consistent with the decreased anxiety phenotype. These data suggest that the functions of forebrain MR may overlap with GR in hypothalamic-pituitary-adrenal axis regulation, but they dissociate significantly from GR in the modulation of affective responses, with GR overexpression increasing anxiety-like behavior and MR overexpression dampening it. These findings point to the importance of the MR:GR ratio in the control of emotional reactivity.

hVH‐5: A Protein Tyrosine Phosphatase Abundant in Brain that Inactivates Mitogen‐Activated Protein Kinase

Abstract: A novel protein tyrosine phosphatase [homologue of vaccinia virus H1 phosphatase gene clone 5 (hVH‐5)] was cloned; it shared sequence similarity with a subset of protein tyrosine phosphatases that regulate mitogen‐activated protein kinase. The catalytic region of hVH‐5 was expressed as a fusion protein and was shown to hydrolyze p‐nitrophenylphosphate and inactivate mitogen‐activated protein kinase, thus proving that hVH‐5 possessed phosphatase activity. A unique proline‐rich region distinguished hVH‐5 from other closely related protein tyrosine phosphatases. Another feature that distinguished hVH‐5 from related phosphatases was that hVH‐5 was expressed predominantly in the adult brain, heart, and skeletal muscle. In addition, in situ hybridization histochemistry of mouse embryo revealed high levels of expression and a wide distribution in the central and peripheral nervous system. Some specific areas of abundant hVH‐5 expression included the olfactory bulb, retina, layers of the cerebral cortex, and cranial and spinal ganglia. hVH‐5 was induced in PC12 cells upon nerve growth factor and insulin treatment in a manner characteristic of an immediate‐early gene, suggesting a possible role in the signal transduction cascade.

CRH-BP: the regulation and function of a phylogenetically conserved binding protein

Corticotropin Releasing Hormone-Binding Protein (CRH-BP), a 37 kDa secreted glycoprotein, binds both CRH and urocortin with high affinity and is structurally unrelated to the CRH receptors. CRH-BP orthologues have been identified in multiple invertebrate and vertebrate species. It is strongly conserved throughout evolution, suggesting the maintenance of a structural conformation necessary for biological activity. CRH-BP is an important modulator of CRH activity; it inhibits CRH-induced ACTH secretion from pituitary corticotropes and may exert similar actions at central sites of CRH release. While the function of CRH-BP is thought to be primarily inhibitory, recent studies indicate that novel functional roles may exist in both the brain and pituitary. Regulation of CRH-BP expression by stress and metabolic factors are consistent with in vivo models of altered CRH-BP expression. Positive regulation of pituitary CRH-BP by reproductive hormones suggests that additional interactions between the stress and reproductive axes may exist. While recent research has focused on the evolutionary conservation, expanded sites of expression, regulation and in vivo function of CRH-BP, a more complete understanding of the central and peripheral functions of CRH-BP and its mechanisms of action will help elucidate its potential role in the etiology or treatment of disorders of CRH dysregulation.

Genealogy of the Anterior Pituitary Gland: Tracing a Family Tree.

The anterior lobe of the pituitary gland is derived from the oral ectoderm early in gestation. A variety of techniques have been used to understand how early precursor cells differentiate to form the five major cell types that populate the adult anterior lobe. Current evidence suggests that corticotropes arise from a lineage distinct from that of the other four cell types. The cells of the other lineage branch - thyrotropes, gonadotropes, somatotropes and lactotropes - appear to be related because of their dependence on common transcription factors and the frequent occurrence of cells that produce multiple hormones. While thyrotropes arise through two routes, the lineage related to somatotropes and lactotropes appears to be the most important for hormone production. Each cell type can populate the organ and function in the absence of the other cell types, except for lactotropes, which have a strong dependence on somatotropes. Our current knowledge of anterior pituitary cell lineage relationships may contribute to a better understanding of the origin of pituitary adenomas and tumors.

MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF THE MOUSE BRAIN CORTICOTROPIN-RELEASING HORMONE-BINDING PROTEIN

A 37 kDa corticotropin-releasing hormone-binding protein (CRH-BP), distinct from the CRH receptor, is expressed in rat anterior pituitary corticotrophs and many regions of the brain, suggesting that CRH-BP may modulate the biological activity of CRH. In these studies a mouse brain CRH-BP (mCRH-BP) cDNA has been isolated and characterized. The 1666 nucleotide mCRH-BP cDNA is expressed in brain and pituitary and encodes a 322 amino acid protein that is highly homologous to human and rat CRH-BPs. Recombinant mCRH-BP, expressed in cultured mammalian cells, binds human CRH (Kd(app) = 0.56 nM and Ki(app) = 0.37 nM) and the alpha-helical (9-41) CRH antagonist (Ki(app) = 0.28 nM) with high affinity, but exhibits much weaker affinity for ovine CRH (Ki(app) = 206 nM). Recombinant mCRH-BP also blocks CRH-induced adrenocorticotropin release from AtT-20 cells. Additional biochemical characterization of the binding activity of mCRH-BP indicates that CRH-BP and CRH receptor utilize different molecular interactions to bind CRH.

Glucocorticoid and mineralocorticoid receptor expression in the human hippocampus in major depressive disorder

Approximately 50% of mood disorder patients exhibit hypercortisolism. Cortisol normally exerts its functions in the CNS via binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Both MR and GR are highly expressed in human hippocampus and several studies have suggested that alterations in the levels of MR or GR within this region may contribute to the dysregulation in major depressive disorder (MDD). Studies have also shown functional heterogeneity across the hippocampus, with posterior hippocampus preferentially involved in cognitive processes and anterior hippocampus involved in stress, emotion and affect. We therefore hypothesize that GR and MR expression in hippocampus of control and MDD patients may vary not only with disease, but also with regional specificity along the anterior/posterior axis. Students t-test analysis showed decreased expression of MR in the MDD group compared to controls in the anterior, but not the posterior hippocampus, with no significant changes in GR. Linear regression analysis showed a marked difference in MR:GR correlation between suicide and non-suicide patients in the posterior hippocampus. Our findings are consistent with previous reports of hippocampal corticosteroid receptor dysregulation in mood disorders, but extend those findings by analysis across the anterior/posterior axis of the hippocampus. A decrease in MR in the anterior but not posterior hippocampus of MDD patients emphasizes the important functional role of the anterior hippocampus in neuroendocrine regulation in humans.

Overexpressing the Glucocorticoid Receptor in Forebrain Causes an Aging-Like Neuroendocrine Phenotype and Mild Cognitive Dysfunction

Repeated stress enhances vulnerability to neural dysfunction that is cumulative over the course of the lifespan. This dysfunction contributes to cognitive deficits observed during aging. In addition, aging is associated with dysregulation of the limbic–hypothalamic–pituitary–adrenal (LHPA) axis, leading to a delayed termination of the stress response. This delay, in turn, increases exposure to glucocorticoids and exacerbates the likelihood of neural damage. Here we asked whether similar effects could emerge at an early age as a result of genetic variations in the level or function of the brain glucocorticoid receptor (GR). We investigated the effect of forebrain-specific overexpression of GR on LHPA axis activity. Transgenic mice with GR overexpression in forebrain (GRov) display normal basal circulating adrenocorticotropic hormone and corticosterone levels. However, young GRov mice exhibit a number of LHPA alterations, including a blunted initial response to acute restraint stress followed by a delayed turn-off of the stress response. This deficit in negative feedback is paradoxical in the face of elevated GR levels, resembles the stress response in aged animals, and continues to worsen as GRov mice age. The neuroendocrine dysregulation in young GRov mice is coupled with a mild cognitive deficit, also consistent with the accelerated aging hypothesis. The molecular basis of this phenotype was examined using microarray analysis of the hippocampus, which revealed a broad downregulation of glutamate receptor signaling in GRov mice. Thus, even in the absence of chronic stress, elevation of GR gene expression can lead to an increased allostatic load and result in an “aging-like” phenotype in young animals.

Cre-mediated recombination in the pituitary gland.

Summary: Organ‐specific expression of a cre recombinase transgene allows for the analysis of gene function in a particular tissue or cell type. Using a 4.6 kb promoter from the mouse glycoprotein hormone α‐subunit (αGSU or Cga) gene, we have generated and characterized a line of transgenic mice that express cre recombinase in the anterior and intermediate lobes of the pituitary gland. Utilizing a cre‐responsive reporter transgene, αGSU‐cre transgene expression was detected in the pituitary primordium and in all five cell types of the adult anterior pituitary. αGSU‐cre transgene activity was also detected in the cardiac and skeletal muscle. Little or no activity was evident in the gonads, adrenal glands, brain, ventromedial hypothalamus, or kidneys. The αGSU‐cre transgenic mice characterized here will be a valuable tool for examining gene function in the pituitary gland. genesis 28:167–174, 2000.

Nitric Oxide/Cyclic Guanosine Monophosphate Pathway in the Peripheral and Central Auditory System of the Rat

The neuronal isoform of nitric oxide synthase (nNOS) and soluble guanylate cyclase (sGC) were localized in the cochlea, the cochlear nucleus (CN), and the superior olivary complex (SOC) of Fisher 344 rats. In the cochlea, nNOS was identified in spiral ganglion cells by using nicotinamide adenine dinucleotide phosphate (NADPH)‐diaphorase histochemistry and in situ hybridization. NADPH‐diaphorase staining also was detected in blood vessels of the modiolus. By using immunohistochemistry against cyclic guanosine monophosphate, cochlear sGC activity was localized to pericytes in the spiral ligament as well as nerve fibers innervating outer hair cells. In the lower auditory brainstem, nNOS was localized to principal cells of the medial nucleus of the trapezoid body (MNTB) with NADPH‐diaphorase histochemistry and in situ hybridization. NADPH‐diaphorase activity also was observed in the lateral and medial superior olive (LSO and MSO, respectively), the superior periolivary nucleus (SPN), the ventral and lateral nuclei of the trapezoid body (VNTB and LNTB, respectively), and the ventral cochlear nucleus (VCN). Transcripts of the β‐subunit of sGC were localized in rat brainstem by using in situ hybridization. mRNA for sGC was expressed in neurons within the SPN, LSO, MSO, LNTB, MNTB, VNTB, and VCN. Highest levels of sGC expression were seen in the SPN. These results suggest that the NO/cGMP pathway is involved in both the ascending and descending pathways of the auditory brainstem. J. Comp. Neurol. 404:52–63, 1999.