Relative Number and Distribution of Murine Hypothalamic Proopiomelanocortin Neurons Innervating Distinct Target Sites

Proopiomelanocortin (POMC) neurons send projections widely throughout the brain consistent with their role in regulating numerous homeostatic processes and mediating analgesia and reward. Recent data suggest that POMC neurons located in the rostral and caudal extents of the arcuate nucleus of the hypothalamus may mediate selective actions, however it is not clear if POMC neurons in these regions of the arcuate nucleus innervate specific target sites. In the present study, fluorescent microspheres and cholera toxin B were used to retrogradely label POMC neurons in POMC-DsRed transgenic mice. The number and location of POMC cells projecting to the supraoptic nucleus, periaqueductal gray, ventral tegmental area, paraventricular nucleus, lateral hypothalamic nucleus, amygdala and the dosal vagal complex was determined. Tracer injected unilaterally labeled POMC neurons in both sides of the arcuate nucleus. While the total number of retrogradely labeled cells in the arcuate nucleus varied by injection site, less than 10% of POMC neurons were labeled with tracer injected into any target area. Limited target sites appear to be preferentially innervated by POMC neurons that reside in the rostral or caudal extremes of the arcuate nucleus, whereas the majority of target sites are innervated by diffusely distributed POMC neurons. The modest number of cells projecting to each target site indicates that relatively few POMC neurons may mediate potent and specific physiologic responses and therefore disturbed signaling in a very few POMC neurons may have significant consequences

Separate GABA Afferents to Dopamine Neurons Mediate Acute Action of Opioids, Development of Tolerance, and Expression of Withdrawal

SummaryGABA release from interneurons in VTA, projections from the nucleus accumbens (NAc), and rostromedial tegmental nucleus (RMTg) was selectively activated in rat brain slices. The inhibition induced by μ-opioid agonists was pathway dependent. Morphine induced a 46% inhibition of IPSCs evoked from the RMTg, 18% from NAc, and IPSCs evoked from VTA interneurons were almost insensitive (11% inhibition). In vivo morphine treatment resulted in tolerance to the inhibition of RMTg, but not local interneurons or NAc, inputs. One common sign of opioid withdrawal is an increase in adenosine-dependent inhibition. IPSCs evoked from the NAc were potently inhibited by activation of presynaptic adenosine receptors, whereas IPSCs evoked from RMTg were not changed. Blockade of adenosine receptors selectively increased IPSCs evoked from the NAc during morphine withdrawal. Thus, the acute action of opioids, the development of tolerance, and the expression of withdrawal are mediated by separate GABA afferents to dopamine neurons

Somato-Dendritic Localization and Signaling by Leptin Receptors in Hypothalamic POMC and AgRP Neurons

Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP)/Neuropeptide Y (NPY)/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb). Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM), confocal-laser scanning microscopy (CLSM), and electron microscopy (EM) to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Leprb+/+ mice and in Leprbdb/db mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin’s central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms

A Transgenic Marker for Newly Born Granule Cells in Dentate Gyrus

Neurogenesis in the dentate gyrus continues into adulthood, yet little is known about the function of newly born neurons or how they integrate into an existing network of mature neurons. We made transgenic mice that selectively and transiently express enhanced green fluorescent protein (EGFP) in newly born granule cells of the dentate gyrus under the transcriptional control of proopiomelanocortin (POMC) genomic sequences. Analysis of transgenic pedigrees with truncation or deletion mutations indicated that EGFP expression in the dentate gyrus required cryptic POMC promoter regions dispensable for arcuate hypothalamic or pituitary expression. Unlike arcuate neurons, dentate granule cells did not express the endogenous POMC gene. EGFP-positive neurons had immature properties, including short spineless dendrites and small action potentials. Colocalization with bromodeoxyuridine indicated that EGFP-labeled granule cells were 2 weeks postmitotic. EGFP-labeled cells expressed markers for immature granule cells but not the glial marker GFAP. The number of EGFP-labeled neurons declined with age and increased with exercise, paralleling neurogenesis. Our results indicate that POMC-EGFP marks immature granule cells and that adult-generated granule cells integrate quite slowly into the hippocampal circuitry

Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk

Pituitary tumor development involves clonal expansion stimulated by hormones and growth factors/cytokines. Using mRNA differential display, we found that the bone morphogenetic protein (BMP inhibitor noggin is down-regulated in prolactinomas from dopamine D2-receptor-deficient mice. BMP-4 is overexpressed in prolactinomas taken from dopamine D2-receptor-deficient female mice, but expression of the highly homologous BMP-2 does not differ in normal pituitary tissue and prolactinomas. BMP-4 is overexpressed in other prolactinoma models, including estradiol-induced rat prolactinomas and human prolactinomas, compared with normal tissue and other pituitary adenoma types (Western blot analysis of 48 tumors). BMP-4 stimulates, and noggin blocks, cell proliferation and the expression of c-Myc in human prolactinomas, whereas BMP-4 has no action in other human pituitary tumors. GH3 cells stably transfected with a dominant negative of Smad4 (Smad4dn; a BMP signal cotransducer) or noggin have reduced tumorigenicity in nude mice. Tumor growth recovered in vivo when the Smad4dn expression was lost, proving that BMP-4/Smad4 are involved in tumor development in vivo. BMP-4 and estrogens act through overlapping intracellular signaling mechanisms on GH3 cell proliferation and c-myc expression: they had additive effects at low concentrations but not at saturating doses, and their action was inhibited by blocking either pathway with the reciprocal antagonist (i.e., BMP-4 with ICI 182780 or 17β-estradiol with Smad4dn). Furthermore, coimmunoprecipitation studies demonstrate that under BMP-4 stimulation Smad4 and Smadl physically interact with the estrogen receptor. This previously undescribed prolactinoma pathogenesis mechanism may participate in tumorigenicity in other cells where estrogens and the type β transforming growth factor family have important roles.Facultad de Ciencias ExactasInstituto de Investigaciones Bioquímicas de La Plat

Rostral-to-caudal distribution of POMC neurons that project to distinct target sites.

<p>(A) Atlas diagrams modified from Paxinos and Franklin (2nd edition, © 2001) depicting the cut-offs used to define rostral, mid and caudal regions of the arcuate nucleus. Values are distance from Bregma. (B) Bar graph showing the percent of POMC-DsRed cells that contained CTB in the rostral, mid and caudal portions of the arcuate nucleus after CTB was injected into various target sites. Numbers in parentheses indicate the number of animals in each group. Asterisks indicate p<0.05 compared to rostral sections for that injection site. All data are mean ± SEM. Abbreviations: VMH, ventromedial hypothalamic nucleus; 3V, third ventricle; Arc, arcuate nucleus (MP, medial posterior; LP, lateroposterior); sox, supraoptic decussation; opt, optic tract; LH, lateral hypothalamus.</p

Percent of POMC cells with CTB compared across rostral to caudal regions.

<p>Mean difference for the percent of POMC cells that contain the retrograde tracer (CTB) between rostral (Rost), middle (Mid) and caudal (Caud) regions of the arcuate nucleus with 95% confidence intervals in parentheses for tracer injections into each of the indicated target sites. Asterisks denote P<0.01.</p

CTB selectively labels cell in distinct brain regions.

<p>(A) A portion of the atlas page (Lateral 0.24) from Paxinos and Franklin (2nd edition, © 2001) corresponding to the sagittal section shown in (B) and (C). The dotted line in (A) depicts where the images in (B) and (C) are cropped. CTB was injected into the VTA and a low-power image containing the arcuate nucleus was taken to reveal the distribution of CTB (B). Fluorescence from the injection site can be seen at the upper right edge of the photo in (B, marked with asterisk). (C) The same field of view as in (B) imaged to reveal the POMC-Ds-Red labeled cells in the arcuate and some of their prominent fibers rostral and caudal to the arcuate nucleus. Abbreviations: ac, anterior commissure; PaAP, paraventricular hypothalamic nucleus-anterior parvocellular; PaLM, paraventricular hypothalamic nucleus-lateral magnocellular; PaMM, paraventricular hypothalamic nucleus-medial magnocellular; PaDC, paraventricular hypothalamic nucleus-dorsal cap; PaPo, paraventricular hypothalamic nucleus-posterior; PH, posterior hypothalamic area; MPA, medial preoptic area; AHA, anterior hypothalamic area; LA, lateroanterior hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus; DM, dorsomedial hypothalamic nucleus; Arc, arcuate hypothalamic nucleus, MM, medial mammilary nucleus; IPF, interpeduncular fossa; OX, optic chiasm; VTA, ventral tegmental area.</p