Arquitectura subcelular del sistema endocannabinoide en el núcleo ventromedial del hipotálamo de ratón

356 p. : il., graf. Nota: Contiene versión resumida en inglés p.237-256 con el título “Subcellular architecture of the endocannabinoid system in the mouse ventromedial nucleus of the hypothalamus”El sistema endocannabinoide (eCB) es un complejo sistema endógeno de señalización, que participa en la modulación de la transmisión sináptica en múltiples circuitos cerebrales, incluyendo los que regulan el apetito, ingesta y balance energético. Por su parte, el hipotálamo, y en concreto el núcleo ventromedial (VMH), también participa en estas funciones. Esta Tesis Doctoral se ha centrado en el estudio de la localización subcelular de elementos clave del sistema eCB en el VMH de ratón, como el receptor CB1 y las enzimas de síntesis y degradación de los principales endocannabinoides (NAPE-PLD y FAAH para la anandamida y DAGL-¿ y MAGL para el 2-AG). Para ello, se han empleado ratones silvestres y mutantes condicionales junto con técnicas inmunocitoquímicas de alta resolución para microscopía electrónica.En resumen, el receptor CB1 en el VMH se distribuye en el 20% de las terminales sinápticas, sobre todo GABAérgicas, así como en terminales glutamatérgicas mayoritariamente de origen subcortical y, en menor medida, de origen cortical, mostrando una densidad similar en las terminales excitadoras e inhibidoras (0,4-0,5 partículas/¿m). Además, el 40% de los astrocitos del VMH son inmunopositivos para CB1. En lo que respecta a las enzimas, NAPE-PLD está presente a nivel presináptico y postsináptico pero con una distribución preferente en dendritas, al ser la mitad de ellas inmunopositivas; mientras que FAAH presenta una clara localización postsináptica siendo el 60% de las dendritas positivas. En cuanto al 2-AG, DAGL-¿ tiene una franca localización postsináptica en las membranas del 54% de las dendritas y del 44% de las espinas dendríticas; mientras que MAGL se distribuye en perfiles presinápticos y postsinápticos mayoritariamente en dendritas, al ser el 84% de ellas inmunopositivas. Por último, la densidad de marcado de FAAH en dendritas (8,6 partículas/¿m2) es mucho mayor que la de las otras enzimas (1,9-3,9 partículas/¿m2), y la densidad de todas ellas es más homogénea en las terminales sinápticas (3,0-3,7 partículas/¿m2). Estos resultados anatómicos contribuyen a un mejor entendimiento de la compleja modulación mediada por el sistema endocannabinoide de las funciones reguladas por el núcleo ventromedial del hipotálamo, incluyendo el control del apetito, la ingesta y el balance energético.Financiada con beca predoctoral para la formación de investigadores del Gobierno Vasco. Referencia: BFI07.28

Localization and Function of the Cannabinoid CB1 Receptor in the Anterolateral Bed Nucleus of the Stria Terminalis

11 p.Background: The bed nucleus of the stria terminalis (BNST) is involved in behaviors related to natural reward, drug addiction and stress. In spite of the emerging role of the endogenous cannabinoid (eCB) system in these behaviors, little is known about the anatomy and function of this system in the anterolateral BNST (alBNST). The aim of this study was to provide a detailed morphological characterization of the localization of the cannabinoid 1 (CB1) receptor a necessary step toward a better understanding of the physiological roles of the eCB system in this region of the brain. Methodology/Principal Findings: We have combined anatomical approaches at the confocal and electron microscopy level to ex-vivo electrophysiological techniques. Here, we report that CB1 is localized on presynaptic membranes of about 55% of immunopositive synaptic terminals for the vesicular glutamate transporter 1 (vGluT1), which contain abundant spherical, clear synaptic vesicles and make asymmetrical synapses with alBNST neurons. About 64% of vGluT1 immunonegative synaptic terminals show CB1 immunolabeling. Furthermore, 30% and 35% of presynaptic boutons localize CB1 in alBNST of conditional mutant mice lacking CB1 mainly from GABAergic neurons (GABA-CB1-KO mice) and mainly from cortical glutamatergic neurons (Glu-CB1-KO mice), respectively. Extracellular field recordings and whole cell patch clamp in the alBNST rat brain slice preparation revealed that activation of CB1 strongly inhibits excitatory and inhibitory synaptic transmission. Conclusions/Significance: This study supports the anterolateral BNST as a potential neuronal substrate of the effects of cannabinoids on stress-related behaviors.Dr. Pedro Grandes' laboratory is supported by The Basque Country Government grant GIC07/70-IT-432-07, by Red de Trastornos Adictivos (RETICS), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (MICINN), grant RD07/0001/2001 and MICINN grant SAF2009-07065. Nagore Puente is supported by a Basque Country University grant for PhD Researcher's Specialization. Leire Reguero is in receipt of a predoctoral fellowship from the Basque Country Government. Dr. Olivier J. Manzoni's laboratory is supported by INSERM, ANR Neurosciences “Neurologie et Psychiatrie ANR-06-NEURO-043-01” and Région Aquitaine. Dr. Giovanni Marsicano's laboratory is supported by AVENIR/INSERM (with the Fondation Bettencourt-Schueller), by ANR (ANR-06-NEURO-043-01), by European Foundation for the Study of Diabetes (EFSD), by the EU-FP7 (REPROBESITY, contract number HEALTH-F2-2008-223713) and European Commission Coordination Action ENINET (contract number LSHM-CT-2005-19063). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Monosynaptic and polysynaptic feed-forward inputs to mitral cells from olfactory sensory neurons.

Olfactory sensory neurons (OSNs) expressing the same odorant receptor converge in specific glomeruli where they transmit olfactory information to mitral cells. Surprisingly, synaptic mechanisms underlying mitral cell activation are still controversial. Using patch-clamp recordings in mouse olfactory bulb slices, we demonstrate that stimulation of OSNs produces a biphasic postsynaptic excitatory response in mitral cells. The response was initiated by a fast and graded monosynaptic input from OSNs and followed by a slower component of feedforward excitation, involving dendro-dendritic interactions between external tufted, tufted and other mitral cells. The mitral cell response occasionally lacked the fast OSN input when few afferent fibers were stimulated. We also show that OSN stimulation triggers a strong and slow feedforward inhibition that shapes the feedforward excitation but leaves unaffected the monosynaptic component. These results confirm the existence of direct OSN to mitral cells synapses but also emphasize the prominence of intraglomerular feedforward pathways in the mitral cell response.journal articleresearch support, non-u.s. gov't2011 Jun 15importe

Altered glial expression of the cannabinoid 1 receptor in the subiculum of a mouse model of Alzheimer's disease.

The alteration of the endocannabinoid tone usually associates with changes in the expression and/or function of the cannabinoid CB1 receptor. In Alzheimer's disease (AD), amyloid beta (Aβ)-containing aggregates induce a chronic inflammatory response leading to reactivity of both microglia and astrocytes. However, how this glial response impacts on the glial CB1 receptor expression in the subiculum of a mouse model of AD, a brain region particularly affected by large accumulation of plaques and concomitant subcellular changes in microglia and astrocytes, is unknown. The CB1 receptor localization in both glial cells was investigated in the subiculum of male 5xFAD/CB2EGFP/f/f (AD model) and CB2EGFP/f/f mice by immuno-electron microscopy. The findings revealed that glial CB1 receptors suffer remarkable changes in the AD mouse. Thus, CB1 receptor expression increases in reactive microglia in 5xFAD/CB2EGFP/f/f, but remains constant in astrocytes with CB1 receptor labeling rising proportionally to the perimeter of the reactive astrocytes. Not least, the CB1 receptor localization in microglial processes in the subiculum of controls and closely surrounding amyloid plaques and dystrophic neurites of the AD model, supports previous suggestions of the presence of the CB1 receptor in microglia. These findings on the correlation between glial reactivity and the CB1 receptor expression in microglial cells and astrocytes, contribute to the understanding of the role of the endocannabinoid system in the pathophysiology of Alzheimer's disease.post-print4763 K

GABAergic and Cortical and Subcortical Glutamatergic Axon Terminals Contain CB1 Cannabinoid Receptors in the Ventromedial Nucleus of the Hypothalamus

Background: Type-1 cannabinoid receptors (CB1R) are enriched in the hypothalamus, particularly in the ventromedial hypothalamic nucleus (VMH) that participates in homeostatic and behavioral functions including food intake. Although CB1R activation modulates excitatory and inhibitory synaptic transmission in the brain, CB1R contribution to the molecular architecture of the excitatory and inhibitory synaptic terminals in the VMH is not known. Therefore, the aim of this study was to investigate the precise subcellular distribution of CB1R in the VMH to better understand the modulation exerted by the endocannabinoid system on the complex brain circuitries converging into this nucleus. Methodology/Principal Findings: Light and electron microscopy techniques were used to analyze CB1R distribution in the VMH of CB1R-WT, CB1R-KO and conditional mutant mice bearing a selective deletion of CB1R in cortical glutamatergic (Glu-CB1R-KO) or GABAergic neurons (GABA-CB1R-KO). At light microscopy, CB1R immunolabeling was observed in the VMH of CB1R-WT and Glu-CB1R-KO animals, being remarkably reduced in GABA-CB1R-KO mice. In the electron microscope, CB1R appeared in membranes of both glutamatergic and GABAergic terminals/preterminals. There was no significant difference in the percentage of CB1R immunopositive profiles and CB1R density in terminals making asymmetric or symmetric synapses in CB1R-WT mice. Furthermore, the proportion of CB1R immunopositive terminals/preterminals in CB1R-WT and Glu-CB1R-KO mice was reduced in GABA-CB1R-KO mutants. CB1R density was similar in all animal conditions. Finally, the percentage of CB1R labeled boutons making asymmetric synapses slightly decreased in Glu-CB1R-KO mutants relative to CB1R-WT mice, indicating that CB1R was distributed in cortical and subcortical excitatory synaptic terminals. Conclusions/Significance: Our anatomical results support the idea that the VMH is a relevant hub candidate in the endocannabinoid-mediated modulation of the excitatory and inhibitory neurotransmission of cortical and subcortical pathways regulating essential hypothalamic functions for the individual's survival such as the feeding behavior.L. Reguero is in receipt of a Predoctoral Fellowship from the Basque Country Government (BFI 07.286); I. Buceta is in receipt of a Predoctoral Fellowship from the Basque Country University. Dr. Pedro Grandes' laboratory is supported by The Basque Country Government grant GIC07/70-IT-432-07, by Ministerio de Ciencia e Innovacion (SAF2009-07065) and by Red de Trastornos Adictivos, RETICS, Instituto de Salud Carlos III, MICINN, grant RD07/0001/2001. Dr. Giovanni Marsicano's laboratory is supported by AVENIR/INSERM (with the Fondation Bettencourt-Schueller), by ANR (ANR-06-NEURO-043-01), by European Foundation for the Study of Diabetes (EFSD), by the EU-FP7 (REPROBESITY, contract number HEALTH-F2-2008-223713) and European Commission Coordination Action ENINET (contract number LSHM-CT-2005-19063). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The endocannabinoid system controls food intake via olfactory processes

Comment in Sensory systems: the hungry sense. [Nat Rev Neurosci. 2014] Inhaling: endocannabinoids and food intake. [Nat Neurosci. 2014]; International audience; Hunger arouses sensory perception, eventually leading to an increase in food intake, but the underlying mechanisms remain poorly understood. We found that cannabinoid type-1 (CB1) receptors promote food intake in fasted mice by increasing odor detection. CB1 receptors were abundantly expressed on axon terminals of centrifugal cortical glutamatergic neurons that project to inhibitory granule cells of the main olfactory bulb (MOB). Local pharmacological and genetic manipulations revealed that endocannabinoids and exogenous cannabinoids increased odor detection and food intake in fasted mice by decreasing excitatory drive from olfactory cortex areas to the MOB. Consistently, cannabinoid agonists dampened in vivo optogenetically stimulated excitatory transmission in the same circuit. Our data indicate that cortical feedback projections to the MOB crucially regulate food intake via CB1 receptor signaling, linking the feeling of hunger to stronger odor processing. Thus, CB1 receptor-dependent control of cortical feedback projections in olfactory circuits couples internal states to perception and behavior

Arquitectura subcelular del sistema endocannabinoide en el núcleo ventromedial del hipotálamo de ratón

356 p. : il., graf. Nota: Contiene versión resumida en inglés p.237-256 con el título “Subcellular architecture of the endocannabinoid system in the mouse ventromedial nucleus of the hypothalamus”El sistema endocannabinoide (eCB) es un complejo sistema endógeno de señalización, que participa en la modulación de la transmisión sináptica en múltiples circuitos cerebrales, incluyendo los que regulan el apetito, ingesta y balance energético. Por su parte, el hipotálamo, y en concreto el núcleo ventromedial (VMH), también participa en estas funciones. Esta Tesis Doctoral se ha centrado en el estudio de la localización subcelular de elementos clave del sistema eCB en el VMH de ratón, como el receptor CB1 y las enzimas de síntesis y degradación de los principales endocannabinoides (NAPE-PLD y FAAH para la anandamida y DAGL-¿ y MAGL para el 2-AG). Para ello, se han empleado ratones silvestres y mutantes condicionales junto con técnicas inmunocitoquímicas de alta resolución para microscopía electrónica.En resumen, el receptor CB1 en el VMH se distribuye en el 20% de las terminales sinápticas, sobre todo GABAérgicas, así como en terminales glutamatérgicas mayoritariamente de origen subcortical y, en menor medida, de origen cortical, mostrando una densidad similar en las terminales excitadoras e inhibidoras (0,4-0,5 partículas/¿m). Además, el 40% de los astrocitos del VMH son inmunopositivos para CB1. En lo que respecta a las enzimas, NAPE-PLD está presente a nivel presináptico y postsináptico pero con una distribución preferente en dendritas, al ser la mitad de ellas inmunopositivas; mientras que FAAH presenta una clara localización postsináptica siendo el 60% de las dendritas positivas. En cuanto al 2-AG, DAGL-¿ tiene una franca localización postsináptica en las membranas del 54% de las dendritas y del 44% de las espinas dendríticas; mientras que MAGL se distribuye en perfiles presinápticos y postsinápticos mayoritariamente en dendritas, al ser el 84% de ellas inmunopositivas. Por último, la densidad de marcado de FAAH en dendritas (8,6 partículas/¿m2) es mucho mayor que la de las otras enzimas (1,9-3,9 partículas/¿m2), y la densidad de todas ellas es más homogénea en las terminales sinápticas (3,0-3,7 partículas/¿m2). Estos resultados anatómicos contribuyen a un mejor entendimiento de la compleja modulación mediada por el sistema endocannabinoide de las funciones reguladas por el núcleo ventromedial del hipotálamo, incluyendo el control del apetito, la ingesta y el balance energético.Financiada con beca predoctoral para la formación de investigadores del Gobierno Vasco. Referencia: BFI07.28

Visualization by high resolution immunoelectron microscopy of the transient receptor potential vanilloid-1 at inhibitory synapses of the mouse dentate gyrus.

We have recently shown that the transient receptor potential vanilloid type 1 (TRPV1), a non-selective cation channel in the peripheral and central nervous system, is localized at postsynaptic sites of the excitatory perforant path synapses in the hippocampal dentate molecular layer (ML). In the present work, we have studied the distribution of TRPV1 at inhibitory synapses in the ML. With this aim, a preembedding immunogold method for high resolution electron microscopy was applied to mouse hippocampus. About 30% of the inhibitory synapses in the ML are TRPV1 immunopositive, which is mostly localized perisynaptically (∼60% of total immunoparticles) at postsynaptic dendritic membranes receiving symmetric synapses in the inner 1/3 of the layer. This TRPV1 pattern distribution is not observed in the ML of TRPV1 knock-out mice. These findings extend the knowledge of the subcellular localization of TRPV1 to inhibitory synapses of the dentate molecular layer where the channel, in addition to excitatory synapses, is present

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