Distribución del sistema endocannabinoide durante el desarrollo postnatal temprano de la corteza cerebelosa de la rata. Efecto de la ausencia del receptor de cannabinoides CB1 en la sinapsis fibra paralela-célula de Purkinje

189 p.La corteza cerebelosa experimenta profundos cambios morfológicos y moleculares durante el período postnatal. La morfogénesis de las células granulares (CG) es uno de ellos e implica todos los procesos neuronales del desarrollo. Existen evidencias de la participación del sistema endocannabinoide (SEC) en los procesos del desarrollo. Además, se conoce que tanto el receptor CB1 como los enzimas de síntesis y degradación del eCB 2-AG, el DAGL-¿ y MAGL, respectivamente, están presentes en la corteza cerebelosa en la segunda semana postnatal; sin embargo, se desconoce su localización específica en las edades más tempranas. Nuestros resultados obtenidos mediante técnicas inmunohistoquímicas para microscopía convencional y microscopía electrónica, demuestran la presencia de estos tres integrantes del SEC en los días postnatales 0 (P0), P5, P12 en los procesos axónicos de las fibras paralelas (FP) de las CG en la capa granular externa y la capa molecular, sugiriendo su participación en los procesos del desarrollo neuronal acaecidos en esa franja del desarrollo postnatal. Habida cuenta de la participación del SEC en el desarrollo, quisimos determinar si la ausencia del mismo podría tener algún impacto en las características fenotípicas típicas de las terminales de las FP (TFP) en los animales adultos que carecen del gen para CB1. Para ellos, estudiamos diversos parámetros ultraestructurales de las sinapsis TFP-células de Purkinje. Centramos el estudio en los lóbulos 5 y 10 de los ratones adultos CB1-WT y CB1-KO debido a la distinta sensibilidad que presentan al daño en situaciones patológicas y, por tanto, la ausencia del receptor CB1 podría tener consecuencias diferentes. Demostramos que existen diferencias evidentes, centradas en la arquitectura presináptica en estas sinapsis. Demostramos además, que la distinta sensibilidad descrita para las divisiones cerebelosas estudiadas tiene una base anatómica ultraestructural

Environmental Enrichment Rescues Endocannabinoid-Dependent Synaptic Plasticity Lost in Young Adult Male Mice after Ethanol Exposure during Adolescence

Binge drinking (BD) is a serious health concern in adolescents as high ethanol (EtOH) consumption can have cognitive sequelae later in life. Remarkably, an enriched environment (EE) in adulthood significantly recovers memory in mice after adolescent BD, and the endocannabinoid, 2-arachydonoyl-glycerol (2-AG), rescues synaptic plasticity and memory impaired in adult rodents upon adolescent EtOH intake. However, the mechanisms by which EE improves memory are unknown. We investigated this in adolescent male C57BL/6J mice exposed to a drinking in the dark (DID) procedure four days per week for a duration of 4 weeks. After DID, the mice were nurtured under an EE for 2 weeks and were subjected to the Barnes Maze Test performed the last 5 days of withdrawal. The EE rescued memory and restored the EtOH-disrupted endocannabinoid (eCB)-dependent excitatory long-term depression at the dentate medial perforant path synapses (MPP-LTD). This recovery was dependent on both the cannabinoid CB1 receptor and group I metabotropic glutamate receptors (mGluRs) and required 2-AG. Also, the EE had a positive effect on mice exposed to water through the transient receptor potential vanilloid 1 (TRPV1) and anandamide (AEA)-dependent MPP long-term potentiation (MPP-LTP). Taken together, EE positively impacts different forms of excitatory synaptic plasticity in water- and EtOH-exposed brains.This research was funded by ISCIII (“RD16/0017/0012” to P.G.), co-funded by ERDF/ESF, “Investing in your future”; The Basque Government (IT1230-19 to P.G.); Ministry of Science and Innovation (PID2019-107548RB-I00 to P.G.); Ph.D. contract from MINECO (BES-2013-065057 to S.P.); Ph.D. contract from UPV/EHU (PIF 18/315 to L.L.), and Ph.D. contract from UPV/EHU (PIF 19/164 to M.S.)

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

Distribución del sistema endocannabinoide durante el desarrollo postnatal temprano de la corteza cerebelosa de la rata. Efecto de la ausencia del receptor de cannabinoides CB1 en la sinapsis fibra paralela-célula de Purkinje

189 p.La corteza cerebelosa experimenta profundos cambios morfológicos y moleculares durante el período postnatal. La morfogénesis de las células granulares (CG) es uno de ellos e implica todos los procesos neuronales del desarrollo. Existen evidencias de la participación del sistema endocannabinoide (SEC) en los procesos del desarrollo. Además, se conoce que tanto el receptor CB1 como los enzimas de síntesis y degradación del eCB 2-AG, el DAGL-¿ y MAGL, respectivamente, están presentes en la corteza cerebelosa en la segunda semana postnatal; sin embargo, se desconoce su localización específica en las edades más tempranas. Nuestros resultados obtenidos mediante técnicas inmunohistoquímicas para microscopía convencional y microscopía electrónica, demuestran la presencia de estos tres integrantes del SEC en los días postnatales 0 (P0), P5, P12 en los procesos axónicos de las fibras paralelas (FP) de las CG en la capa granular externa y la capa molecular, sugiriendo su participación en los procesos del desarrollo neuronal acaecidos en esa franja del desarrollo postnatal. Habida cuenta de la participación del SEC en el desarrollo, quisimos determinar si la ausencia del mismo podría tener algún impacto en las características fenotípicas típicas de las terminales de las FP (TFP) en los animales adultos que carecen del gen para CB1. Para ellos, estudiamos diversos parámetros ultraestructurales de las sinapsis TFP-células de Purkinje. Centramos el estudio en los lóbulos 5 y 10 de los ratones adultos CB1-WT y CB1-KO debido a la distinta sensibilidad que presentan al daño en situaciones patológicas y, por tanto, la ausencia del receptor CB1 podría tener consecuencias diferentes. Demostramos que existen diferencias evidentes, centradas en la arquitectura presináptica en estas sinapsis. Demostramos además, que la distinta sensibilidad descrita para las divisiones cerebelosas estudiadas tiene una base anatómica ultraestructural

Statistical analysis of CB₁R in the mouse VMH processed by a preembedding immunogold method.

<p>A: 20.5±1.3% of the synaptic terminals/preterminals are CB₁R immunopositive in <i>CB₁R</i>-WT mice. Similar proportion is in Glu-<i>CB₁R</i>-KO (20.8±0.5%, x² = 0.00024, p = 0.9876), lower in GABA-<i>CB₁R</i>-KO (12.4±1.2%, x² = 8.593, p = 0.0034) and virtually disappears in <i>CB₁R</i>-KO mice (3.9±0.6%, x² = 48.61, p<0.0001). A similar area was analyzed for each animal condition (1,467 µm² in <i>CB₁R</i>-WT; 1,562 µm² in Glu-<i>CB₁R</i>-KO; 1,646 µm² in GABA-<i>CB₁R</i>-KO and 1,519 µm² in <i>CB₁R</i>-KO mice). B: CB₁R immunoparticle density after subtraction of background labeling (0.015±0.003 particles/ µm in the VMH of <i>CB₁R</i>-KO) is very close in <i>CB₁R</i>-WT (0.49±0.07), Glu-<i>CB₁R</i>-KO (0.42±0.02, P = 0.7000) and GABA-<i>CB₁R</i>-KO (0.45±0.03, P = 0.7000) mice. C: There is no statistically significant difference between the percentage of CB₁R immunopositive asymmetric synapses in the VMH of <i>CB₁R</i>-WT (27.2±0.7%) and Glu-<i>CB₁R</i>-KO (21.3±2.5%, x² = 0.4189, p = 0.5175) mice. This value practically disappears in <i>CB₁R</i>-KO mice (2.9±2.9%, x² = 15.47, p<0.0001). A similar area was analyzed for each animal condition (1,352 µm² in <i>CB₁R</i>-WT; 1,547 µm² in Glu-<i>CB₁R</i>-KO and 1,274 µm² in <i>CB₁R</i>-KO mice).</p

CB₁R immunostaining in the mouse VMH.

<p>Preembedding immunoperoxidase method for light microscopy. VMH (oval circle in A-D) shows a moderate punctate CB₁R staining in <i>CB₁R</i>-WT (A, A') and Glu-<i>CB₁R</i>-KO (B, B') mice. However, the CB₁R immunoreaction product decreases in the VMH of GABA-<i>CB₁R</i>-KO mice, particularly in the dorsomedial part (C, C'). The CB₁R pattern disappears in <i>CB₁R</i>-KO tissue (D, D'). Framed areas in A, B, C, D are enlarged in A', B', C', D'. Scale bars: 100 µm (A, B, C, D), 50 µm (A', B', C', D').</p

Ultrastructural localization of CB₁R in the mouse VMH.

<p>Preembedding immunogold method for electron microscopy. A, B: In <i>CB₁R</i>-WT, CB₁R immunoparticles (arrows) are localized on membranes of presynaptic terminals (Ter) making asymmetric (white arrowheads) and symmetric synapses (black arrowheads) with dendritic spines (Sp) or dendrites (Den). C-E: In Glu-<i>CB₁R</i>-KO, CB₁R immunoparticles (arrows) localize to asymmetric synaptic terminals (Ter) presumably of excitatory subcortical neurons (observe the thick postsynaptic density marked with white arrowheads in D) as well as in inhibitory terminals (Ter) with symmetric synapses (black arrowheads in E). Notice CB₁R immunonegative axon terminals (Ter) establishing asymmetric synapses (white arrowheads in C, E) with a dendrite (Den) or a spine (Sp). F-H: In GABA-<i>CB₁R</i>-KO, CB₁R immunolabeling (arrows) is in excitatory synaptic terminals (Ter) (see asymmetric synapses with white arrowheads in F and G) impinging on dendritic elements (Den). Observe in H a CB₁R immunonegative synaptic terminal (Ter) making a symmetric synapse (black arrowheads) with a dendrite (Den). I, J: CB₁R immunolabeling disappears in <i>CB₁R</i>-KO mice indicating the specificity of the CB₁R antibody used in the study. Note CB₁R immunonegative synaptic terminals (Ter) making asymmetric (white arrowheads in I) and symmetric (black arrowheads in J) synapses with a dendritic spine (Sp) and a soma (Som), respectively. Scale bars: 0.4 µm.</p

Statistical analysis of CB₁R in terminals forming asymmetric and symmetric synapses in the VMH of <i>CB₁R</i>-WT mice processed by a preembedding immunogold method.

<p>A: 24.0±2.9% of synaptic terminals making asymmetric and 28.9±7.5% of terminals with symmetric synapses are CB₁R immunopositive. No statistically significant difference is detected (x² = 0.5946, p = 0.4406, analyzed area: 2,376 µm²). B: CB₁R density after subtraction of background labeling (0.015±0.003 particles/ µm in the VMH of <i>CB₁R</i>-KO) is pretty similar in synaptic terminals making asymmetric or symmetric synapses (0.42±0.03 and 0.47±0.09 immunoparticles/ µm respectively, p = 0.6553).</p