Bed nucleus of the stria terminalis alpha(1)-adrenoceptor modulates baroreflex cardiac component in unanesthetized rats

The bed nucleus of stria terminalis (BST) has a tonic modulating role on the baroreflex parasympathetic component. In the present study, we verified that local BST-adrenoceptors modulate baroreflex-evoked bradycardiac responses in unanesthetized rats. Bilateral microinjection of the selective alpha(1)-adrenoceptor antagonist WB4101 (15 nmol/100 nL) into the BST increased the gain of reflex bradycardia in response to mean arterial pressure increases caused by intravenous (i.v.) infusion of phenylephrine, suggesting that BST alpha(1)-adrenoceptors modulate baroreflex bradycardiac response. Bilateral microinjection of either the selective alpha(2)-adrenoceptor antagonist RX821002 (15 nmol/100 nL) or the non-selective beta-adrenoceptor antagonist propranolol (15 nmol/100 nL) into the BST had not affected baroreflex bradycardia. Animals were pretreated intravenously with the cholinergic muscarinic receptor antagonist homatropine methyl bromide (HMB, 1.5 mg/Kg) to test the hypothesis that activation of alpha(1)-adrenoceptors in the BST would modulate the baroreflex parasympathetic component. Baroreflex bradycardiac responses evoked before and after BST treatment with WB4101 were no longer different when rats were pretreated with HMB. These results suggest that parasympathetic activation accounts for the effects saw after BST pharmacological manipulation and ruling out the possibility of a sympathetic withdraw. In conclusion, our data point out that local alpha(1)-adrenoceptors mediate the BST tonic influence on the baroreflex bradycardiac response modulating parasympathetic cardiac activity. (C) 2008 Elsevier B.V. All rights reserved.

Dorsal and ventral medullary catecholamine cell groups contribute differentially to systemic interleukin-1 beta-induced hypothalamic pituitary adrenal axis responses

Medial parvocellular paraventricular corticotropin-releasing hormone (mPVN CRH) cells are critical in generating hypothalamic-pituitary-adrenal (HPA) axis responses to systemic interleukin-1 beta (IL-1 beta). However, although it is understood that catecholamine inputs are important in initiating mPVN CRH cell responses to IL-1 beta, the contributions of distinct brainstem catecholamine cell groups are not known. We examined the role of nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM) catecholamine cells in the activation of mPVN CRH, hypothalamic oxytocin (OT) and central amygdala cells in response to IL-1 beta (1 mug/kg, i.a.). Immunolabelling for the expression of c-fos was used as a marker of neuronal activation in combination with appropriate cytoplasmic phenotypic markers. First we confirmed that PVN 6-hydroxydopamine lesions, which selectively depleted catecholaminergic terminals, significantly reduced IL-1 beta -induced mPVN CRH cell activation. The contribution of VLM (A1/C1 cells) versus NTS (A2 cells) catecholamine cells to mPVN CRH cell responses was then examined by placing ibotenic acid lesions in either the VLM or NTS. The precise positioning of these lesions was guided by prior retrograde tracing studies in which we mapped the location of IL-1 beta -activated VLM and NTS cells that project to the mPVN. Both VLM and NTS lesions reduced the mPVN CRH and OT cell responses to IL-1 beta. Unlike VLM lesions, NTS lesions also suppressed the recruitment of central amygdala neurons. These studies provide novel evidence that both the NTS and VLM catecholamine cells have important, but differential, contributions to the generation of IL-1 beta -induced HPA axis responses. Copyright (C) 2001 S. Karger AG, Basel.

Topographic organization of V1 projections through the corpus callosum in humans.

The visual cortex in each hemisphere is linked to the opposite hemisphere by axonal projections that pass through the splenium of the corpus callosum. Visual-callosal connections in humans and macaques are found along the V1/V2 border where the vertical meridian is represented. Here we identify the topography of V1 vertical midline projections through the splenium within six human subjects with normal vision using diffusion-weighted MR imaging and probabilistic diffusion tractography. Tractography seed points within the splenium were classified according to their estimated connectivity profiles to topographic subregions of V1, as defined by functional retinotopic mapping. First, we report a ventral-dorsal mapping within the splenium with fibers from ventral V1 (representing the upper visual field) projecting to the inferior-anterior corner of the splenium and fibers from dorsal V1 (representing the lower visual field) projecting to the superior-posterior end. Second, we also report an eccentricity gradient of projections from foveal-to-peripheral V1 subregions running in the anterior-superior to posterior-inferior direction, orthogonal to the dorsal-ventral mapping. These results confirm and add to a previous diffusion MRI study (Dougherty et al., 2005) which identified a dorsal/ventral mapping of human splenial fibers. These findings yield a more detailed view of the structural organization of the splenium than previously reported and offer new opportunities to study structural plasticity in the visual system.

Subcolumnar Dendritic and Axonal Organization of Spiny Stellate and Star Pyramid Neurons within a Barrel in Rat Somatosensory Cortex

Excitatory neurons at the level of cortical layer 4 in the rodent somatosensory barrel field often display a strong eccentricity in comparison with layer 4 neurons in other cortical regions. In rat, dendritic symmetry of the 2 main excitatory neuronal classes, spiny stellate and star pyramid neurons (SSNs and SPNs), was quantified by an asymmetry index, the dendrite-free angle. We carefully measured shrinkage and analyzed its influence on morphological parameters. SSNs had mostly eccentric morphology, whereas SPNs were nearly radially symmetric. Most asymmetric neurons were located near the barrel border. The axonal projections, analyzed at the level of layer 4, were mostly restricted to a single barrel except for those of 3 interbarrel projection neurons. Comparing voxel representations of dendrites and axon collaterals of the same neuron revealed a close overlap of dendritic and axonal fields, more pronounced in SSNs versus SPNs and considerably stronger in spiny L4 neurons versus extragranular pyramidal cells. These observations suggest that within a barrel dendrites and axons of individual excitatory cells are organized in subcolumns that may confer receptive field properties such as directional selectivity to higher layers, whereas the interbarrel projections challenge our view of barrels as completely independent processors of thalamic input.

Layer-specific programs of development in neocortical projection neurons.

How are long-range axonal projections from the cerebral cortex orchestrated during development? By using both passively and actively transported axonal tracers in fetal and postnatal ferrets, we have analyzed the development of projections from the cortex to a number of thalamic nuclei. We report that the projections of a cortical area to its corresponding thalamic nuclei follow highly cell-specific programs of development. Axons from cells in the deepest layers of the cerebral cortex (layer 6 and superficial subplate neurons) appear to grow very slowly and be delayed for several weeks in the cerebral white matter, reaching the thalamus over a protracted period. Neurons of layer 5, on the other hand, develop their projections much faster; despite being born after the neurons of deeper layers, layer 5 neurons are the first to extend their axons out of the cortical hemisphere and innervate the thalamus. Layer 5 projections are massive in the first postnatal weeks but may become partly eliminated later in development, being overtaken in number by layer 6 cells that constitute the major corticothalamic projection by adulthood. Layer 5 projections are area-specific from the outset and arise as collateral branches of axons directed to the brainstem and spinal cord. Our findings show that the early development of corticofugal connections is determined not by the sequence of cortical neurogenesis but by developmental programs specific for each type of projection neuron. In addition, they demonstrate that in most thalamic nuclei, layer 5 neurons (and not subplate or layer 6 neurons) establish the first descending projections from the cerebral cortex.

rst Transcriptional Activity Influences kirre mRNA Concentration in the Drosophila Pupal Retina during the Final Steps of Ommatidial Patterning

Background: Drosophila retinal architecture is laid down between 24-48 hours after puparium formation, when some of the still uncommitted interommatidial cells (IOCs) are recruited to become secondary and tertiary pigment cells while the remaining ones undergo apoptosis. This choice between survival and death requires the product of the roughest (rst) gene, an immunoglobulin superfamily transmembrane glycoprotein involved in a wide range of developmental processes. Both temporal misexpression of Rst and truncation of the protein intracytoplasmic domain, lead to severe defects in which IOCs either remain mostly undifferentiated and die late and erratically or, instead, differentiate into extra pigment cells. Intriguingly, mutants not expressing wild type protein often have normal or very mild rough eyes. Methodology/Principal Findings: By using quantitative real time PCR to examine rst transcriptional dynamics in the pupal retina, both in wild type and mutant alleles we showed that tightly regulated temporal changes in rst transcriptional rate underlie its proper function during the final steps of eye patterning. Furthermore we demonstrated that the unexpected wild type eye phenotype of mutants with low or no rst expression correlates with an upregulation in the mRNA levels of the rst paralogue kin-of-irre (kirre), which seems able to substitute for rst function in this process, similarly to their role in myoblast fusion. This compensatory upregulation of kirre mRNA levels could be directly induced in wild type pupa upon RNAi-mediated silencing of rst, indicating that expression of both genes is also coordinately regulated in physiological conditions. Conclusions/Significance: These findings suggest a general mechanism by which rst and kirre expression could be fine tuned to optimize their redundant roles during development and provide a clearer picture of how the specification of survival and apoptotic fates by differential cell adhesion during the final steps of retinal morphogenesis in insects are controlled at the transcriptional level.

Transformation of external sensilla to chordotonal sensilla in the cut mutant of Drosophila assessed by single-cell marking in the embryo and larva

The cut gene of Drosophila melanogaster is an identity selector gene that establishes the program of development and differentiation of external sense organs. Mutations in the cut gene cause a transformation of the external sense organs into chordotonal organs, originally assessed by the use of immunostaining methods [Bodmer et al. (1987): Cell, 51:293-307]. Because of evidence that axonal projections of the transformed neurons within the central nervous system are not completely switched in cut mutants, the transformation of the four cells making up a sense organ was reassessed using single-cell staining with fluorescent dye and differential interface contrast (DIC) microscopy of the embryo and larva. The results provide strong evidence that all cells of the sense organs are completely transformed, exhibiting the morphologies and organelles characteristic of chordotonal sense organs. A comparison of the structures of external sense organs and chordotonal organs indicates that a number of the differences could be due to the degree of development of common structures, and that cut or downstream genes modulate effector genes that are normally utilized in both receptor types. The possible derivation of insect chordotonal and external sense organs from a receptor type found in crustaceans is discussed in the light of arthropod phylogenetics and the molecular genetics of sense organ development. (C) 1997 Wiley-Liss, Inc.

Precise Temporal Regulation of roughest is Required for Correct Salivary Gland Autophagic Cell Death in Drosophila

The Drosophila roughest (rst) locus encodes an immunoglobulin superfamily transmembrane glycoprotein implicated in a variety of embryonic and postembryonic developmental processes. Here we demonstrate a previously unnoticed role for this gene in the autophagic elimination of larval salivary glands during early pupal stages by showing that overexpression of the Rst protein ectodomain in early pupa leads to persistence of salivary glands up to at least 12 hours after head eversion, although with variable penetrance. The same phenotype is observed in individuals carrying the dominant regulatory allele rst(D), but not in loss of function alleles. Analysis of persistent glands at the ultrastructural level showed that programmed cell death starts at the right time but is arrested at an early stage of the process. Finally we describe the expression pattern and intracellular distribution of Rst in wild type and rstD mutants, showing that its downregulation in salivary glands at the beginning of pupal stage is an important factor in the correct implementation of the autophagic program of this tissue in space and time. genesis 47:492-504, 2009. (C) 2009 Wiley-Liss, Inc.

Involvement of hypothalamic paraventricular nucleus non-N-methyl-d-aspartate receptors in the pressor response to noradrenaline microinjected into the bed nucleus of the stria terminalis of unanesthetized rats

Microinjection of noradrenaline into the bed nucleus of the stria terminalis (BST) has been reported to cause a pressor response in unanesthetized rats, which was shown to be mediated by acute vasopressin release into the systemic circulation. In the present study we verified the involvement of magnocellular neurons of the hypothalamic paraventricular (PVN) or supraoptic (SON) nuclei and the local neurotransmitter involved in the pressor response to noradrenaline microinjection into the BST. The PVN pretreatment with the non-selective neurotransmission blocker CoCl(2) (1 nmol/100 nL) inhibited the noradrenaline-evoked pressor response. However, responses were not affected by SON treatment with CoCl(2). Further experiments were carried out to test if glutamatergic neurotransmission in the PVN mediates the pressor response evoked by noradrenaline microinjection into the BST. Pretreatment of the PVN with the selective N-methyl-d-aspartate (NMDA) receptor antagonist LY235959 (2 nmol/100 nL) did not affect the noradrenaline-evoked pressor response. However, PVN pretreatment with the selective non-NMDA receptor antagonist NBQX (2 nmol/100 nL) significantly reduced the pressor response to noradrenaline microinjection into the BST. In conclusion, our results suggest that pressor responses to noradrenaline microinjection into the BST are mediated by PVN magnocellular neurons without involvement of SON neurons. They also suggest that a glutamatergic neurotransmission through non-NMDA glutamate receptors in the PVN mediates the response.

White matter maturation reshapes structural connectivity in the late developing human brain.

From toddler to late teenager, the macroscopic pattern of axonal projections in the human brain remains largely unchanged while undergoing dramatic functional modifications that lead to network refinement. These functional modifications are mediated by increasing myelination and changes in axonal diameter and synaptic density, as well as changes in neurochemical mediators. Here we explore the contribution of white matter maturation to the development of connectivity between ages 2 and 18 y using high b-value diffusion MRI tractography and connectivity analysis. We measured changes in connection efficacy as the inverse of the average diffusivity along a fiber tract. We observed significant refinement in specific metrics of network topology, including a significant increase in node strength and efficiency along with a decrease in clustering. Major structural modules and hubs were in place by 2 y of age, and they continued to strengthen their profile during subsequent development. Recording resting-state functional MRI from a subset of subjects, we confirmed a positive correlation between structural and functional connectivity, and in addition observed that this relationship strengthened with age. Continuously increasing integration and decreasing segregation of structural connectivity with age suggests that network refinement mediated by white matter maturation promotes increased global efficiency. In addition, the strengthening of the correlation between structural and functional connectivity with age suggests that white matter connectivity in combination with other factors, such as differential modulation of axonal diameter and myelin thickness, that are partially captured by inverse average diffusivity, play an increasingly important role in creating brain-wide coherence and synchrony.

ALDH1A3 loss of function causes bilateral anophthalmia/microphthalmia and hypoplasia of the optic nerve and optic chiasm.

The major active retinoid, all-trans retinoic acid, has long been recognized as critical for the development of several organs, including the eye. Mutations in STRA6, the gene encoding the cellular receptor for vitamin A, in patients with Matthew-Wood syndrome and anophthalmia/microphthalmia (A/M), have previously demonstrated the importance of retinol metabolism in human eye disease. We used homozygosity mapping combined with next-generation sequencing to interrogate patients with anophthalmia and microphthalmia for new causative genes. We used whole-exome and whole-genome sequencing to study a family with two affected brothers with bilateral A/M and a simplex case with bilateral anophthalmia and hypoplasia of the optic nerve and optic chiasm. Analysis of novel sequence variants revealed homozygosity for two nonsense mutations in ALDH1A3, c.568A>G, predicting p.Lys190*, in the familial cases, and c.1165A>T, predicting p.Lys389*, in the simplex case. Both mutations predict nonsense-mediated decay and complete loss of function. We performed antisense morpholino (MO) studies in Danio rerio to characterize the developmental effects of loss of Aldh1a3 function. MO-injected larvae showed a significant reduction in eye size, and aberrant axonal projections to the tectum were noted. We conclude that ALDH1A3 loss of function causes anophthalmia and aberrant eye development in humans and in animal model systems.

Modulation de l’expression du transporteur vésiculaire du glutamate : implication dans la plasticité des neurones dopaminergiques

De nombreuses études ont établi que la majorité des neurones libèrent plus qu’une substance chimique. Il est bien connu que les neurones peuvent co-exprimer et co-libérer des neuropeptides en plus de leur neurotransmetteur, mais des évidences de la co-libération de deux petits neurotransmetteurs à action rapide se sont accumulées récemment. Des enregistrements électrophysiologiques ont aussi montré que des neurones sérotoninergiques et dopaminergiques isolés peuvent libérer du glutamate quand ils sont placés en culture. De plus, la présence de glutamate et de glutaminase a été détectée dans des neurones sérotoninergiques, dopaminergiques et noradrénergiques par immunomarquage sur des tranches de cerveau. Malheureusement, en considérant le rôle métabolique du glutamate, sa détection immunologique n’est pas suffisante pour assurer le phénotype glutamatergique d’un neurone. Récemment, la découverte de trois transporteurs vésiculaires du glutamate (VGLUT1-3) a grandement facilité l’identification des neurones glutamatergiques. Ces transporteurs sont nécessaires pour la libération de glutamate et constituent les premiers marqueurs morphologiques du phénotype glutamatergique. Il a été démontré que des neurones noradrénergiques expriment VGLUT2 et que des neurones sérotoninergiques expriment VGLUT3. Mais aucune évidence d’expression d’un des sous-types de VGLUT n’a été reportée pour les neurones dopaminergiques. Le but de notre travail était d’identifier quel sous-type de VGLUT est exprimé par les neurones dopaminergiques mésencéphaliques, et de déterminer si le phénotype glutamatergique de ces neurones peut être modulé dans des conditions particulières. Premièrement, nous avons utilisé des microcultures pour isoler les neurones dopaminergiques et des doubles marquages immunocytochimiques pour observer l’expression de VGLUT dans les neurones positifs pour la tyrosine hydroxylase (TH). Nous avons montré que la majorité (80%) des neurones TH+ isolés exprime spécifiquement VGLUT2. Cette expression est précoce au cours du développement in vitro et limitée aux projections axonales des neurones dopaminergiques. Toutefois, cette forte expression in vitro contraste avec la non-détection de ce transporteur dans les rats adultes in vivo. Nous avons décidé ensuite de regarder si l’expression de VGLUT2 pouvait être régulée pendant le développement cérébral de jeunes rats et sous des conditions traumatiques, par double hybridation in situ. Entre 14 et 16 jours embryonnaires, les marquages de VGLUT2 et de TH montraient une superposition significative qui n’était pas retrouvée à des stades ultérieurs. Dans le mésencéphale de jeunes rats postnataux, nous avons détecté l’ARNm de VGLUT2 dans environs 1-2% des neurones exprimant l’ARNm de TH dans la substance noire et l’aire tegmentaire ventrale (ATV). Pour explorer la régulation de l’expression de VGLUT2 dans des conditions traumatiques, nous avons utilisé la 6-hydroxydopamine (6-OHDA) pour léser les neurones dopaminergiques dans les jeunes rats. Dix jours après la chirurgie, nous avons trouvé que 27% des neurones dopaminergiques survivants dans l’ATV exprimaient l’ARNm de VGLUT2 dans les rats 6-OHDA. Finalement, nous avons observé la colocalisation de la protéine VGLUT2 dans les terminaisons TH positives par microscopie électronique. Dans les rats normaux, la protéine VGLUT2 est retrouvée dans 28% des terminaisons axonales TH dans le noyau accumbens. Dans les rats lésés à la 6-OHDA, nous avons observé une diminution considérable des terminaisons TH positives, et une augmentation dans la proportion (37%) des terminaisons dopaminergiques présentant du VGLUT2. Nos résultats suggèrent que le phénotype glutamatergique des neurones dopaminergiques est régulé au cours du développement, peut être réactivé dans des états pathologiques, et que ces neurones peuvent libérer du glutamate dans conditions spécifiques.

Impact de l'haploinsuffisance du gène Sim1 sur le développement et la fonction du noyau paraventriculaire de l'hypothalamus

L’obésité provient d’un déséquilibre de l’homéostasie énergétique, c’est-à-dire une augmentation des apports caloriques et/ou une diminution des dépenses énergétiques. Plusieurs données, autant anatomiques que physiologiques, démontrent que l’hypothalamus est un régulateur critique de l’appétit et des dépenses énergétiques. En particulier, le noyau paraventriculaire (noyau PV) de l’hypothalamus intègre plusieurs signaux provenant du système nerveux central (SNC) et/ou de la périphérie, afin de contrôler l’homéostasie énergétique via des projections axonales sur les neurones pré-ganglionnaires du système autonome situé dans le troc cérébral et la moelle épinière. Plusieurs facteurs de transcription, impliqués dans le développement du noyau PV, ont été identifiés. Le facteur de transcription SIM1, qui est produit par virtuellement tous les neurones du noyau PV, est requis pour le développement du noyau PV. En effet, lors d’une étude antérieure, nous avons montré que le noyau PV ne se développe pas chez les souris homozygotes pour un allèle nul de Sim1. Ces souris meurent à la naissance, probablement à cause des anomalies du noyau PV. Par contre, les souris hétérozygotes survivent, mais développent une obésité précoce. De façon intéressante, le noyau PV des souris Sim1+/- est hypodéveloppé, contenant 24% moins de cellules. Ces données suggèrent fortement que ces anomalies du développement pourraient perturber le fonctionnement du noyau PV et contribuer au développement du phénotype d’obésité. Dans ce contexte, nous avons entrepris des travaux expérimentaux ayant pour but d’étudier l’impact de l’haploinsuffisance de Sim1 sur : 1) le développement du noyau PV et de ses projections neuronales efférentes; 2) l’homéostasie énergétique; et 3) les voies neuronales physiologiques contrôlant l’homéostasie énergétique chez les souris Sim1+/-. A cette fin, nous avons utilisé : 1) des injections stéréotaxiques combinées à des techniques d’immunohistochimie afin de déterminer l’impact de l’haploinsuffisance de Sim1 sur le développement du noyau PV et de ses projections neuronales efférentes; 2) le paradigme des apports caloriques pairés, afin de déterminer l’impact de l’haploinsuffisance de Sim1 sur l’homéostasie énergétique; et 3) une approche pharmacologique, c’est-à-dire l’administration intra- cérébroventriculaire (i.c.v.) et/ou intra-péritonéale (i.p.) de peptides anorexigènes, la mélanotane II (MTII), la leptine et la cholécystokinine (CCK), afin de déterminer l’impact de l’haploinsuffisance de Sim1 sur les voies neuronales contrôlant l’homéostasie énergétique. Dans un premier temps, nous avons constaté une diminution de 61% et de 65% de l’expression de l’ARN messager (ARNm) de l’ocytocine (Ot) et de l’arginine-vasopressine (Vp), respectivement, chez les embryons Sim1+/- de 18.5 jours (E18.5). De plus, le nombre de cellules produisant l’OT et la VP est apparu diminué de 84% et 41%, respectivement, chez les souris Sim1+/- adultes. L’analyse du marquage axonal rétrograde des efférences du noyau PV vers le tronc cérébral, en particulier ses projections sur le noyau tractus solitaire (NTS) aussi que le noyau dorsal moteur du nerf vague (X) (DMV), a permis de démontrer une diminution de 74% de ces efférences. Cependant, la composition moléculaire de ces projections neuronales reste inconnue. Nos résultats indiquent que l’haploinsuffisance de Sim1 : i) perturbe spécifiquement le développement des cellules produisant l’OT et la VP; et ii) abolit le développement d’une portion importante des projections du noyau PV sur le tronc cérébral, et notamment ses projections sur le NTS et le DMV. Ces observations soulèvent donc la possibilité que ces anomalies du développement du noyau PV contribuent au phénotype d’hyperphagie des souris Sim1+/-. En second lieu, nous avons observé que la croissance pondérale des souris Sim1+/- et des souris Sim1+/+ n’était pas significativement différente lorsque la quantité de calories présentée aux souris Sim1+/- était la même que celle consommée par les souris Sim1+/+. De plus, l’analyse qualitative et quantitative des tissus adipeux blancs et des tissus adipeux bruns n’a démontré aucune différence significative en ce qui a trait à la taille et à la masse de ces tissus chez les deux groupes. Finalement, au terme de ces expériences, les souris Sim1+/--pairées n’étaient pas différentes des souris Sim1+/+ en ce qui a trait à leur insulinémie et leur contenu en triglycérides du foie et des masses adipeuses, alors que tous ces paramètres étaient augmentés chez les souris Sim1+/- nourries ad libitum. Ces résultats laissent croire que l’hyperphagie, et non une diminution des dépenses énergétiques, est la cause principale de l’obésité des souris Sim1+/-. Par conséquent, ces résultats suggèrent que : i) l’haploinsuffisance de Sim1 est associée à une augmentation de l’apport calorique sans toutefois moduler les dépenses énergétiques; ii) l’existence d’au moins deux voies neuronales issues du noyau PV : l’une qui régule la prise alimentaire et l’autre la thermogénèse; et iii) l’haploinsuffisance de Sim1 affecte spécifiquement la voie neuronale qui régule la prise alimentaire. En dernier lieu, nous avons montré que l’injection de MTII, de leptine ainsi que de CCK induit une diminution significative de la consommation calorique des souris des deux génotypes, Sim1+/+ et Sim1+/-. De fait, la consommation calorique cumulative des souris Sim1+/- et Sim1+/+ est diminuée de 37% et de 51%, respectivement, durant les 4 heures suivant l’administration i.p. de MTII comparativement à l’administration d’une solution saline. Lors de l’administration i.c.v. de la leptine, la consommation calorique cumulative des souris Sim1+/- et Sim1+/+ est diminuée de 47% et de 32%, respectivement. Finalement, l’injection i.p. de CCK diminue la consommation calorique des souris Sim1+/- et Sim1+/+ de 52% et de 36%, respectivement. L’ensemble des résultats suggère ici que l’haploinsuffisance de Sim1 diminue l’activité de certaines voies neuronales régulant l’homéostasie énergétique, et particulièrement de celles qui contrôlent la prise alimentaire. En résumé, ces travaux ont montré que l’haploinsuffisance de Sim1 affecte plusieurs processus du développement au sein du noyau PV. Ces anomalies du développement peuvent conduire à des dysfonctions de certains processus physiologiques distincts régulés par le noyau PV, et notamment de la prise alimentaire, et contribuer ainsi au phénotype d’obésité. Les souris hétérozygotes pour le gène Sim1 représentent donc un modèle animal unique, où l’hyperphagie, et non les dépenses énergétiques, est la principale cause de l’obésité. En conséquence, ces souris pourraient représenter un modèle expérimental intéressant pour l’étude des mécanismes cellulaires et moléculaires en contrôle de la prise alimentaire.

The Role of Antennapedia in Anteroposterior Patterning of the Drosophila Melanogaster Ventral Nervous System

A major unresolved question in developmental neurobiology is how the nervous system is adapted to the specific needs of the organism at different life stages. In the holometabolous insect Drosophila melanogaster, the larval ventral nervous system (VNS) is comprised of similar repeating segments, as opposed to the adult VNS, which varies greatly from segment to segment both in number and types of neurons. The adult-specific neurons of each segment are generated by 25 distinct types of neuronal progenitor cells called neuroblasts (NBs) that appear in a stereotyped array (Truman et al., 2004). Each NB divides repeatedly to produce a distinct set of daughter cells termed a lineage, which is bilaterally symmetric but present to varying degrees in each segment. These daughter cells can be distinguished by their position within the nervous system as well as by their axonal projections. Each of the 25 NBs produces neurons; if both daughter cells are present in a lineage then both sibling populations survived, whereas if only one projection is seen cell death occurred, leaving a hemilineage (half lineage). In some lineages, the same sibling type survives in all segments in which the lineage appears, but in others, the surviving sibling type varies across segments, resulting in a different morphology for the same lineage in different segments. How are these differences in survival and morphology controlled? The Hox genes provide positional information for developing structures along the anterior-posterior (AP) axis of animals. They encode transcription factors, thereby controlling the activity of genes down stream. In the postembryonic VNS, each NB lineage features its own characteristic expression pattern of Hox genes Antp and Ubx, which can vary from segment-to-segment, and can thereby cause variation in the number of neural cells and axonal projections that survive. This study defines the wild-type expression pattern of Antp and elucidates the role of Antp in gain of function studies. These studies are possible due to the MARCM (Mosaic Analysis with a Repressible Cell Marker) method, which allows the genetically manipulated cells to be specifically labeled in an otherwise normal, unlabeled organism. The results indicate that Antp is expressed in a segment-, lineage-, and hemilineage-specific manner. Antp is sufficient for both anterior and posterior transformations of particular lineages, including promotion of cell death and/or survival as well as axon guidance.

POU domain transcription factor Brn3b is critical for the normal development and survival of retinal ganglion cells

The POU domain transcription factor Brn3b/POU4F2 plays a critical role regulating gene expression in mouse retinal ganglion cells (RGCs). Previous investigations have shown that Brn3b is not required for initial cell fate specification or migration; however, it is essential for normal RGC differentiation. In contrast to wild type axons, the mutant neurites were phenotypically different: shorter, rougher, disorganized, and poorly fasciculated. Wild type axons stained intensely with axon specific marker tau-1, while mutant projections were weakly stained and the mutant projections showed strong labeling with dendrite specific marker MAP2. Brn-3b mutant axonal projections contained more microtubules and fewer neurofilaments, a dendritic characteristic, than the wild type. The mutant neurites also exhibited significantly weaker staining of neurofilament low-molecular-weight (NF-L) in the axon when compared to the wild type, and NF-L accumulation in the neuron cell body. The absence of Brn-3b results in an inability to form normal axons and enhanced apoptosis in RGCs, suggesting that Brn-3b may control a set of genes involved in axon formation. ^ Brn3b contains several distinct sequence motifs: a glycine/serine rich region, two histidine rich regions, and a fifteen amino acid conserved sequence shared by all Brn3 family members in the N-terminus and a POU specific and POU homeodomain in the C-terminus. Brn3b activates a Luciferase reporter over 25 fold in cell culture when binding to native brn3 binding sites upstream of a minimal promoter. When fused to the Gal4 DNA Binding domain (DBD) and driven by either a strong (CMV) or weaker (pAHD) promoter, the N-terminal of Brn3b is capable of similar activation when binding to Gal4 UAS sites, indicating a presumptive activator of transcription. Both full length Brn3b or the C-terminus fused to the Gal4DBD and driven by pCMV repressed a Luciferase reporter downstream of UAS binding sites. Lower levels of expression of the fusion protein driven by pADH resulted in an alleviation of repression. This repression appears to be a limitation of this system of transcriptional analysis and a potential pitfall in conventional pCMV based transfection assays. ^