Neurons of the Dentate Molecular Layer in the Rabbit Hippocampus

The molecular layer of the dentate gyrus appears as the main entrance gate for information into the hippocampus, i.e., where the perforant path axons from the entorhinal cortex synapse onto the spines and dendrites of granule cells. A few dispersed neuronal somata appear intermingled in between and probably control the flow of information in this area. In rabbits, the number of neurons in the molecular layer increases in the first week of postnatal life and then stabilizes to appear permanent and heterogeneous over the individuals’ life span, including old animals. By means of Golgi impregnations, NADPH histochemistry, immunocytochemical stainings and intracellular labelings (lucifer yellow and biocytin injections), eight neuronal morphological types have been detected in the molecular layer of developing adult and old rabbits. Six of them appear as interneurons displaying smooth dendrites and GABA immunoreactivity: those here called as globoid, vertical, small horizontal, large horizontal, inverted pyramidal and polymorphic. Additionally there are two GABA negative types: the sarmentous and ectopic granular neurons. The distribution of the somata and dendritic trees of these neurons shows preferences for a definite sublayer of the molecular layer: small horizontal, sarmentous and inverted pyramidal neurons are preferably found in the outer third of the molecular layer; vertical, globoid and polymorph neurons locate the intermediate third, while large horizontal and ectopic granular neurons occupy the inner third or the juxtagranular molecular layer. Our results reveal substantial differences in the morphology and electrophysiological behaviour between each neuronal archetype in the dentate molecular layer, allowing us to propose a new classification for this neural population

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