Keywords: Système endocannabinoïde, synapses, microscopie électronique, hippocampe, souris mutantes Endocannabinoid system, synapses, electron microscopy, hippocampus, mutant rodents

The Cannabinoid Type I receptor protein (CB1) expression in the hippocampus of rescue mice modified to express the gene exclusively in specific brain cell types : such as dorsal telencephalic glutamatergic neurons, or GABAergic neurons have been analysed. Furthermore, aiming at knowing the exact anatomical distribution of the astroglial CB1 receptors with respect to the excitatory and inhibitory synapses, the CB1 receptor expression in astrocytes of mouse expressing CB1 receptor only in astrocytes and mutant mouse expressing the protein hrGFP into astrocytes (that allows for better detection of the astrocytic processes) have been also investigated. The results showed that the majority of the hippocampal synapses surrounded by CB1 receptor immunopositive astrocytes in the 400-800 nm range are of excitatory nature. Moreover, the CB1 receptor rescue mutant mice characterized in this Doctoral Thesis have proven 1) to express CB1 receptors in specific brain cell types; 2) the re-expression is limited to the particular brain cell populations; 3) the endogenous levels of CB1 receptors are maintained in the brain cell types re-expressing the receptor. Which makes this mutant mice excellent tools for functional and translational investigations on the role of the CB1 receptors in the normal and diseased brain.

Titre :

Caractérisation anatomique des récepteurs cannabinoïdes de type 1 dans des populations de cellules cérébrales spécifiques de souris mutantes.

Résumé : Dans cette thèse l'expression du récepteur CB1 dans l'hippocampe de souris mutantes ré-exprimant spécifiquement le gène spécifiquement dans certains types cellulaires du cerveau tels que : les neurones glutamatergiques du télencéphale dorsal, et les neurones GABAergiques a été analysé. De plus, dans le but de connaître la distribution anatomique exacte des récepteurs CB1 astrogliaux par rapport aux synapses excitatrices et inihibitrices, j'ai étudié l'expression des récepteurs CB1 dans les astrocytes de souris exprimant le récepteur CB1 seulement dans les astrocytes et une souris mutante ciblée pour exprimer la protéine cytoplasmique hrGFP diffusible dans les cellules astrogliales ce qui permet une meilleure détection des prolongements astrocytaires. Les conclusions de ce travail de thèse sont les suivantes: La distance la plus commune entre le récepteur CB1 astroglial et la synapse la plus proche est de 400 à 800 nm. La majorité des synapses entourées par des astrocytes immunopositifs pour le récepteur CB1 dans l'hippocampe, est de nature excitatrice. Les souris mutantes réexprimants le récepteur CB1 caractérisées dans ce travail de thèse montre : 1) expression du récepteur CB1 dans différents types cellulaires, 2) la réexpression est limitée à une population neuronale particulière ou aux astrocytes, 3) les niveaux endogènes de récepteurs CB1 sont maintenus dans les différents types cellulaires ré-exprimants le récepteur CB1. De façon générale, ces résultats nous montrent que les souris mutantes ré-exprimants le récepteur CB1 sont d'excellents outils pour l'étude fonctionnelle et translationnelles sur le rôle de ce récepteur dans le cerveau sain ou pathologique.

FRENCH SUMMARY

INTRODUCTION

Le système endocannabinoïde est principalement formé par des ligands endogènes lipides (endocannabinoïdes), le Récepteur au cannabinoïde de type 1 (CB1) et de type 2 (CB2), ainsi que leurs protéines de synthèse, de transport et de dégradation [START_REF] Piomelli | The molecular logic of endocannabinoid signaling[END_REF][START_REF] Marsicano | Neuromodulatory functions of the endocannabinoid system[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] Pertwee | Endocannabinoids and Their Pharmacological Actions[END_REF][START_REF] Lu | An Introduction to the Endogenous Cannabinoid System[END_REF]. Ce système est impliqué dans plusieurs fonctions cérébrales, le développement prénatal du cerveau et de nombreuses fonctions cognitives [START_REF] Marsicano | Anatomical Distribution of Receptors, Ligands and Enzymes in the Brain and in the Spinal Cord: Circuitries and Neurochemistry[END_REF][START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF]. L'expression du récepteur CB1 est très élevée dans les neurones inhibiteurs GABAergiques concentrée sur leurs terminaisons synaptiques [START_REF] Katona | Presynaptically Located CB1 Cannabinoid Receptors Regulate GABA Release from Axon Terminals of Specific Hippocampal Interneurons[END_REF][START_REF] Kawamura | The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum[END_REF][START_REF] Ludányi | Downregulation of the CB1 cannabinoid receptor and related molecular elements of the endocannabinoid system in epileptic human hippocampus[END_REF][START_REF] Marsicano | Anatomical Distribution of Receptors, Ligands and Enzymes in the Brain and in the Spinal Cord: Circuitries and Neurochemistry[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] De-May | Cell type-specific regulation of inhibition via cannabinoid type 1 receptors in rat neocortex[END_REF][START_REF] Steindel | Neuron-type specific cannabinoid-mediated G protein signaling in mouse hippocampus[END_REF][START_REF] Hu | Distribution of the Endocannabinoid System in the Central Nervous System[END_REF] et faible dans les neurones glutamatergiques avec une localisation rare des récepteurs sur leurs boutons axonaux [START_REF] Marsicano | CB1 cannabinoid receptors and on-demand defense against excitotoxicity[END_REF][START_REF] Domenici | Cannabinoid receptor type 1 located on presynaptic terminals of principal neurons in the forebrain controls glutamatergic synaptic transmission[END_REF][START_REF] Takahashi | The CB1 cannabinoid receptor mediates glutamatergic synaptic suppression in the hippocampus[END_REF][START_REF] Katona | Molecular composition of the endocannabinoid system at glutamatergic synapses[END_REF][START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF][START_REF] Kamprath | Endocannabinoids mediate acute fear adaptation via glutamatergic neurons independently of corticotropin-releasing hormone signaling[END_REF][START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF][START_REF] Puente | Polymodal activation of the endocannabinoid system in the extended amygdala[END_REF][START_REF] Reguero | GABAergic and cortical and subcortical glutamatergic axon terminals contain CB1 cannabinoid receptors in the ventromedial nucleus of the hypothalamus[END_REF][START_REF] Steindel | Neuron-type specific cannabinoid-mediated G protein signaling in mouse hippocampus[END_REF][START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF]. Les récepteurs CB1 sont aussi localisés dans les membranes mitochondriales dans le cerveau et les muscles [START_REF] Bénard | Mitochondrial CB₁ receptors regulate neuronal energy metabolism[END_REF]Hebert-Chatelain et al., 2014a;b;[START_REF] Koch | Hypothalamic POMC neurons promote cannabinoid-induced feeding[END_REF] et dans les astrocytes où ils jouent un rôle clé dans le contexte de la synapse tripartite (Navarrete andAraque, 2008, 2010;[START_REF] Perea | Tripartite synapses: astrocytes process and control synaptic information[END_REF][START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF][START_REF] Gómez-Gonzalo | Endocannabinoids Induce Lateral Long-Term Potentiation of Transmitter Release by Stimulation of Gliotransmission[END_REF][START_REF] Navarrete | Astrocytes in endocannabinoid signaling[END_REF][START_REF] Metna-Laurent | Rising stars: Modulation of brain functions by astroglial type-1 cannabinoid receptors[END_REF][START_REF] Viader | Metabolic Interplay between Astrocytes and Neurons Regulates Endocannabinoid Action[END_REF][START_REF] Oliveira Da Cruz | Astroglial type-1 cannabinoid receptor (CB1): A new player in the tripartite synapse[END_REF]. En raison de la très grande différence de niveau d'expression entre les différents types de cellules [START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF], il est extrêmenent difficile d'identifier les faibles niveaux du récepteur CB1 dans ces types cellulaires et/ou dans les compartiments subcellulaires de cerveaux de souris de type sauvage [START_REF] Busquets-Garcia | Dissecting the cannabinergic control of behavior: The where matters[END_REF]. En effet, auparavant, de faibles niveaux d'expression du récepteur CB1 ont été considérés comme bruit de fond [START_REF] Freund | Role of endogenous cannabinoids in synaptic signaling[END_REF]. Pour établir correctement la distribution anatomique des récepteurs CB1 dans des types cellulaires spécifiques, indépendamment des niveaux d'expression endogènes, de nouvelles techniques d'analyse sont nécessaires. Par conséquent, la stratégie génétique de "sauvetage" (rescue) basée sur l'utilisation du système Cre/loxP [START_REF] Balthasar | Divergence of melanocortin pathways in the control of food intake and energy expenditure[END_REF] est apparue comme un excellent outil pour la détermination de l'expression du récepteur CB1 endogène dans des types cellulaires spécifiques [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF][START_REF] De Salas-Quiroga | Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons[END_REF]. Ceci est particulièrement utile pour élucider les niveaux réels des récepteurs CB1 dans des types cellulaires spécifiques exprimant de faibles niveaux de la protéine CB1, ce qui élimine les interférences causées par des cellules qui expriment des niveaux élevés de récepteurs CB1. Cependant, il est très important de vérifier que la réexpression est limitée aux types de cellules recherchées et qu'elle maintient les niveaux des récepteurs CB1 endogènes, où les séquences régulatrices du récepteur sont normalement activées. J'ai examiné dans ma thèse l'expression du récepteur CB1 dans l'hippocampe de souris mutantes ré-exprimant spécifiquement le gène spécifiquement dans certains types cellulaires du cerveau tels que : les neurones glutamatergiques du télencéphale dorsal (souris Glu-CB 1 -RS), et les neurones GABAergiques (souris GABA-CB 1 -RS). De plus, dans le but de connaître la distribution anatomique exacte des récepteurs CB1 astrogliaux par rapport aux synapses excitatrices et inihibitrices, j'ai également étudié l'expression des récepteurs CB1 dans les astrocytes de souris GFAP-CB 1 -RS (exprimant le récepteur CB1 seulement dans les astrocytes) et une souris mutante ciblée pour exprimer la protéine cytoplasmique hrGFP diffusible dans les cellules astrogliales (GFAPhrGFP-CB 1 -WT) ce qui permet une meilleure détection des prolongements astrocytaires.

MATERIEL ET METHODES

Souris: CB 1 -WT, CB 1 -KO (absence des récepteurs CB1), GABA-CB 1 -RS, Glu-CB1-RS, GABA-CB 1 -KO (portant une délétion sélective du récepteur CB1 dans les neurons GABAergiques), Glu-CB 1 -KO (portant une délétion sélective du récepteur CB1 dans les neurons glutamatergiques du télencéphale dorsal), GFAP-CB1-RS, GFAP-CB1-KO (portant une délétion sélective du récepteur CB1 dans les astrocytes), GFAPhrGFP-CB 1 -WT et GFAPhrGFP-CB 1 -KO (absence des récepteurs CB1 dans les astrocytes). Les animaux ont été profondément anesthésiés et perfusés par voie transcardiaque.

Une méthode de pré-enrobage immunogold pour la microscopie électronique a été appliquée aux coupes hippocampales des souris CB 1 -WT, CB 1 -KO, GABA-CB 1 -RS, Glu-CB 1 -RS, GABA-CB 1 -KO et Glu-CB 1 -KO. Egalement, une méthode combinant un pré-enrobage immunogold et une immunoperoxidase a été utilisée pour les coupes hippocampales des souris GFAP-CB 1 -RS, GFAP-CB 1 -KO, GFAPhrGFP-CB 1 -WT et GFAPhrGFP-CB 1 -KO. Les expériences ont toujours été réalisées sur trois animaux différents pour chaque condition. Afin de déterminer statistiquement la distribution des récepteurs CB1 dans les différents types de cellules et compartiments cellulaires étudiés, un test de normalité a été appliqué (test de normalité de Kolmogorov-Smirnov) avant l'exécution des analyses statistiques. Les données ont été analysées à l'aide de tests non-paramétriques (Man-Whitney U test lorsque k=2 ou Kruskal-Wallis test lorsque k>2).

RESULTATS

La proportion de récepteurs CB1 immunopositifs au niveau des terminaisons synaptiques excitatrices du stratum radiatum de la région CA1 de l'hippocampe des Glu-CB 1 -RS (21.89% ± 1.21), CB 1 -WT et GABA-CB 1 -KO n'est pas statistiquement différente. Cependant, le pourcentage de terminaisons immunopositives disparait virtuellement chez les animaux Glu-CB 1 -KO, GABA-CB 1 -RS et CB 1 -KO. En outre, aucune différence statistique n'est trouvée pour la densité de récepteur CB1, exprimée comme étant le nombre de particules par µm du périmètre de la cellule (particules/µm), entre les terminaisons synaptiques excitatrices des Glu-CB 1 -RS (0.45 ± 0.02), CB 1 -WT et GABA-CB 1 -KO.

L'analyse du tiers interne de la couche dentée moléculaire montre des pourcentages de récepteurs immuno-positifs au niveau des terminaisons synaptiques excitatrices comparables chez les Glu-CB 1 -RS (53.19% ± 2.89), CB 1 -WT et GABA-CB 1 -KO. Cette proportion est quasi nulle chez les Glu-CB 1 -KO, GABA-CB 1 -RS et CB 1 -KO. La densité d'immunoparticules de récepteurs CB1 est statistiquement similaire chez les Glu-CB 1 -RS (0.39 ± 0.02) CB 1 -WT et GABA-CB 1 -KO.

Le nombre de récepteurs CB1 immunopositifs au niveau des terminaisons neuronales des synapses inhibitrices chez les GABA-CB 1 -RS (77.92% ± 2.63), CB 1 -WT et Glu-CB 1 -KO est maintenu dans le stratum radiatum de CA1. Cependant, la proportion disparait chez les GABA-CB 1 -KO, Glu-CB 1 -RS et CB 1 -KO. Il n'existe pas de différence significative pour la densité des immunoparticules du récepteur CB1 entre les terminaisons des synapses inhibitrices des GABA-CB 1 -RS (4.33 ± 0.11), CB 1 -WT et Glu-CB 1 -KO.

La proportion de récepteurs CB1 immunopositifs au niveau des terminaisons des synapses inhibitrices du tiers interne de la couche dentée moléculaire est similaire entre les GABA-CB 1 -RS (85.07% ± 1.76), CB 1 -WT et Glu-CB 1 -KO. Ces valeurs disparaissent quasiment chez les GABA-CB 1 -KO, Glu-CB 1 -RS et CB 1 -KO. Il n'existe pas de différence significative pour la densité de récepteurs CB1 au niveau des synapses inhibitrices des GABA-CB 1 -RS (7.47 ± 0.14), CB 1 -WT et Glu-CB 1 -KO.

La proportion de récepteurs CB1 immunopositifs au niveau des prolongements astrocytaires du stratum radiatum n'est pas significativement différente entre les animaux CB 1 -WT et GFAP-CB 1 -RS (37.12% ± 3.79) Chez les animaux GFAPhrGFP-CB 1 -WT, la proportion d'éléments astrocytaires immunomarqués pour le récepteur CB1 augmente de 59.91 % ± 3.29, devenant statistiquement différent comparé au CB 1 -WT. A l'inverse, seuls de rares récepteurs CB1 sont marqués dans les astrocytes des souris GFAP-CB 1 -KO, CB 1 -KO et chez les souris GFAPhrGFP-CB 1 -KO. La densité d'immunoparticule pour le récepteur CB1 au niveau de la membrane astrocytaire (particules/µm) est également analysée. Nos données montrent qu'il n'y a pas de différence significative entre les CB 1 -WT et les GFAP-CB 1 -RS (0.128 ± 0.020), alors que nous avons une plus forte densité chez les GFAPhrGFP-CB 1 -WT (0.384 ± 0.039).

L'analyse du tiers interne de la couche dentée moléculaire montre des pourcentages de récepteurs immuno-positifs au niveau des prolongements astrocytaires comparables chez les GFAP-CB 1 -RS (39.84% ± 3.50) et les CB 1 -WT alors que cette proportion augmente de manière significative (59.99% ± 3.37) chez les GFAPhrGFP-CB 1 -WT. Seuls des particules résiduelles sont observées chez les souris GFAP-CB 1 -KO, GFAPhrGFP-CB 1 -KO et CB 1 -KO. Enfin, aucune différence significative de densité du récepteur CB1 n'est observée dans les prolongements astrocytaires des souris CB 1 -WT et GFAP-CB 1 -RS (0.138 ± 0.016). On observe une différence significative de densité du récepteur CB1 dans les prolongements astrocytaires des souris GFAPhrGFP-CB 1 -WT comparée au CB 1 -WT (0.334 ± 0.033).

Grâce à une meilleure détection des astrocytes obtenue avec la protéine hr-GFP en microscopie électronique, nous avons mesuré la distance entre les immunoparticules du récepteur CB1 astroglial et le point central de la synapse la plus proche entourée par l'élément astrocytaire immunopositif chez les GFAPhrGFP-CB 1 -WT. Dans le CA1, 51.07% ± 3.79 de toutes les synapses entourées par un prolongement astrocytaire se trouvent à une distance de 400 à 800 nm de la particule astrocytaire la plus proche. Parmi celles-ci, 43.33% ± 1.01 sont des synapses excitatrices alors que 9.91%+/-1.93 sont des synapses inhibitrices. Dans la couche moléculaire dentée, 57.25% ± 3.19 des synapses entourées par un prolongement astrocytaire se trouvent entre 400 et 800 nm de la particule du récepteur CB1 astroglial. 46.67% ± 2.17 sont au niveau de synapses excitatrices alors que 10.59% ± 2.18 se trouvent au niveau de synapses inhibitrices.

CONCLUSIONS

Les conclusions de ce travail de thèse sont les suivantes:

1. Le niveau d'expression et la localisation des récepteurs CB1 des neurones glutamatergiques de l'hippocampe de souris Glu-CB 1 -RS est similaire à celui de souris CB 1 -WT.

2. Le niveau d'expression et la localisation des récepteurs CB1 des neurones GABAergiques de l'hippocampe de souris GABA-CB 1 -RS est similaire à celui des CB 1 -WT. 3. Le niveau d'expression et la localisation des récepteurs CB1 dans les astrocytes de l'hippocampe de souris GFAP-CB 1 -RS est similaire à celui des CB 1 -WT. 4. La détection des récepteurs CB1 dans les astrocytes de l'hippocampe de souris GFAPhrGFP-CB 1 -WT est significativement supérieure comparée aux animaux CB 1 -WT avec un marqueur GFAP.

5. La distance la plus commune entre le récepteur CB1 astroglial et la synapse la plus proche est de 400 à 800 nm. 6. La majorité des synapses entourées par des astrocytes immunopositifs pour le récepteur CB1 dans la région CA1 et de la couche moléculaire dentée, est de nature excitatrice. 7. Les souris mutantes réexprimants le récepteur CB1 caractérisées dans ce travail de thèse montre: 1) expression du récepteur CB1 dans différents types cellulaires, 2) la réexpression est limitée à une population neuronale particulière ou aux astrocytes, 3) les niveaux endogènes de récepteurs CB1 sont maintenus dans les différents types cellulaires ré-exprimants le récepteur CB1. 8. De façon générale, ces résultats nous montrent que les souris mutantes réexprimants le récepteur CB1 sont d'excellents outils pour l'étude fonctionnelle et translationnelles sur le rôle de ce récepteur dans le cerveau sain ou pathologique.

SPANISH SUMMARY

INTRODUCCIÓN

El sistema endocannabinoide está compuesto, principalmente, por un grupo de ligandos lipídicos endógenos denominados endocannabinoides, los Receptores Cannabinoide de Tipo 1 (CB1) y Tipo 2 (CB2), así como las proteínas que participan en la síntesis, transporte y la degradación de los endocannabinoides [START_REF] Piomelli | The molecular logic of endocannabinoid signaling[END_REF][START_REF] Marsicano | Neuromodulatory functions of the endocannabinoid system[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] Pertwee | Endocannabinoids and Their Pharmacological Actions[END_REF][START_REF] Lu | An Introduction to the Endogenous Cannabinoid System[END_REF]. Este sistema se encuentra involucrado en numerosos mecanismos cerebrales, en el desarrollo del sistema nervioso prenatal y en diversas funciones cognitivas [START_REF] Marsicano | Anatomical Distribution of Receptors, Ligands and Enzymes in the Brain and in the Spinal Cord: Circuitries and Neurochemistry[END_REF]Bellocchio y coils., 2010;[START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF]. Respecto a la expresión del receptor CB1, se ha descrito como los terminales inhibidores de neuronas GABAérgicas presentan elevados niveles de expresión de este receptor (Katona y coils., 1999;Kawamura y coils., 2006;Ludányi y coils., 2008;[START_REF] Marsicano | Anatomical Distribution of Receptors, Ligands and Enzymes in the Brain and in the Spinal Cord: Circuitries and Neurochemistry[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] De-May | Cell type-specific regulation of inhibition via cannabinoid type 1 receptors in rat neocortex[END_REF]Steindel y coils., 2013;[START_REF] Hu | Distribution of the Endocannabinoid System in the Central Nervous System[END_REF] mientras en los terminales axónicos de neuronas glutamatérgicas encontramos niveles de expresión más bajos (Marsicano y coils., 2003;Domenici y coils., 2006;[START_REF] Takahashi | The CB1 cannabinoid receptor mediates glutamatergic synaptic suppression in the hippocampus[END_REF] Las diferencias existentes en los niveles de expresión de este receptor entre los diversos tipos celulares [START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF], hacen que resulte extremadamente difícil detectar su expresión en tipos de células y / o en compartimentos sub-celulares donde su expresión es fisiológicamente muy baja (Busquets-Garcia y coils., 2015). En ese sentido, en el pasado, la presencia de niveles bajos de receptor CB1 fue interpretada erróneamente como mera tinción de fondo en análisis anatómicos de ratones de silvestres (CB 1 -WT) (Freund y coils., 2003).

De esta manera, para la correcta caracterización anatómica de la distribución subcelular de los receptores CB1 en cualquier tipo celular, independientemente de sus niveles fisiológicos de expresión, se hace necesarias mejores técnicas de análisis. En ese sentido, las estrategias de "rescate" genético mediante el uso del sistema Cre/LoxP (Balthasar y coils., 2005) suponen una gran herramienta de estudio, ya que permiten analizar la expresión del receptor CB1 endógeno exclusivamente en tipos celulares determinados (Ruehle y coils., 2013; Soria-Gómez y coils., 2014; de Salas-Quiroga y coils.). En concreto, esta estrategia resulta particularmente útil para determinar el patrón de expresión del receptor CB1 en tipos específicos de células donde sus niveles de expresión son bajos y puedan encontrarse enmascarados por la enorme expresión de este receptor en otros tipos de células. Sin embargo, es de gran importancia comprobar que la reexpresión del receptor CB1 mantiene sus niveles endógenos y se limita a las células deseadas.

En mi tesis doctoral se pretende examinar la expresión receptor CB1 en el hipocampo de ratones rescatados genéticamente los cuales expresan el gen del receptor CB1 exclusivamente en determinados tipos celulares: neuronas glutamatérgicas telencefálicas dorsales (ratones Glu-CB 1 -RS) y neuronas GABAérgicas (ratones GABA-CB 1 -RS).

Así mismo, con el objetivo de conocer la distribución del receptor CB1 de astrocitos, respecto a las sinapsis excitadoras e inhibidoras, se realizará el análisis de la expresión del CB1 en ratones que expresan el receptor CB1 únicamente en astrocitos, (GFAP-CB 1 -RS) y en ratones transgénicos que expresan en astrocitos la proteína citoplasmática difusible hrGFP (ratones GFAPhrGFP-CB 1 -WT) que permite una mejor detección de la superficie de los astrocitos.

MATERIAL Y MÉTODOS

Ratones: CB1-WT, CB 1 -KO (no expresan receptor CB1), GABA-CB 1 -RS, Glu-CB1-RS, GABA-CB 1 -KO (no expresan el receptor CB1 en neuronas GABAérgicas), Glu-CB1-KO (no expresan el receptor CB1 en neuronas glutamatérgicas telencefálicas dorsales), GFAP-CB 1 -RS, GFAP-CB 1 -KO (no expresan el receptor CB1 en astrocitos), GFAPhrGFP-CB 1 -WT y GFAPhrGFP-CB 1 -KO (no expresan el receptor CB1), fueron profundamente anestesiados y sometidos a perfusión transcardiaca.

La técnica de inmuno-oro preinclusión para Microscopía Electrónica fue aplicada a secciones del hipocampo de ratones CB 1 -WT, CB 1 -KO, GABA-CB 1 -RS, Glu-CB 1 -RS, GABA-CB 1 -KO y Glu-CB 1 -KO. Mientras que a las secciones de hipocampo de ratones GFAP-CB 1 -RS, GFAP-CB 1 -KO, GFAPhrGFP-CB 1 -WT y GFAPhrGFP-CB 1 -KO se les aplicó la técnica de inmuno-oro e inmunoperoxidasa preinclusión para Microscopía Electrónica. En todos los casos los análisis se llevaron a cabo utilizando tres animales de cada tipo. En estos animales se analizó la distribución anatómica del receptor CB1 en los diferentes tipos celulares y compartimentos subcelulares. Los datos obtenidos fueron analizados aplicando primero la prueba de normalidad de Kolmogorov-Smirnov para posteriormente analizar estadísticamente los datos mediante test no paramétricos de contraste de medias (prueba de Man-Whitney cuando k = 2 o la prueba de Kruskal-Wallis cuando k> 2).

RESULTADOS

La comparación del porcentaje de terminales excitadores marcados con CB1 en el stratum radiatum del CA1 en el ratón Glu-CB 1 -RS (21,89% ± 1,21) en el ratón CB 1 -WT y en el ratón GABA-CB 1 -KO no fue estadísticamente significativo. Sin embargo, este valor prácticamente desaparece en los ratones Glu-CB 1 -KO, GABA-CB 1 -RS and CB 1 -KO. Además, no se encontraron diferencias significativas entre el valor de la densidad, expresada como el número de partículas del receptor CB1 por µm del perímetro de la membrana (partículas/µm), entre los terminales excitadores de Glu-CB 1 -RS (0.45 ± 0.02), CB 1 -WT y GABA-CB 1 -KO.

En el tercio interno de la capa molecular del giro dentado similares porcentajes de terminales excitadores que presentan receptor CB1 se encontraron en Glu-CB 1 -RS (53.19% ± 2.89), CB 1 -WT y GABA-CB 1 -KO. Desapareciendo prácticamente esta proporción en los ratones Glu-CB 1 -KO, GABA-CB 1 -RS and CB 1 -KO. La densidad de inmunopartículas de CB1 fue estadísticamente similar entre Glu-CB 1 -RS (0.39 ± 0.02) CB 1 -WT y GABA-CB 1 -KO.

El valor del porcentaje de terminales inhibidores marcados positivamente para el receptor CB1 en los ratones GABA-CB 1 -RS (77.92% ± 2.63), CB 1 -WT y Glu-CB 1 -KO se mantuvo en el stratum radiatum de la región CA1. Sin embargo, esta proporción prácticamente desaparece en los animales GABA-CB 1 -KO, Glu-CB 1 -RS y CB 1 -KO. No se encontraron diferencias significativas tras la comparación de la densidad de partículas de CB1 en los terminales inhibidores de los ratones GABA-CB 1 -RS (4.33 ± 0.11 partículas/µm), CB 1 -WT y Glu-CB 1 -KO.

En el tercio interno de la capa molecular del giro dentado, no se obtuvieron diferencias significativas comparando los valores obtenidos para el porcentaje de terminales inhibidores marcados con CB1 en ratones GABA-CB 1 -RS (85.07% ± 1.76), CB 1 -WT y Glu-CB 1 -KO. Solamente valores residuales del receptor CB1 se hallaron en animales GABA-CB 1 -KO, Glu-CB 1 -RS y CB 1 -KO. No hubo diferencias significativas tras el análisis estadístico de la densidad de partículas de CB1 en los terminales inhibidores entre los ratones GABA-CB 1 -RS (7.47 ± 0.14), CB 1 -WT y Glu-CB 1 -KO En el stratum radiatum de la región CA1 el análisis de los procesos astrocíticos que presentaban un marcaje positivo para CB1 no resulto estadísticamente significativo al comparar los valores obtenidos en el ratón CB 1 -WT y el ratón GFAP-CB 1 -RS (37.12% ± 3.79). Sin embargo, la proporción de procesos astrocíticos positivos para CB1 en el ratón GFAPhrGFP-CB 1 -WT aumentó hasta alcanzar el valor de 59.91 % ± 3.29, un aumento que resultó estadísticamente significativo al compararlo con CB 1 -WT. Por otra parte, sólo niveles residuales del receptor CB1 se encontraron en los astrocitos de los animales GFAP-CB 1 -KO, CB 1 -KO y GFAPhrGFP-CB 1 -KO.

Así mismo, la densidad de partículas de CB1 en la membrana de los procesos astrocíticos (partículas/µm) fue similar entre los valores del ratón CB 1 -WT y del ratón GFAP-CB 1 -RS (0.128 ± 0.020). Por otro lado, un gran aumento estadísticamente significativo en la densidad del receptor se obtuvo en el ratón GFAPhrGFP-CB 1 -WT (0.384 ± 0.039).

En la capa molecular del giro dentado del hipocampo, tras analizar el porcentaje de procesos astrocíticos positivos para CB1 no se obtuvieron diferencias significativas entre los valores del ratón GFAP-CB 1 -RS (39.84% ± 3.50) y el ratón CB 1 -WT, mientras que la proporción aumentó significativamente hasta ser del 59.99% ± 3.37 en el ratón GFAPhrGFP-CB 1 -WT. Por otra parte, solamente se encontraron partículas de oro residuales en los ratones GFAP-CB 1 -KO, GFAPhrGFP-CB 1 -KO y CB 1 -KO. Además, no hubo diferencias significativas tras la comparación de la densidad del receptor en los astrocitos del ratón CB 1 -WT y el ratón GFAP-CB 1 -RS (0.138 ± 0.016). Sin embargo, se obtuvieron diferencias significativas al comparar estos valores con el obtenido en el ratón GFAPhrGFP-CB 1 -WT (0.334 ± 0.033).

Una vez demostrado que una mejor detección del CB1 en astrocitos es posible al utilizar la proteína hr-GFP como marcador en el ratón GFAPhrGFP-CB 1 -WT, se analizó la distancia existente entre el receptor CB1 astroglial y el punto medio de la sinapsis más cercana. Así, en el stratum radiatum de CA1, el 51,07% ± 3,79 de las sinapsis totales que rodean a un proceso astrocítico estaban situadas a una distancia entre 400 y 800 nm del receptor CB1 astroglial. Por otra parte, el 43% ± 1,01 de estas sinapsis fueron identificados como sinapsis excitadoras mientras que sólo el 9,91% ± 1,93 eran inhibidoras. En la capa molecular del giro dentado el 57,25% ± 3,19 del total de las sinapsis que rodean a un proceso astrocítico se encontraron entre 400 y 800 nm de distancia del receptor CB1 de los astrocitos.

De estas sinapsis el 46,67% ± 2,17 fueron identificadas como excitadoras mientras que el 10,59% ± 2,18 eran sinapsis inhibidoras.

CONCLUSIONES

Las conclusiones obtenidas en esta Tesis son las que siguen:

1-En el ratón Glu-CB 1 -RS se mantienen los mismos niveles de expresión de los receptores CB1 hipocampal que en los ratones tipo silvestre CB 1 -WT en las neuronas glutamatérgicas del telencéfalo dorsal. 2-En el ratón GABA-CB 1 -RS se mantienen los mismos niveles de expresión de los receptores CB1 hipocampal que en los ratones tipo silvestre CB 1 -WT únicamente en neuronas GABAergicas. 3-En el ratón GFAP-CB 1 -RS se mantienen los mismos niveles de expresión de los receptores CB1 hipocampal que en procesos astrocíticos de los ratones tipo silvestre CB 1 -WT.

4-La utilización de hrGFP como marcador en el ratón mutante GFAPhrGFP-CB 1 -WT permitió la detección de niveles significativamente mayores de expresión del receptor CB1 en los procesos astrocíticos hipocampales que los encontrados en los ratones tipo silvestre CB 1 -WT utilizando como marcador GFAP. 5-En el hipocampo, la distancia más común entre el receptor CB1 de astrocitos y la sinapsis más cercana se encuentra en el rango de los 400-800 nm, siendo la mayoría de ellas excitadoras.

6-La mayoria de las sinapsis observadas en el CA1 y en la capa molecular del giro dentado rodeadas de astrocitos inmunopositivos para el receptor CB1 en el rango de 400-800 nm son de naturaleza excitatoria. 7-El estudio de los ratones mutantes rescatados para el receptor CB1 caracterizados en esta Tesis Doctoral ha demostrado que: 1) la expresión de los receptores CB1 se produce en tipos celulares específicos; 2) la reexpresión se encuentra limitada a poblaciones neurales concretas o a astrocitos; 3) los niveles endógenos de receptors CB1 se mantienen en los tipos celulares que re-expresan el receptor. 8-En conjunto, los ratones mutantes estudiados rescatos para el receptor CB1 son herramientas excelentes para la realización de estudios funcionales y translacionales relativas al papel de los receptores CB1 en el cerebro en condiciones normales y patológicas.

THE ENDOCANNABINOID SYSTEM

The Cannabis plant (Cannabis sativa L.) has been used since antiquity, mostly for the manufacturing and fabrication of cordages but also by its psychotropic, analgesic and antispasmodic effects [START_REF] Piomelli | The molecular logic of endocannabinoid signaling[END_REF]. Cannabis plants produce a mixture of chemical constituents, which are collectively referred as phytocannabinoids. Cannabinoids have been shown to produce a unique plethora of effects on the behavior of humans and animals which include euphoria, excitement, cognitive impairments, hypothermia, errors of time and space, enhancement of the sense of hearing, fluctuations of emotions, discoordination, illusions and hallucinations, disruption of short-term memory, relaxation, tachycardia, a decreased ability to focus attention, and sleepiness [START_REF] Block | Acute effects of marijuana on cognition: relationships to chronic effects and smoking techniques[END_REF][START_REF] Chait | Acute and residual effects of alcohol and marijuana, alone and in combination, on mood and performance[END_REF][START_REF] Heishman | Comparative effects of alcohol and marijuana on mood, memory, and performance[END_REF][START_REF] Court | Cannabis and brain function[END_REF][START_REF] Mechoulam | The Endocannabinoid System and the Brain[END_REF]. Among these compounds, delta (9) -tetrahydrocannabinol (Δ 9 -THC or THC) was first isolated in 1964 [START_REF] Gaoni | Isolation, structure and partial synthesis of an active constituent of hashish[END_REF], and was shown to account for the psychotropic effects of cannabis. Δ 9 -THC was described to couple with a group of receptors later named as the cannabinoid receptors, which consequently represent the target of a type of endogenous lipidic messengers, the endocannabinoids. Thus, the endocannabinoid system is comprised by the cannabinoid receptors, their endogenous lipid ligands (the endocannabinoids) and also the enzymes for their synthesis and degradation [START_REF] Piomelli | The molecular logic of endocannabinoid signaling[END_REF][START_REF] Piomelli | More surprises lying ahead. The endocannabinoids keep us guessing[END_REF][START_REF] De Petrocellis | The endocannabinoid system: a general view and latest additions[END_REF][START_REF] Marsicano | Neuromodulatory functions of the endocannabinoid system[END_REF][START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] Pertwee | Endocannabinoids and Their Pharmacological Actions[END_REF][START_REF] Lu | An Introduction to the Endogenous Cannabinoid System[END_REF]. This system is involved in many brain processes ranging from food intake to cognition [START_REF] Marsicano | Anatomical Distribution of Receptors, Ligands and Enzymes in the Brain and in the Spinal Cord: Circuitries and Neurochemistry[END_REF][START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF]. More specifically, endocannabinoids participate in the complex cellular and molecular mechanisms involved in endocannabinoid-mediated synaptic plasticity [START_REF] Castillo | Presynaptic LTP and LTD of excitatory and inhibitory synapses[END_REF] which provide important computational properties to brain circuits, such as coincidence detection and input specificity, critical for high brain functions [START_REF] Castillo | Endocannabinoid signaling and synaptic function[END_REF].

ENDOCANNABINOID COMPOUNDS

Endocannabinoids are endogenous lipid compounds synthesized from the cellular membranes. Among them, there are two main endocannabinoids which are better identified and characterized, i.e. N-arachidonoylethanolamine (anandamide, AEA) [START_REF] Devane | Isolation and structure of a brain constituent that binds to the cannabinoid receptor[END_REF] and 2-arachidonoylglycerol (2-AG) [START_REF] Mechoulam | Identification of an endogenous 2monoglyceride, present in canine gut, that binds to cannabinoid receptors[END_REF]. AEA is known by triggering the "tetrad" effects (i.e., catalepsy, antinociception, hypolocomotion, and hypothermia) of cannabinoids in rodents [START_REF] Fride | Pharmacological activity of the cannabinoid receptor agonist, anandamide, a brain constituent[END_REF] whereas 2-AG plays a key role in most of the Cannabinoid Type I (CB1) receptor mediation of synaptic transmission [START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF].

Endocannabinoids are synthesized and released on demand following physiological and pathological stimuli and, for the best of our knowledge no evidence have been found for their storage in secretory vesicles (De Petrocellis and Di Marzo, 2009a;[START_REF] Pertwee | International Union of Basic and Clinical Pharmacology . LXXIX . Cannabinoid Receptors and Their Ligands : Beyond CB 1 and CB 2[END_REF]. Interestingly, other N-acyletanolamines such as oleoylethanolamine (OEA) [START_REF] Fu | Oleylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-alpha[END_REF][START_REF] Wang | Oleoylethanolamide excites vagal sensory neurones, induces visceral pain and reduces short-term food intake in mice via capsaicin receptor TRPV1[END_REF][START_REF] Thabuis | Biological functions and metabolism of oleoylethanolamide[END_REF] and palmitoylethanolamine (PEA) (Di [START_REF] Marzo | Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells[END_REF][START_REF] Costa | The endogenous fatty acid amide, palmitoylethanolamide, has anti-allodynic and anti-hyperalgesic effects in a murine model of neuropathic pain: involvement of CB1, TRPV1 and PPARgamma receptors and neurotrophic factors[END_REF] exhibit a variety of similar biological activities and share with the endocannabinoids certain metabolic pathways. However, it has not been well established if these molecules also share with endocannabinoids their molecular targets (De Petrocellis and Di Marzo, 2009a).

METABOLISM OF ENDOCANNABINOIDS

A wide variety of biochemical pathways for the synthesis, transport, release and degradation of endocannabinoids have been described to date [START_REF] Piomelli | More surprises lying ahead. The endocannabinoids keep us guessing[END_REF]. In this sense, neuronal membrane depolarization or the activation of Gq-coupled GPCRs triggers the synthesis of 2-AG [START_REF] Hashimotodani | Phospholipase Cbeta serves as a coincidence detector through its Ca2+ dependency for triggering retrograde endocannabinoid signal[END_REF][START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF]. It is thought that phospholipase C, β or δ hydrolyses membrane phospholipids to obtain diacylglycerols (DGs), and then the degradation of these DGs by diacylglycerol lipases (DGLs) is driven to the synthesis 2-AG [START_REF] Gao | liu[END_REF][START_REF] Tanimura | The endocannabinoid 2-arachidonoylglycerol produced by diacylglycerol lipase alpha mediates retrograde suppression of synaptic transmission[END_REF].

Similarly, it has been described that the Ca 2+ -dependent enzyme N-acyltransferase (NAT) drives the transfer of arachidonic acid (AA) from phosphatidylcholine to phosphatidylethanolamine, consequently forming the AEA precursor Narachidonoyl phosphatidylethanolamine (NAPE) [START_REF] Cadas | Biosynthesis of an endogenous cannabinoid precursor in neurons and its control by calcium and cAMP[END_REF]. Afterwards, the N-acyl phosphatidylethanolamine specific phospholipase D (NAPE-PLD) [START_REF] Okamoto | Molecular characterization of a phospholipase D generating anandamide and its congeners[END_REF] catalyzes the release of AEA. Nevertheless, an alternative pathway for the releasement of AEA includes the hydrolysis of NAPE to phosphoanandamide by the action of the Phospholipase C (PLC) followed by dephosphorylation mediated by phosphatase, such as PTPN22 [START_REF] Liu | A biosynthetic pathway for anandamide[END_REF]. Conversely, monoacylglicerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) represent the two main catabolic enzymes for the hydrolysis of 2-AG and AEA respectively [START_REF] Dinh | Brain monoglyceride lipase participating in endocannabinoid inactivation[END_REF][START_REF] Ueda | Endocannabinoid hydrolases[END_REF]. In this sense, MAGL is mainly found on presynaptic terminals, where it hydrolyses 2-AG molecules bound to presynaptic CB1 receptors [START_REF] Straiker | Monoacylglycerol lipase limits the duration of endocannabinoidmediated depolarization-induced suppression of excitation in autaptic hippocampal neurons[END_REF][START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF]. Furthermore, two other hydrolases, ABHD6 and ABHD12 (α/β-hydrolase domain containing 6 and 12) are also capable of hydrolyzing 2-AG [START_REF] Blankman | A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol[END_REF]. On the other hand, FAAH showing overlap with CB1 in many brain regions [START_REF] Egertová | Comparative analysis of fatty acid amide hydrolase and cb1 cannabinoid receptor expression in the mouse brain: evidence of a widespread role for fatty acid amide hydrolase in regulation of endocannabinoid signaling[END_REF][START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF]. Moreover, FAAH has also been described to be involved in the metabolism of other bioactive N-acylethanolamines such as OEA and PEA (Di [START_REF] Marzo | Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells[END_REF].

N-arachidonoylethanolamine 2-arachidonoylglycerol

CANNABINOID RECEPTORS

Endocannabinoids receptors are known to be present in many vertebrate species, including rodents and primates, both monkeys and humans, [START_REF] Elphick | The phylogenetic distribution and evolutionary origins of endocannabinoid signaling[END_REF]. To date, two cannabinoid receptors have been cloned, i.e. the Type I Cannabinoid receptor (CB1 receptor) [START_REF] Matsuda | Localization of cannabinoid receptor mRNA in rat brain[END_REF] and the Type II Cannabinoid receptor (CB2 receptor) [START_REF] Munro | Molecular characterization of a peripheral receptor for cannabinoids[END_REF]. The CB1 and CB2 cannabinoid receptors belonging to the G-protein-coupled receptors (GPCRs) superfamily, the largest family of trans-membrane proteins in the human genome, and crucial for many essential physiological processes. GPCRs are characterized by seven hydrophobic transmembrane segments connected by intracellular and extracellular loops, an N-terminal extracellular domain that possesses glycosylation sites and a C-terminal intracellular domain coupled to a Gi/o protein [START_REF] Howlett | International Union of Pharmacology. XXVII. Classification of Cannabinoid Receptors[END_REF].

In the cell, the activation of CB1 or CB2 receptor by endocannabinoids triggers intracellular signaling events coupled to these two G-protein-coupled receptors (GPCRs) [START_REF] Pertwee | Pharmacology of cannabinoid CB1 and CB2 receptors[END_REF]. Firstly, stimulation of CB1 or CB2 receptor leads to the inhibition of stimulus-induced adenylate cyclase (AC) and, consequently, modulates protein kinase-A-dependent cascades. Moreover, the activation of these receptors provokes the stimulation of mitogen-activated protein kinase signaling. More specifically, the activation of CB1 receptor also exerts the modulation of ion channels, inducing inhibition of P, Q and N-type voltage-sensitive Ca 2+ channels and stimulation of rectifying G-protein-coupled K + channels. Also, just in the case of CB1 receptors, the stimulation of this receptor induces the activation of PLC-Ɣ, by the βƔ subunits of Gi/o proteins stimulates phosphatidylinositol 3-kinase and intracellular Ca 2+ mobilization [START_REF] Devane | Determination and characterization of a cannabinoid receptor in rat brain[END_REF][START_REF] Howlett | The cannabinoid receptor: biochemical, anatomical and behavioral characterization[END_REF][START_REF] Howlett | International Union of Pharmacology. XXVII. Classification of Cannabinoid Receptors[END_REF][START_REF] De Petrocellis | The endocannabinoid system: a general view and latest additions[END_REF][START_REF] Pertwee | Endocannabinoids and Their Pharmacological Actions[END_REF]. CB1 receptor is predominantly expressed in the central nervous system (CNS) particularly in brain cortex, basal ganglia, hippocampus, and cerebellum [START_REF] Hu | Distribution of the Endocannabinoid System in the Central Nervous System[END_REF]. Besides, minor levels of expression of CB1 receptors have been also document in various peripheral tissues [START_REF] Pertwee | Cannabinoid receptor ligands: clinical and neuropharmacological considerations, relevant to future drug discovery and development[END_REF][START_REF] Pertwee | Cannabinoid receptors and pain[END_REF]. On the contrary, CB2 receptor are localized primarily in immune cells and tissues derived from the immune system [START_REF] Ameri | The effects of cannabinoids on the brain[END_REF][START_REF] Cabral | Endocannabinoids and the Immune System in Health and Disease. Endocannabinoids[END_REF]. Although CB1 and CB2 are well stablished as endocannabionid receptors, there exist other receptors that could be mediating the effects of endocannabinoids [START_REF] Pertwee | Endocannabinoids and Their Pharmacological Actions[END_REF], such as the Transient Receptor Potential Vanilloid 1, also known as TRPV1, wich can be activated by anandamide [START_REF] Maccarrone | Anandamide inhibits metabolism and physiological actions of 2arachidonoylglycerol in the striatum[END_REF]De Petrocellis and Di Marzo, 2009b;[START_REF] Tóth | Anandamide and the vanilloid receptor (TRPV1)[END_REF][START_REF] Alhouayek | Controlling 2-arachidonoylglycerol metabolism as an anti-inflammatory strategy[END_REF][START_REF] Rossi | The interplay between inflammatory cytokines and the endocannabinoid system in the regulation of synaptic transmission[END_REF], as well as for Transient Receptor Potential Ankyrin 1 (TRPA1) receptors [START_REF] De Petrocellis | Plant-derived cannabinoids modulate the activity of transient receptor potential channels of ankyrin type-1 and melastatin type-8[END_REF], Peroxisome Proliferator-Activated Receptors, namely PPAR-α [START_REF] Sun | Cannabinoids and PPARalpha signaling[END_REF][START_REF] Alhouayek | Controlling 2-arachidonoylglycerol metabolism as an anti-inflammatory strategy[END_REF] and non-CB1/CB2 GPCRs such as G protein-coupled receptor 55 (GPR55) [START_REF] Ryberg | The orphan receptor GPR55 is a novel cannabinoid receptor[END_REF].

PHYSIOLOGICAL ACTIONS OF ENDOCANNABINOIDS

Endocannabinoids are released from the postsynaptic neurons after its depolarization and/or receptor activation. Once released, endocannabinoids bind to the presynaptically located CB1 [START_REF] Robbe | Endogenous cannabinoids mediate long-term synaptic depression in the nucleus accumbens[END_REF][START_REF] Chevaleyre | Heterosynaptic LTD of hippocampal GABAergic synapses: a novel role of endocannabinoids in regulating excitability[END_REF][START_REF] Kano | Control of synaptic function by endocannabinoid-mediated retrograde signaling[END_REF]. Thus, the binding of the endocannabinoid to its receptor, is able to suppress the elevation of the intracellular free Ca 2+ concentration induced by the depolarization of the postsynaptic neuron [START_REF] Sugiura | Inhibition by 2-arachidonoylglycerol, a novel type of possible neuromodulator, of the depolarization-induced increase in intracellular free calcium in neuroblastoma x glioma hybrid NG108-15 cells[END_REF] and consequently regulate the release of different neurotransmitters such as: glutamate [START_REF] Shen | Cannabinoid receptor agonists inhibit glutamatergic synaptic transmission in rat hippocampal cultures[END_REF], and gamma-Aminobutyric acid (GABA) [START_REF] Katona | Presynaptically Located CB1 Cannabinoid Receptors Regulate GABA Release from Axon Terminals of Specific Hippocampal Interneurons[END_REF], acetylcholine (Gifford et al., 1997a;b) or noradrenaline [START_REF] Schlicker | Cannabinoid CB1 receptormediated inhibition of noradrenaline release in the human and guinea-pig hippocampus[END_REF]. The transient suppression of transmitter release on the presynaptic neuron following the release of endocannabinoid from postsynaptic neurons is known as endocannabinoid-mediated short-term depression or eCB-STD. The transient suppression has been consistently demonstrated to mediate both, depolarizationinduced suppression of inhibition (DSI) [START_REF] Ohno-Shosaku | Endogenous Cannabinoids Mediate Retrograde Signals from Depolarized Postsynaptic Neurons to Presynaptic Terminals[END_REF][START_REF] Wilson | Endogenous cannabinoids mediate retrograde signaling at hippocampal synapses[END_REF] and of excitation (DSE) [START_REF] Kreitzer | Retrograde Inhibition of Presynaptic Calcium Influx by Endogenous Cannabinoids at Excitatory Synapses onto Purkinje Cells[END_REF], mechanisms by which short-term depression of GABA or glutamate release from axon terminals is induced by the depolarization of a postsynaptic neuron [START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] Kano | Control of synaptic function by endocannabinoid-mediated retrograde signaling[END_REF].

On the other hand, long-term depression (LTD) of GABA or glutamate release is mediated by CB1 receptor in response to long-lasting synaptic activity, which triggers postsynaptic endocannabinoid releasement and retrograde signaling (endocannabinoid-mediated long-term depression or eCB-LTD) in different types of synapses throughout nervous system [START_REF] Chevaleyre | Heterosynaptic LTD of hippocampal GABAergic synapses: a novel role of endocannabinoids in regulating excitability[END_REF][START_REF] Chevaleyre | Endocannabinoid-mediated synaptic plasticity in the CNS[END_REF][START_REF] Castillo | Presynaptic LTP and LTD of excitatory and inhibitory synapses[END_REF].

In the past years, endocannabinoid signaling have been demonstrated to play a key role in the potentiation of glutamatergic transmission through neuron-glia communication, also known as tripartite synapses, that includes the activation of astroglial CB1 receptors (Navarrete andAraque, 2008, 2010;[START_REF] Navarrete | Astrocyte calcium signal and gliotransmission in human brain tissue[END_REF][START_REF] Metna-Laurent | Rising stars: Modulation of brain functions by astroglial type-1 cannabinoid receptors[END_REF][START_REF] Oliveira Da Cruz | Astroglial type-1 cannabinoid receptor (CB1): A new player in the tripartite synapse[END_REF].

In general, most of the actions of (endo)cannabinoids are mediated by CB1 receptor ability to modulate synaptic transmission [START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF][START_REF] Castillo | Presynaptic LTP and LTD of excitatory and inhibitory synapses[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] Kano | Control of synaptic function by endocannabinoid-mediated retrograde signaling[END_REF]. More specifically, the regulation of synaptic plasticity mediated by CB1 receptor has been related to the memoryimpairment effects of cannabinoids by means of the induction of LTD and the inhibition of LTP in the hippocampus [START_REF] Stella | A second endogenous cannabinoid that modulates long-term potentiation[END_REF][START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF].

In addition, endocannabinoids have been described to also be involved in cell survival and differentiation processes [START_REF] Galve-Roperh | Cannabinoid receptor signaling in progenitor/stem cell proliferation and differentiation[END_REF]. In this sense, during embryonary development, the activation of CB1 and CB2 receptors participate in the regulation of neuronal progenitor-stem cells self-renewal, proliferation and differentiation. Particularly, the activation of these receptors have been reported to trigger many different signaling pathways, such as mitogenactivated protein kinase signaling pathways, focal adhesion kinase cascade, phosphatidylinositol 3-kinase, extracellular-signaling regulated protein kinase, and ceramide signaling, among others (Bouaboula et al., 1995b;[START_REF] Derkinderen | Regulation of a neuronal form of focal adhesion kinase by anandamide[END_REF][START_REF] Aguado | The endocannabinoid system drives neural progenitor proliferation[END_REF][START_REF] Ortega-Gutiérrez | Effect of anandamide uptake inhibition in the production of nitric oxide and in the release of cytokines in astrocyte cultures[END_REF][START_REF] Galve-Roperh | Cannabinoid receptor signaling in progenitor/stem cell proliferation and differentiation[END_REF].

Endocannabinoids are also able to develop neuroprotective roles acting through CB1 cannabinoid receptors. In this sense, it has been shown that the role developed by the eCBs in retrograde signaling system in glutamatergic synapses (Ohno-Shosaku and [START_REF] Kano | Control of synaptic function by endocannabinoid-mediated retrograde signaling[END_REF] in the brain could be the origin of its protective role [START_REF] Wallace | The endogenous cannabinoid system regulates seizure frequency and duration in a model of temporal lobe epilepsy[END_REF][START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF]. Importantly, the eCB system would be a pharmacological target for the treatment of diseases characterized by abnormal glutamate homeostasis, such as neurodegenerative disorders [START_REF] Fernández-Ruiz | The endocannabinoid system as a target for the treatment of motor dysfunction[END_REF][START_REF] Fernández-Ruiz | Endocannabinoids and Neurodegenerative Disorders: Parkinson's Disease, Huntington's Chorea, Alzheimer's Disease, and Others[END_REF].

EXPRESSION OF CB 1 RECEPTOR IN THE BRAIN

The CB1 receptor is the main endocannabinoid signaling protein regarding the CNS [START_REF] Pertwee | Emerging strategies for exploiting cannabinoid receptor agonists as medicines[END_REF], and, more specifically, the CB1 receptors represent one of the most abundant GPCRs in the brain [START_REF] Herkenham | Neuronal localization of cannabinoid receptors and second messengers in mutant mouse cerebellum[END_REF][START_REF] Tsou | Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system[END_REF][START_REF] Moldrich | Localization of the CB1 cannabinoid receptor in the rat brain. An immunohistochemical study[END_REF]. Its expression is widespread and heterogeneous, developing crucial roles in brain function, dysfunction and cognition [START_REF] Marsicano | The endogenous cannabinoid system controls extinction of aversive memories[END_REF][START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF][START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF][START_REF] Puente | Polymodal activation of the endocannabinoid system in the extended amygdala[END_REF][START_REF] Castillo | Presynaptic LTP and LTD of excitatory and inhibitory synapses[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] Steindel | Neuron-type specific cannabinoid-mediated G protein signaling in mouse hippocampus[END_REF][START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF], 2015;[START_REF] Hu | Distribution of the Endocannabinoid System in the Central Nervous System[END_REF][START_REF] Katona | Cannabis and Endocannabinoid Signaling in Epilepsy[END_REF][START_REF] Martín-García | Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors[END_REF], and it has been reported to palliate numerous pathologies, including Alzheimer's disease, pain, obesity, and cancer, [START_REF] Pertwee | Emerging strategies for exploiting cannabinoid receptor agonists as medicines[END_REF]. More specifically, the highest levels of CB1 receptors are present in the substantia nigra, globus pallidus, cerebellum, hippocampus and cortex [START_REF] Howlett | The cannabinoid receptor: biochemical, anatomical and behavioral characterization[END_REF]. At the ultrastructural level, CB1 receptor expression is very high in inhibitory GABAergic synaptic terminals [START_REF] Katona | Presynaptically Located CB1 Cannabinoid Receptors Regulate GABA Release from Axon Terminals of Specific Hippocampal Interneurons[END_REF][START_REF] Kawamura | The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum[END_REF][START_REF] Ludányi | Downregulation of the CB1 cannabinoid receptor and related molecular elements of the endocannabinoid system in epileptic human hippocampus[END_REF][START_REF] Marsicano | Anatomical Distribution of Receptors, Ligands and Enzymes in the Brain and in the Spinal Cord: Circuitries and Neurochemistry[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] De-May | Cell type-specific regulation of inhibition via cannabinoid type 1 receptors in rat neocortex[END_REF][START_REF] Steindel | Neuron-type specific cannabinoid-mediated G protein signaling in mouse hippocampus[END_REF][START_REF] Hu | Distribution of the Endocannabinoid System in the Central Nervous System[END_REF] but, to a lower extent, CB1 receptors are also present in glutamatergic neurons [START_REF] Marsicano | CB1 cannabinoid receptors and on-demand defense against excitotoxicity[END_REF][START_REF] Domenici | Cannabinoid receptor type 1 located on presynaptic terminals of principal neurons in the forebrain controls glutamatergic synaptic transmission[END_REF][START_REF] Takahashi | The CB1 cannabinoid receptor mediates glutamatergic synaptic suppression in the hippocampus[END_REF][START_REF] Katona | Molecular composition of the endocannabinoid system at glutamatergic synapses[END_REF][START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF][START_REF] Kamprath | Endocannabinoids mediate acute fear adaptation via glutamatergic neurons independently of corticotropin-releasing hormone signaling[END_REF][START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF][START_REF] Puente | Polymodal activation of the endocannabinoid system in the extended amygdala[END_REF][START_REF] Reguero | GABAergic and cortical and subcortical glutamatergic axon terminals contain CB1 cannabinoid receptors in the ventromedial nucleus of the hypothalamus[END_REF][START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF] and in brain astrocytes (Navarrete andAraque, 2008, 2010;[START_REF] Stella | Cannabinoid and cannabinoid-like receptors in microglia, astrocytes, and astrocytomas[END_REF][START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF][START_REF] Bosier | Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes[END_REF][START_REF] Metna-Laurent | Rising stars: Modulation of brain functions by astroglial type-1 cannabinoid receptors[END_REF][START_REF] Viader | Metabolic Interplay between Astrocytes and Neurons Regulates Endocannabinoid Action[END_REF][START_REF] Oliveira Da Cruz | Astroglial type-1 cannabinoid receptor (CB1): A new player in the tripartite synapse[END_REF]. CB1 receptors are also constitutively expressed by oligodendrocytes (Molina-Holgado et al., 2002a;[START_REF] Benito | Cannabinoid CB1 and CB2 receptors and fatty acid amide hydrolase are specific markers of plaque cell subtypes in human multiple sclerosis[END_REF][START_REF] Garcia-Ovejero | The endocannabinoid system is modulated in response to spinal cord injury in rats[END_REF][START_REF] Mato | CB1 cannabinoid receptor-dependent and -independent inhibition of depolarization-induced calcium influx in oligodendrocytes[END_REF][START_REF] Gomez | The constitutive production of the endocannabinoid 2arachidonoylglycerol participates in oligodendrocyte differentiation[END_REF]. Inside the cells, CB1 receptors, can be subcellularly localized at plasma membranes and intracellular organelles, such as brain mitochondria [START_REF] Bénard | Mitochondrial CB₁ receptors regulate neuronal energy metabolism[END_REF]Hebert-Chatelain et al., 2014a;b;[START_REF] Koch | Hypothalamic POMC neurons promote cannabinoid-induced feeding[END_REF].

Because of the hugely different levels of expression among different cell types [START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF], it is extremely difficult to identify low CB1 receptor expression in cell types and/or in subcellular compartments of wild-type brains [START_REF] Busquets-Garcia | Dissecting the cannabinergic control of behavior: The where matters[END_REF]. Actually, former anatomical studies often misinterpreted low CB1 receptor expression as background staining [START_REF] Freund | Role of endogenous cannabinoids in synaptic signaling[END_REF].

Pharmacological blockade or genetic ablation of the receptor and its eventual behavioral and neuronal consequences are clear indications of the role of endocannabinoids in the brain. Hence, based on methodological "loss of function" approaches, the use of mutant mice lacking CB1 receptors in specific cell populations helped to identify the necessary role of the receptor in different brain functions [START_REF] Marsicano | CB1 cannabinoid receptors and on-demand defense against excitotoxicity[END_REF][START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF][START_REF] Monory | Cannabinoid CB1 receptor calibrates excitatory synaptic balance in the mouse hippocampus[END_REF][START_REF] Martín-García | Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors[END_REF][START_REF] Zimmer | Genetic Manipulation of the Endocannabinoid System[END_REF]. Moreover, neuroanatomical analysis of these mutant animal models determined the presence of the CB1 receptor at sites previously not known (e.g. cortical glutamatergic neurons or neuronal mitochondria). However, these approaches do not provide definitive information concerning the possibly sufficient role of the cell-type expression of CB1 receptors for a given function. Indeed, distinguishing between low levels (wild-type) and no-expression (mutant mice lacking CB1 receptors in specific cell populations) is very difficult. To establish precise causal relationships and to identify low levels of expression, "rescue" strategies utilizing the endogenous genetic locus are needed in order to analyze the function of endogenous CB1 receptor expression exclusively in specific cell types. To reach this aim the Cre/LoxP system have been previously applied to restore CB1 receptor expression in a CB1 receptor-null mutant only in specific cell types [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF][START_REF] De Salas-Quiroga | Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons[END_REF]. In this sense, the rescue approach was first applied to the hypothalamic expression of the melanocortin 4 receptor [START_REF] Balthasar | Divergence of melanocortin pathways in the control of food intake and energy expenditure[END_REF] using the Cre/loxP system, and was recently applied to the CB 1 gene [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF][START_REF] De Salas-Quiroga | Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons[END_REF]. Briefly, a "stop cassette" flanked by LoxP sites was introduced immediately upstream of the coding sequence of the CB 1 gene by homologous recombination to generate a mutant mouse line. The mice obtained by this approach bore no expression of the CB1 receptor protein [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF][START_REF] De Salas-Quiroga | Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons[END_REF], making these mice phenotypically very similar to conventional CB1 knock-out mice (CB 1 -KO). However, the crossing of these mice with Cre-expressing transgenic mouse lines or the local injection of viruses expressing the Cre recombinase induces a Cre-mediated deletion of the "stop cassette", causing a specific rescue of expression of the protein in specific cell populations and/or brain regions [START_REF] Balthasar | Divergence of melanocortin pathways in the control of food intake and energy expenditure[END_REF][START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF][START_REF] De Salas-Quiroga | Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons[END_REF]. Moreover, this re-expression is limited to the cell types where the regulatory sequences of the CB1 receptor are normally activated and the expression levels are the same as in wild-type animals [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF][START_REF] De Salas-Quiroga | Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons[END_REF]. Furthermore, these rescue mice allowed determining the sufficient causal role of the CB1 receptor in the anterior olfactory cortex area as well as in dorsal telencephalic glutamatergic neurons [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF][START_REF] De Salas-Quiroga | Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons[END_REF]. However, no studies have addressed so far the subcellular localization of CB1 receptor in these mutant brains at the electron microscopic level. This is particularly interesting due to the huge expression of CB1 receptors in specific cell types (e.g. hippocampal GABAergic interneurons) which hinder to appreciate the detailed pattern of expression of the CB1 receptor in other cell types (e.g. hippocampal glutamatergic pyramidal neurons) in wild-type mouse samples [START_REF] Freund | Role of endogenous cannabinoids in synaptic signaling[END_REF]. Therefore, the conditional rescue mutants emerge as key tools for the detailed anatomical characterization of the subcellular distribution of the receptor in specific cell types, independently of its level of expression.

ASTROCYTES AND THE CB 1 RECEPTOR

Presence of CB1 receptors in astrocytes has been long debated. Its presence in astrocytes was firstly reported in human and rodent cell cultures (Bouaboula et al., 1995a;[START_REF] Sánchez | Δ 9 -Tetrahydrocannabinol induces apoptosis in C6 glioma cells[END_REF]Molina-Holgado et al., 2002b;[START_REF] Sheng | Synthetic cannabinoid WIN55,212-2 inhibits generation of inflammatory mediators by IL-1beta-stimulated human astrocytes[END_REF]. However, incongruous results were obtained in mice [START_REF] Sagan | Anandamide and WIN 55212-2 inhibit cyclic AMP formation through G-proteincoupled receptors distinct from CB1 cannabinoid receptors in cultured astrocytes[END_REF][START_REF] Walter | Endothelin-1 increases 2-arachidonoyl glycerol (2-AG) production in astrocytes[END_REF] suggesting that CB1 receptor expression in astrocytes could vary among different animal species, strains or different cell culture conditions. In addition, low levels of expression of astrocytic CB1 receptor made it remarkably arduous to detect astroglial CB 1 mRNA or protein expression by means of in situ hybridization or immunohistochemistry. Fortunately, electron microscopy immunodetection allowed to reveal the presence of astrocytic CB1 receptor [START_REF] Rodriguez | Ultrastructural Localization of the CB1 Cannabinoid Receptor in micro-Opioid Receptor Patches of the Rat Caudate Putamen Nucleus[END_REF][START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF][START_REF] Bosier | Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes[END_REF]. Moreover, the enzymes for synthesis and degradation of the main endocannabinoids co-localize with Glial Fibrillary Acidic Protein (GFAP) [START_REF] Suárez | Endocannabinoid system in the adult rat circumventricular areas: an immunohistochemical study[END_REF]. Finally, in light of

CB 1 STOP CB 1 PROTEIN CB 1 CB 1 PROTEIN

Cre recombinase

Lox-P Lox-P Lox-P many different works published during the last years (Navarrete andAraque, 2008, 2010;[START_REF] Stella | Cannabinoid and cannabinoid-like receptors in microglia, astrocytes, and astrocytomas[END_REF][START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF][START_REF] Bosier | Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes[END_REF][START_REF] Gómez-Gonzalo | Endocannabinoids Induce Lateral Long-Term Potentiation of Transmitter Release by Stimulation of Gliotransmission[END_REF][START_REF] Navarrete | Astrocytes in endocannabinoid signaling[END_REF][START_REF] Metna-Laurent | Rising stars: Modulation of brain functions by astroglial type-1 cannabinoid receptors[END_REF][START_REF] Viader | Metabolic Interplay between Astrocytes and Neurons Regulates Endocannabinoid Action[END_REF][START_REF] Oliveira Da Cruz | Astroglial type-1 cannabinoid receptor (CB1): A new player in the tripartite synapse[END_REF] the presence of the CB1 receptor in astrocytes and its functional importance have been demonstrated.

In this sense, different authors have proposed various signaling pathways in astrocytes after the CB1 receptor activation. Thus, some authors have proposed the coupling of astrocytic CB1 to Gαi/o-protein [START_REF] Guzmán | Control of the cell survival/death decision by cannabinoids[END_REF] whereas other authors suggest that astrocytic CB1 receptor is able to modulate Ca 2+ levels in astrocytes and associated signaling by means of Gαq proteins activation [START_REF] Navarrete | Endocannabinoids mediate neuron-astrocyte communication[END_REF][START_REF] Navarrete | Astrocytes in endocannabinoid signaling[END_REF]. In this sense, the development of different intracellular astrocytic signaling pathways in response to endocannabinoids provides to endocannabinoid-mediated responses with a particular important flexibility in astrocytes (Metna-Laurent and Marsicano, 2015).

Otherwise, astrocytes have long been known for their role as protective elements against injury in the central nervous system and as energetics providers for neurons [START_REF] Magistretti | Cellular mechanisms of brain energy metabolism and their relevance to functional brain imaging[END_REF][START_REF] Bélanger | The role of astroglia in neuroprotection[END_REF][START_REF] Yi | A role for astrocytes in the central control of metabolism[END_REF], not taking a relevant part in the transmission of information for being nonelectrically excitable cells. However, this last consideration has already changed, owing to the fact that many signaling systems that are capable to modulate neuronal activity have been described to also regulate astrocytic-neuronal communication [START_REF] Araque | Tripartite synapses: glia, the unacknowledged partner[END_REF][START_REF] Haydon | Astrocyte control of synaptic transmission and neurovascular coupling[END_REF][START_REF] Perea | Tripartite synapses: astrocytes process and control synaptic information[END_REF][START_REF] Gómez-Gonzalo | Endocannabinoids Induce Lateral Long-Term Potentiation of Transmitter Release by Stimulation of Gliotransmission[END_REF]. Moreover, these findings lead to the establishment of a new concept: the "tripartite synapse", which is formed by a presynaptic and a postsynaptic neuronal elements and by the surrounding astroglial processes [START_REF] Araque | Tripartite synapses: glia, the unacknowledged partner[END_REF] (Fig. 6). In this context, astrocytic CB1 receptors have been demonstrated to play a key role in the communication between neurons and astrocytes (Navarrete andAraque, 2008, 2010;[START_REF] Navarrete | Astrocyte calcium signal and gliotransmission in human brain tissue[END_REF][START_REF] Navarrete | Astrocytes in endocannabinoid signaling[END_REF][START_REF] Gómez-Gonzalo | Endocannabinoids Induce Lateral Long-Term Potentiation of Transmitter Release by Stimulation of Gliotransmission[END_REF]. Moreover, glutamate release is promoted in neurons by the activation of CB1 receptors in astrocytes [START_REF] Navarrete | Endocannabinoids mediate neuron-astrocyte communication[END_REF], and therefore this activation could be improving synaptic transmission [START_REF] Navarrete | Endocannabinoids potentiate synaptic transmission through stimulation of astrocytes[END_REF]. Thus, in this context, it has been suggested that the CB1 receptor would mediate in the development of epileptic seizures [START_REF] Coiret | Neuron to astrocyte communication via cannabinoid receptors is necessary for sustained epileptiform activity in rat hippocampus[END_REF]. In this sense, it has been recently demonstrated that the CB1 receptor expression in astrocytes is increased in sclerotic hippocampus, which is characterized by refractory temporal lobe epilepsy and both neuronal loss and astrocyte proliferation [START_REF] Meng | Astrocytic expression of cannabinoid type 1 receptor in rat and human sclerotic hippocampi[END_REF].

In astrocytes, GFAP is the principal intermediate filament protein [START_REF] Eng | Glial fibrillary acidic protein (GFAP): the major protein of glial intermediate filaments in differentiated astrocytes[END_REF][START_REF] Eng | Glial Fibrillary Acidic Protein: GFAP-Thirty-One Years (1969-2000)[END_REF][START_REF] Hol | Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system[END_REF]. However, due to its filament nature the immunolabeling of GFAP may not show the complete surface of the astrocyte in traditional microscopic preparations. Thus, other methodological approaches have been developed for staining astrocytes in microscopic preparations. In this sense, Green Fluorescence Protein, also known as GFP [START_REF] Shimomura | Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan,Aequorea[END_REF][START_REF] Chalfie | GREEN FLUORESCENT PROTEIN[END_REF], is a cytoplasmically expressed diffusible protein. When inserted in astrocytes, this protein appears distributed throughout the whole cytoplasm, allowing its immunolabeling to be used for a better detection of the whole astrocytic surface [START_REF] Nolte | GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue[END_REF]. More particularly, GFP protein from the sea pansy Renilla reniformis (Ward et al., 1978;[START_REF] Ward | An energy transfer protein in coelenterate bioluminescence. Characterization of the Renilla green-fluorescent protein[END_REF] and its humanized isoform, known as hrGFP, has been used as a marker in a wide variety of studies [START_REF] Navarro-Galve | Gene marking of human neural stem/precursor cells using green fluorescent proteins[END_REF][START_REF] Francesco | Neuroanatomical and functional characterization of CRF neurons of the amygdala using a novel transgenic mouse model[END_REF][START_REF] Kerr | The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2[END_REF]. More specifically, it has been used as an astroglial marker [START_REF] Hadaczek | Transduction of nonhuman primate brain with adeno-associated virus serotype 1: vector trafficking and immune response[END_REF].

HIPPOCAMPUS

DEFINITION OF THE HIPPOCAMPUS

The hippocampus is a structure that includes distinct brain regions differentiated by its cytoarchitecture. The main reason to include these areas under the same anatomical structure is that they are largely connected via unidirectional projections (the perforant path, mossy fibers and Schaffer collaterals). However, as I will describe later, two-way connections may also exist in this region.

The hippocampus can be divided in two cortical structures i.e. the hippocampal formation and the parahippocampal region (Fig. 7). On the one hand, the hippocampal formation is a C-shaped structure localized in the caudal part of the brain. It is comprised by three areas cytoarchitectonically differentiated: the dentate gyrus, the hippocampus properly speaking which is subdivided into three fields (CA1, CA2 and CA3), and the subiculum. On the other hand, the parahippocampal region lies adjacent to the hippocampal formation and comprises five subregions; the perirhinal cortex, the entorhinal cortex, the postrhinal cortex, the presubiculum and the parasubiculum.

The three-dimensional location of the hippocampus in the mouse brain is complex. It appears as an elongated structure, with its septo-temporal C-shaped axis extending towards the temporal lobe in its caudo-ventral end running above and behind the diencephalon. It is delimited by the septal nuclei rostrally and by the lateral ventricles laterally. The longitudinal axis of the hippocampal formation is located in septum-temporal direction, while the transverse or orthogonal axis is extending between temporal lobes. The regions of the hippocampal formation are distributed along the septum-temporal axis so that the dentate gyrus and CA1 and CA3 regions of the hippocampus appear at more septal levels. The subiculum begins to appear in the first third of septotemporal axis, while the presubiculum and parasubiculum are appearing progressively towards the temporal region. Finally, ventrally in the caudal portion of the hippocampus the entorhinal cortex is confined.

DENTATE GYRUS

The dentate gyrus comprises three layers, the molecular layer, the granular cell layer and the hilus.

Molecular layer.

The molecular layer is located edging the hippocampal fissure and contains the perforant path fibers connecting the entorhinal cortex with the dentate gyrus. This layer is mainly occupied by the dendrites of the granule, basket and various polymorphic cells, as well as axon terminals from several sources. Remarkably, synaptic terminals of the commissural/associational system are found in the inner third of the dentate molecular layer, containing a substantial input from the CA3 in addition to the excitatory terminals of the hilar mossy cells [START_REF] Ribak | The development, ultrastructure and synaptic connections of the mossy cells of the dentate gyrus[END_REF][START_REF] Li | The hippocampal CA3 network: An in vivo intracellular labeling study[END_REF][START_REF] Blasco-Ibáñez | Distribution, ultrastructure, and connectivity of calretinin-immunoreactive mossy cells of the mouse dentate gyrus[END_REF][START_REF] Scharfman | The CA3 "backprojection" to the dentate gyrus[END_REF]. multipolar soma and present axons projecting to the granular layer basket plexus cells. They are considered as a type of basket cells [START_REF] Hazlett | Short axon molecular layer neurons in the opossum fascia dentata: a Golgi study[END_REF][START_REF] Ribak | Five types of basket cell in the hippocampal dentate gyrus: a combined Golgi and electron microscopic study[END_REF][START_REF] Witter | The Rat Nervous System[END_REF][START_REF] Jinno | Cellular architecture of the mouse hippocampus: a quantitative aspect of chemically defined GABAergic neurons with stereology[END_REF][START_REF] Witter | The Mouse Nervous System[END_REF].

The other kind of neurons that are localized in the molecular layer are immunoreactive for the neurotransmitter GABA [START_REF] Celio | Calbindin D-28k and parvalbumin in the rat nervous system[END_REF], which indicates that they are modulating the activity of granule cells. They are similar to chandelier cells [START_REF] Kosaka | Axon initial segments of the granule cell in the rat dentate gyrus: synaptic contacts on bundles of axon initial segments[END_REF][START_REF] Somogyi | Identified axo-axonic cells are immunoreactive for GABA in the hippocampus and visual cortex of the cat[END_REF][START_REF] Soriano | A GABAergic axo-axonic cell in the fascia dentata controls the main excitatory hippocampal pathway[END_REF][START_REF] Halasy | Subdivisions in the multiple GABAergic innervation of granule cells in the dentate gyrus of the rat hippocampus[END_REF][START_REF] Witter | The Rat Nervous System[END_REF][START_REF] Witter | The Mouse Nervous System[END_REF]. Their dendrites remain primarily in the molecular layer, while their axons project from the molecular layer into the granular cell layer and contact with initial axonal segments of granule cells.

Granular cell layer

The granule cell layer is situated deeply into the molecular layer. It is constituted mainly of densely packed excitatory glutamatergic neurons known as granule cells.

Their dendritic extensions give the cell a characteristic conical appearance and are oriented towards the molecular layer. Furthermore, they have numerous dendritic spines which are able to contact with many axon terminals (Desmond andLevy, 1982, 1985) and increase in length at more distal positions along the dendrite [START_REF] Stanfield | The development of the hippocampus and dentate gyrus in normal and reeler mice[END_REF][START_REF] Vuksic | 3D-reconstruction and functional properties of GFP-positive and GFP-negative granule cells in the fascia dentata of the Thy1-GFP mouse[END_REF]. On the other hand, their glutamatergic axons known as mossy fibers invade the hilar region where they contact with the mossy cells, CA3 pyramidal neurons and GABAergic interneurons [START_REF] Frotscher | Divergence of hippocampal mossy fibers[END_REF]. No remarkable morphological differences appeared between granule cells taken from the dorsal or ventral portions, except for the more extensive degree of arborisation found in ventral cells [START_REF] Vuksic | 3D-reconstruction and functional properties of GFP-positive and GFP-negative granule cells in the fascia dentata of the Thy1-GFP mouse[END_REF].

Another type of cells in this layer are the GABAergic basket cells [START_REF] Aika | Quantitative analysis of GABA-likeimmunoreactive and parvalbumin-containing neurons in the CA1 region of the rat hippocampus using a stereological method, the disector[END_REF] whose somata are located in the innermost side of the granular cell layer. They owe their name to their axons which form a pericellular plexus surrounding the granule cells bodies [START_REF] Ribak | Five types of basket cell in the hippocampal dentate gyrus: a combined Golgi and electron microscopic study[END_REF][START_REF] Ribak | The development, ultrastructure and synaptic connections of the mossy cells of the dentate gyrus[END_REF] modulating granule cells activity [START_REF] Meyer | In vivo labeling of parvalbumin-positive interneurons and analysis of electrical coupling in identified neurons[END_REF][START_REF] Jinno | Cellular architecture of the mouse hippocampus: a quantitative aspect of chemically defined GABAergic neurons with stereology[END_REF].

Hilus

Also known as the polymorphic layer, the hilus contains a wide variety of cell types. The most common are the glutamatergic mossy cells [START_REF] Frotscher | Divergence of hippocampal mossy fibers[END_REF][START_REF] Blasco-Ibáñez | Distribution, ultrastructure, and connectivity of calretinin-immunoreactive mossy cells of the mouse dentate gyrus[END_REF]. Their large cell bodies are oval and their dendrites extend for long distances within the hilus, sometimes being able to reach out the granule cell layer and the molecular layer (Blasco-Ibáñez and [START_REF] Blasco-Ibáñez | Distribution, ultrastructure, and connectivity of calretinin-immunoreactive mossy cells of the mouse dentate gyrus[END_REF][START_REF] Fujise | Mossy cells in the mouse dentate gyrus: identification in the dorsal hilus and their distribution along the dorsoventral axis[END_REF]. Remarkably, dendrites of the mossy cells do not leave the confines of the hilus to enter the adjacent CA3 field. The most proximal dendrites are covered by the very large and complex spines called "thorny excrescences", which establish synaptic contacts with the granule mossy fibers [START_REF] Ribak | The development, ultrastructure and synaptic connections of the mossy cells of the dentate gyrus[END_REF][START_REF] Frotscher | The mossy cells of the fascia dentata: a comparative study of their fine structure and synaptic connections in rodents and primates[END_REF][START_REF] Blasco-Ibáñez | Distribution, ultrastructure, and connectivity of calretinin-immunoreactive mossy cells of the mouse dentate gyrus[END_REF][START_REF] Fujise | Mossy cells in the mouse dentate gyrus: identification in the dorsal hilus and their distribution along the dorsoventral axis[END_REF]. Importantly, the axon terminals of the hilar mossy cells contain abundant tightly packed synaptic vesicles and end into the inner third of dentate molecular layer forming asymmetric synapses with granule cell dendrites and dendritic spines [START_REF] Ribak | The development, ultrastructure and synaptic connections of the mossy cells of the dentate gyrus[END_REF][START_REF] Blasco-Ibáñez | Distribution, ultrastructure, and connectivity of calretinin-immunoreactive mossy cells of the mouse dentate gyrus[END_REF].

Other cell types located in the hilus are the fusiform cells whose dendrites run parallel to the granular cell layer [START_REF] Bakst | An experimental analysis of the origins of somatostatin-like immunoreactivity in the dentate gyrus of the rat[END_REF][START_REF] Halasy | Subdivisions in the multiple GABAergic innervation of granule cells in the dentate gyrus of the rat hippocampus[END_REF], and the multipolar neurons whose axons and dendrites remain within the hilus [START_REF] Amaral | A Golgi study of cell types in the hilar region of the hippocampus in the rat[END_REF].

HIPPOCAMPUS

As Llorente de Nó proposed (1934) the hippocampus can be divided into three regions, CA1, CA2 and CA3. The mossy fibers inputs that arrive to CA3 from the dentate gyrus not reaching CA1 and CA2 represent the main anatomical difference among these three regions. In addition to the structural characteristics, different patterns of gene expression also exist among the different regions [START_REF] Dong | Genomicanatomic evidence for distinct functional domains in hippocampal field CA1[END_REF][START_REF] Fanselow | Are the Dorsal and Ventral Hippocampus functionally distinct structures[END_REF]) that make a real physiological separation between the three different fields.

The hippocampus shows a trilaminar structure, folded on itself. Each of these layers can be defined depending on the cell types that host and their spatial distribution and density. Remarkably, the cellular distribution is correlated with the performance of specific functions in the hippocampal network [START_REF] Klausberger | Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations[END_REF]. The ventricular surface of the hippocampus is covered by the alveus, which is a thin layer of myelinated fibers.

Stratum oriens

The basal dendritic tree of pyramidal neurons will be part of the stratum oriens, which is a relatively free layer of cells situated in the outer side of the pyramidal layer (Lorente De Nó, 1934).

Pyramidal cell layer

The pyramidal cell layer (Lorente De Nó, 1934), consists of glutamatergic cells (Liu et al., 1989) whose somata have a pyramidal look due to the presence of a prominent apical dendrite. Their basal dendritic trees distribute in the stratum oriens while the apical dendritic tree is oriented in ventromedial direction.

There is also an assorted population of basket cells with their cell bodies confined within the pyramidal cell layer [START_REF] Frotscher | The mossy cells of the fascia dentata: a comparative study of their fine structure and synaptic connections in rodents and primates[END_REF][START_REF] Jinno | Cellular architecture of the mouse hippocampus: a quantitative aspect of chemically defined GABAergic neurons with stereology[END_REF]. Their axons expand transversely from the cell body forming a basket plexus that innervates the pyramidal cell bodies.

Stratum lucidum

This is an acellular CA3 layer just above the pyramidal cell layer that contains the dentate granule mossy fibers axons.

Stratum radiatum

The stratum radiatum is located superficial to stratum lucidum in CA3 and above the pyramidal cell layer in CA2.The majority of the CA3 to CA3 associational fibers and the ipsilateral CA3 to CA1 Schaffer collaterals are found in this layer.

Stratum lacunosum-moleculare

Finally, the stratum lacunosum-moleculare is located in the vicinity of the hippocampal fissure and the dentate gyrus. About 20% of the dendritic tree of the pyramidal cells reaches the stratum lacunoso-moleculare [START_REF] Amaral | Neurons, numbers and the hippocampal network[END_REF]. This layer receives synapses from the entorhinal cortex.

GABAergic cells of the hippocampus

Although there are a wide variety of cells in the stratum oriens, stratum radiatum, and stratum lacunosum-moleculare of the hippocampus, most of them are GABAergic neurons [START_REF] Ribak | Immunocytochemical localization of glutamic acid decarboxylase in neuronal somata following colchicine inhibition of axonal transport[END_REF][START_REF] Aika | Quantitative analysis of GABA-likeimmunoreactive and parvalbumin-containing neurons in the CA1 region of the rat hippocampus using a stereological method, the disector[END_REF]. Moreover, there are eight well defined GABAergic subclasses in the hippocampus based on their colocalization with parvalbumin, calretinin, calbindin, neuropeptide-Y, somatostatin, cholecystokinin, vasoactive intestinal polypeptide (VIP), or nitric oxide synthase (NOS) [START_REF] Morrison | Immunohistochemical distribution of pro-somatostatin-related peptides in cerebral cortex[END_REF][START_REF] Somogyi | Different Populations Cortex and Hippocampus of Gabaergic Neurons in the Somatostatin-Visual or and[END_REF][START_REF] Sloviter | Immunocytochemical localization of GABA-, cholecystokinin-, vasoactive intestinal polypeptide-, and somatostatin-like immunoreactivity in the area dentata and hippocampus of the rat[END_REF][START_REF] Sloviter | Calcium-binding protein (calbindin-D28k) and parvalbumin immunocytochemistry: localization in the rat hippocampus with specific reference to the selective vulnerability of hippocampal neurons to seizure activity[END_REF][START_REF] Gulyás | Subpopulations of GABAergic neurons containing parvalbumin, calbindin D28k, and cholecystokinin in the rat hippocampus[END_REF][START_REF] Gulyás | Calretinin is present in non-pyramidal cells of the rat hippocampus--I. A new type of neuron specifically associated with the mossy fibre system[END_REF][START_REF] Gulyás | Total number and ratio of excitatory and inhibitory synapses converging onto single interneurons of different types in the CA1 area of the rat hippocampus[END_REF][START_REF] Miettinen | Calretinin is present in non-pyramidal cells of the rat hippocampus-II. Coexistence with other calcium binding proteins and gaba[END_REF][START_REF] Tóth | Calbindin D28k-containing nonpyramidal cells in the rat hippocampus: Their immunoreactivity for GABA and projection to the medial septum[END_REF][START_REF] Jinno | Colocalization of parvalbumin and somatostatin-like immunoreactivity in the mouse hippocampus: quantitative analysis with optical dissector[END_REF]. Some studies have shown the relation between chemical phenotypes and electrophysiological properties of the interneurons [START_REF] Jinno | Cellular architecture of the mouse hippocampus: a quantitative aspect of chemically defined GABAergic neurons with stereology[END_REF][START_REF] Klausberger | Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations[END_REF] but a clear correlation between them have not been fully established yet and their function in the hippocampal network is still poorly understood [START_REF] Witter | The Rat Nervous System[END_REF][START_REF] Witter | The Mouse Nervous System[END_REF]

CONNECTIONS OF THE HIPPOCAMPUS

The hippocampal formation is one of the most important functional structures of the mammalian brain. There exists high connectivity with other brain regions to perform its functions properly. These connections allow playing a key role in memory formation, learning, neurogenesis and cognitive function. Also, they keep informed the hippocampal formation of the brain activity. The connections of the hippocampal formation can be divided into two groups: afferent (connections from other regions) and efferent (connections to other regions) projections.

Afferent connections

The hippocampal formation receives main connections from: the neocortex, the septal area, the contralateral hippocampus (commissural fibers), several nuclei of the diencephalon and the reticular formation of the brain stem. The neocortex of the temporal lobe which receives association fibers of different sensory association areas, sends information to the entorhinal cortex of the hippocampal formation.

The entorhinal cortex is a transition structure between the neocortex and hippocampal cortex. Then, the entorhinal cortex sends fibers through the subiculum and intersects the hippocampal sulcus to end in the dentate gyrus forming the so-called perforant pathway. Fewer fibers from the entorhinal cortex form the alveolar via crossing the subcortical white matter as well as the alveus and ending in the hippocampus.

In addition, fibers from the septal nuclei reach the hippocampus through the fornix.

Moreover, other afferent fibers also included in the fornix come from the thalami nuclei, hypothalamus, diencephalon and brainstem regions such as the ventral tegmental area (dopaminergic), the locus coeruleus (noradrenergic) and nuclei raphe (serotonergic).

Efferent connections

The main efferent pathway from the hippocampal formation is the fornix, which is composed by the axons of the CA1 pyramidal neurons and of the subicular cells.

The axons enter the alveus and together constitute the hippocampal fimbria followed by the fornix after leaving the fibers the hippocampus and going into the forebrain.

These glutamatergic fibers travel along the entire fornix which branches near the anterior commissure into two components: precommissural and postcommissural fibers. The precommissural fibers (formed by fibers originating mainly in the hippocampus) extend rostrally, innervating the septal nuclei, the nucleus accumbens and the substantia innominata. Meanwhile, the postcommissural fibers (composed mainly of fibers originating in the subiculum) extend caudally to the diencephalon to finally reach the hypothalamus, where they divide into the medial cortico-hypothalamic tract (that innervates the mammillary bodies, the posterior hypothalamus and other ventromedial hypothalamic nuclei) and the subiculumthalamic tract (reaching the thalamic nuclei) [START_REF] Swanson | Anatomical and electrophysiological evidence for a projection from the medial preoptic area to the "mesencephalic and subthalamic locomotor regions" in the rat[END_REF].

A lot of the fimbria fibers are crossed, before being included into the fornix, in the caudal region of the septal nuclei. Most of these fibers are directed to the contralateral hippocampal formation. However, a few of them are integrated into the fornix to innervate the same structures that receive precommissural and postcommissural projections from the ipsilateral fornix.

The fornix is not the only efferent pathway from the hippocampal formation, since there are significant projections from the subicular complex and entorhinal cortex to cortical areas, such as the perirhinal cortex and the parahippocampal gyrus.

Hippocampal synaptic circuit

It is well characterized that the hippocampus has a unidirectional pathway that connects all its regions sequentially. The start of the hippocampal circuit is the dentate gyrus which receives the perforant pathway from the entorhinal cortex.

Then, the mossy axon fibers of the dentate granule cells project unidirectionally to the proximal dendrites of the CA3 pyramidal cells. In turn, the CA3 pyramidal cell Schaffer collaterals reach the CA1 stratum radiatum. The CA1 hippocampal region projects intensely into the subicular complex which, sends axons back to the entorhinal cortex. Although the circuit forms a loop, each of the hippocampal fields also sends axons out.

However, even though the classical view proposes that the connectivity is unidirectional, several types of retrograde connections also exist in the hippocampal formation [START_REF] Van Strien | The anatomy of memory: an interactive overview of the parahippocampal-hippocampal network[END_REF]. For instance, CA3 pyramidal cells are connected with the hilus and the dentate molecular layer [START_REF] Laurberg | Commissural and intrinsic connections of the rat hippocampus[END_REF][START_REF] Buckmaster | A comparison of rat hippocampal mossy cells and CA3c pyramidal cells[END_REF][START_REF] Witter | The perforant path: projections from the entorhinal cortex to the dentate gyrus[END_REF]. Furthermore, retrograde connections from CA1 to CA3 through interneurons [START_REF] Laurberg | Commissural and intrinsic connections of the rat hippocampus[END_REF][START_REF] Amaral | Organization of CA1 projections to the subiculum: a PHA-L analysis in the rat[END_REF][START_REF] Cenquizca | Spatial organization of direct hippocampal field CA1 axonal projections to the rest of the cerebral cortex[END_REF] and from the subiculum to CA1 [START_REF] Finch | Demonstration of axonal projections of neurons in the rat hippocampus and subiculum by intracellular injection of HRP[END_REF]) have been identified. Also, recurrent collaterals in CA3 [START_REF] Laurberg | Commissural and intrinsic connections of the rat hippocampus[END_REF][START_REF] Amaral | Neurons, numbers and the hippocampal network[END_REF][START_REF] Buckmaster | A comparison of rat hippocampal mossy cells and CA3c pyramidal cells[END_REF][START_REF] Sik | Complete Axon Arborization of a Single CA3 Pyramidal Cell in the Rat Hippocampus, and its Relationship With Postsynaptic Parvalbumin-containing Interneurons[END_REF][START_REF] Siddiqui | CA3 axonal sprouting in kainate-induced chronic epilepsy[END_REF][START_REF] Wittner | Three-dimensional reconstruction of the axon arbor of a CA3 pyramidal cell recorded and filled in vivo[END_REF] and bidirectional connections between the entorhinal cortex and subiculum have been described.

WORKING HYPOTHESIS

As I have already described, the CB1 receptor expression is very high in inhibitory GABAergic synaptic terminals and low in glutamatergic neurons as well as in brain astrocytes. Subcellularly, CB1 receptors are localized in the plasma membranes and in intracellular organelles, such as brain mitochondria. Due to the hugely different levels of expression among different cell types, it is extremely difficult to identify low CB1 receptor expression in cell types and/or in subcellular compartments of wild-type brains. This led in the past to consider low CB1 receptor levels as background staining (see below).

Based on methodological "loss of function" approaches, the use of mutant mice lacking CB1 receptors in specific cell populations helped identifying the necessary role of the CB1 receptor for some brain functions. Moreover, neuroanatomical analyses of these mutants determined the presence of the CB1 receptor at sites previously not known (e.g. cortical glutamatergic neurons or neuronal mitochondria). However, these approaches do not provide definitive information concerning the possibly sufficient role of the cell-type expression of CB1 receptors for a given function. Indeed, distinguishing between low levels (wild-type) and no expression (conditional mutant) is very difficult.

Therefore, to establish precise causal relationships and to identify low levels of expression, "rescue" strategies are needed to analyze the function of endogenous CB1 receptor expression exclusively in specific cell types. Indeed, these rescue mice have allowed already determining the sufficient causal role of the CB1 receptor in the anterior olfactory cortex area as well as in dorsal telencephalic glutamatergic neurons. However, so far no studies have addressed the subcellular localization of the CB1 receptor in these mutant brains in the electron microscope. This is particularly important because the huge expression of the CB1 receptor in specific cell types (e.g. hippocampal GABAergic interneurons) does not appreciate the detailed pattern of expression of the receptor in other cell types (e.g. hippocampal glutamatergic pyramidal neurons) in wild-type mouse samples. Hence, the conditional rescue mutants emerge as key tools for the detailed anatomical characterization of the subcellular distribution of the receptor in specific cell types, independently of its level of expression.

In this study, we have examined the CB1 receptor expression in the hippocampus of rescue mice modified to express the gene exclusively in specific brain cell types, such as dorsal telencephalic glutamatergic neurons (Glu-CB 1 -RS), GABAergic neurons (GABA-CB 1 -RS) and brain astrocytes (GFAP-CB 1 -RS), for three reasons. First, to elucidate the real levels of CB1 receptors in specific cell types expressing very little CB1 protein eliminating the interference of other cells highly expressing CB1 receptors. Second, to verify that the re-expression is limited to the specific cell types and maintains endogenous levels of CB1 receptors where the regulatory sequences of the receptor are normally activated. And third, to know whether the rescue mutant mice are good and reliable tools for functional studies of the endocannabinoid system in the brain. Finally, we have also analyzed the CB1 receptor expression in the hippocampus of transgenic mice expressing the hrGFP protein in astrocytes (GFAPhrGFP-CB 1 -WT and GFAPhrGFP-CB 1 -KO mice) in order to investigate the CB1 receptor distribution in astrocytes relative to the tripartite synapses.

The general goal of this Thesis was the anatomical characterization of new different mutant mice expressing CB1 receptors in specific brain cells. In particular, the study was focused on the hippocampus processed for a preembedding immunogold method for electron microscopy.

The proposed objectives of the present Thesis work are:

-To study the CB1 receptor expression in the hippocampus of mice with the CB1 receptor rescued in dorsal glutamatergic neurons (Glu-CB 1 -RS). -To investigate the CB1 receptor expression in the hippocampus of mice with the CB1 receptor rescued in GABAergic neurons (GABA-CB 1 -RS). -To determine the CB1 receptor expression in the hippocampus of mice with the CB1 receptor rescued in astrocytes (GFAP-CB 1 -RS). -To compare the CB1 receptor expression in hippocampal astrocytes of transgenic mice with GFAP or with hrGFP as the astrocytic marker. -To establish the anatomical distribution of the astrocytic CB1 receptors relative to the surrounding excitatory and inhibitory synapse forming the tripartite synapse.

I have used specific antibodies in combination with an immunocytochemical technique (immunoperoxidase) for light microscopy; a preembedding silver intensified immunogold method and a double preembedding immunogold and immunoperoxidase method for electron microscopy. In addition, I have done the corresponding quantification and statistical analysis of the data obtained in the electron microscopic experiments.

ANTIBODIES

Polyclonal Anti-Cannabinoid Receptor Type-1 (anti-CB1 receptor), monoclonal Anti-Glial Fibrillary Acidic Protein (anti-GFAP) and polyclonal humanized Renilla reniformis Green Fluorescence Protein (anti-hrGFP) antibodies were used (Table 1). The experiments were done under the same conditions. Moreover, a negative control by omitting the primary antibody was performed. Importantly, the CB1 receptor antibodies used were tested in CB 1 -KO brain tissue. Finally, the specificity of the other antibodies has been thoroughly confirmed in previous studies.

CB1:

The sequence of the immunizing peptide used to generate the antibody corresponds with the last 31 amino acids of the C-terminus of the mouse CB1 receptor (NM007726), as provided by the manufacturer (NCBI Reference Sequence: NP_031752.1; 443-473 amino acid residues: MHRAAESCIKSTVKIAKVTMSVSTDTSAEAL). The specificity of the CB1 receptor antibody (Frontier Institute; Goat polyclonal; #CB1-Go-Af450) was assessed in previous publications [START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF][START_REF] Reguero | GABAergic and cortical and subcortical glutamatergic axon terminals contain CB1 cannabinoid receptors in the ventromedial nucleus of the hypothalamus[END_REF][START_REF] Bénard | Mitochondrial CB₁ receptors regulate neuronal energy metabolism[END_REF]Hebert-Chatelain et al., 2014b;Soria-Gómez et al., 2014;[START_REF] Martín-García | Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors[END_REF]. Furthermore, the specificity was tested in this study by applying the antibody to CB 1 -KO hippocampus. No immunostaining was observed in this tissue in the light microscope. Also, the specificity of the CB1 receptor pattern was analyzed in hippocampi of CB 1 -WT and CB 1 -KO processed together for immunoelectron microscopy. Thus, CB1 receptor immunoparticles were localized in the inhibitory and excitatory synaptic terminals of CB 1 -WT mouse while the CB1 receptor pattern disappeared in CB 1 -KO tissue, indicating the high specificity of the antibody used. Moreover, the antibody detects a single protein band of 52 kDa on immunoblots (Manufacturer´s provided information, Frontier Science).

-GFAP: The specificity of the GFAP antibody (Sigma-Aldrich; mouse monoclonal; #G3893) was established in previous publications [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF][START_REF] Bosier | Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes[END_REF]. This antibody has been tested for the GFAP immunolocalization in human, pig and rat tissues, and specifically detects GFAP on immunoblots. In addition, it does not cross react with vimentin, which is frequently co-expressed in glioma cells and some astrocytes (Manufacturer´s provided information Sigma-Aldrich).

-hr-GFP: The humanized Renilla reniformis green fluorescence protein (hr-GFP) antibody (Stratagene; rabbit polyclonal; #240142) has been tested in brain and spinal cord and other tissues previously [START_REF] Sakata | Characterization of a novel ghrelin cell reporter mouse[END_REF][START_REF] Zhang | Projections of nucleus accumbens adenosine A2A receptor neurons in the mouse brain and their implications in mediating sleep-wake regulation[END_REF][START_REF] Kerr | The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2[END_REF]. Moreover, anti-hrGFP antibody was not observed to bind to any cell in wild type mice.

RESEARCH ANIMALS

CB 1 RECEPTOR MUTANT LINES

Generation of the CB 1 -KO mice

CB 1 -KO mice were generated and genotyped as described [START_REF] Marsicano | The endogenous cannabinoid system controls extinction of aversive memories[END_REF]. Conditional CB1 receptor mutant mice were obtained by crossing the respective Cre expressing mouse line with CB1 f/f mice [START_REF] Marsicano | CB1 cannabinoid receptors and on-demand defense against excitotoxicity[END_REF], using a three-step breeding protocol [START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF]. All mice were in a predominant C57BL/6N background.

Generation of conditional mutant mice bearing a selective deletion of the CB 1 receptor in cortical glutamatergic neurons (hereafter Glu-CB 1 -KO)

The helix-loop-helix transcription factor NEX is a marker of embryonic neuronal progenitors, which will develop into mature cortical glutamatergic neurons [START_REF] Wu | Pyramidal neurons of upper cortical layers generated by NEX-positive progenitor cells in the subventricular zone[END_REF]. In the adult brain, NEX is expressed in mature glutamatergic cortical neurons, but not in cortical GABAergic interneurons and to a much lesser extent in subcortical regions [START_REF] Bartholomä | NEX-1: a novel brain-specific helix-loop-helix protein with autoregulation and sustained expression in mature cortical neurons[END_REF]. Cre expression under the control of the regulatory sequences of NEX in mutant mice as generated by knock-in into the NEX locus (NEX-Cre mice), leads to the specific deletion of ''floxed'' alleles in forebrain neurons [START_REF] Kleppisch | Hippocampal cGMP-dependent protein kinase I supports an age-and protein synthesis-dependent component of long-term potentiation but is not essential for spatial reference and contextual memory[END_REF]. Thereby, CB 1 f/f; NEX-Cre mice Glu-CB 1 -KO, [START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF] were obtained by crossing CB 1 f/f; with NEX-Cre mice [START_REF] Schwab | Neuronal Basic Helix-Loop-Helix Proteins (NEX and BETA2/Neuro D) Regulate Terminal Granule Cell Differentiation in the Hippocampus[END_REF][START_REF] Kleppisch | Hippocampal cGMP-dependent protein kinase I supports an age-and protein synthesis-dependent component of long-term potentiation but is not essential for spatial reference and contextual memory[END_REF]. Mutants were obtained by crossing CB 1 f/f females with CB 1 f/f; NEX-Cre males.

Generation of conditional mutant mice bearing a selective deletion of the CB 1 receptor in GABAergic neurons (hereafter GABA-CB 1 -KO)

The Dlx5/Dlx6 genes are homeobox genes that are expressed in differentiating and migrating forebrain GABAergic neurons during embryonic development [START_REF] Stuhmer | Expression from a Dlx Gene Enhancer Marks Adult Mouse Cortical GABAergic Neurons[END_REF]. Dlx5/6-Cre [START_REF] Zerucha | A Highly Conserved Enhancer in the Dlx5/Dlx6 Intergenic Region is the Site of Cross-Regulatory Interactions between Dlx Genes in the Embryonic Forebrain[END_REF] were crossed with CB 1 f/f to obtain CB 1 f/f; Dlx5/6-Cre mice. Thus, the expression of Cre recombinase under the control of the regulatory sequences of Dlx5/Dlx6 genes drives to a recombination of loxP sites in GABAergic neurons (GABA-CB 1 -KO mice, [START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF].

Mutants were obtained by crossing CB 1 f/f females with CB 1 f/f; DLX5/6-Cre males.

Generation of conditional mutant mice bearing a selective deletion of the CB 1 receptor in astrocytes (hereafter GFAP-CB 1 -KO)

Transgenic mice CB1 f/f; GFAP-CreERT2 were obtained from crossing mice carrying CB1 receptor "floxed" sequence [START_REF] Marsicano | CB1 cannabinoid receptors and on-demand defense against excitotoxicity[END_REF] with transgenic mice expressing the inducible version of the Cre recombinase CreERT2 under the control of the human glial fibrillary acid protein promoter, i.e. GFAP-CreERT2 mice [START_REF] Hirrlinger | Temporal Control of Gene Recombination in Astrocytes by Transgenic Expression of the Tamoxifen-Inducible DNA Recombinase Variant CreERT2[END_REF]. This animal model allows the on-demand control of astroglial CB1 receptor recombination in adult mice [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF].

Generation of Glu-CB 1 -RS and GABA-CB 1 -RS

Rescue mice were produced as previously described [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF]. Briefly, Stop-CB1 mice were generated by inserting a loxP-flanked stop cassette into the 5'UTR of the coding exon of the CB 1 gene, 32 nucleotides upstream of the translational start codon to obtain CB 1 stop/stop mice, lacking expression of the CB1 protein [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF]. Conditional rescue mice were obtained by crossing Stop-CB1 with NEX-Cre or Dlx5/6-Cre mice to generate Glu-CB 1 -RS (rescue) and GABA-CB 1 -RS, respectively. Genotyping was performed as previously described [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF], and rescue mutants were obtained by crossing CB 1 stop/stop females with CB 1 stop/stop; Nex-Cre or CB 1 stop/stop; DLX5/6-Cre males, respectively.

Generation of GFAP-CB 1 -RS

To obtain specific CB1 rescue in astrocytes STOP-CB 1 mice [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF] were crossed with GFAP-CreERT2 mice [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF] as previously described [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF]. Three weeks before neuroanatomical characterization, deletion of stop cassette for CB1 rescue was achieved by injection of 4hydroxytamoxifen as previously described [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF].

Generation of GFAPhrGFP-CB 1 -WT and GFAPhrGFP-CB 1 -KO

CB 1 -WT and CB 1 -KO mice received an intrahippocampal injection of a recombinant adeno associated virus expressing humanized Renilla GFP protein under the control of human GFAP promoter [START_REF] Jonquieres G Von | Glial Promoter Selectivity following AAV-Delivery to the Immature Brain[END_REF]. Virus production and purification, as well as injection procedure were performed as previously described [START_REF] Chiarlone | A restricted population of CB1 cannabinoid receptors with neuroprotective activity[END_REF]. After surgery, mice were allowed to recover for at least 4 weeks before anatomical characterization.

ANIMAL TREATMENT

The

TRANSCARDIALLY PERFUSION OF THE ANIMALS

1. C57BL/6N adult mice (between 60 and 90 postnatal days) of either sex were housed under standard conditions (food and water ad libitum; 12h/12h light/dark cycle). CB1 wild type (hereafter CB 1 -WT), Glu-CB 1 -KO, GABA-CB 1 -KO, GFAP-CB 1 -KO, CB 1 -STOP, Glu-CB 1 -RS, GABA-CB 1 -RS, GFAP-CB 1 -RS, CB 1 -KO, GFAPhrGFP-CB 1 -WT and GFAPhrGFP-CB 1 -KO mice (at least three animals of 3 each condition) were deeply anesthetized by intraperitoneal injection of ketamine/xylazine (80/10 mg/kg body weight). 2. Animals were transcardially perfused at room temperature (20-25 ºC) with phosphate buffered saline (0.1 M, pH 7.4) for 20 seconds, followed by the fixative solution made up of 4% formaldehyde (freshly depolymerized from paraformaldehyde), 0.2% picric acid, and 0.1% glutaraldehyde in phosphate buffer (0.1 M Phosphate buffer, pH 7.4) for 10-15 minutes. 3. Brains were carefully removed from the skull and post-fixed in the fixative solution for approximately 1 week at 4 ºC. Finally, brains were stored at 4 ºC in 1:10 diluted fixative solution plus 0.025% sodium azide at 4°C until its processing.

AVIDIN-BIOTIN PEROXIDASE METHOD FOR LIGHT MICROSCOPY

This is the protocol applied:

1. Brain coronal sections were cut at 50 µm in a vibratome and collected in 0.1 M Phosphate buffer at room temperature. 2. Preincubation of the hippocampal sections with blocking solution composed by 10% horse serum, 0.1% sodium azide and 0.5% triton X-100 prepared in Tris-Hydrogen Chloride buffered saline 1X, pH 7.4 for 30 minutes at room temperature.

3. Incubation with the primary anti-CB1 receptor antibody (1:100) prepared in the blocking solution, with continuous gentle shaking for 2 days at 4 ºC. 4. Several washes in 1% horse serum and 0.5% triton X-100 in Tris-Hydrogen Chloride buffered saline for 30 minutes to remove excess of the antibody. 5. Incubation with a secondary biotinylated horse anti-goat Inmunoglobulin-G

(1:200, Vector Laboratories, Burlingame, CA, USA) prepared in the washing solution for 1 hour on a shaker at room temperature.

6. Washes with 1% horse serum and 0.5% triton X-100 in Tris-Hydrogen Chloride buffered saline. 7. Incubation in 1:50 avidin-biotin complex (Avidin-biotin peroxidase complex, Elite, Vector Laboratories, Burlingame, CA, USA) prepared in the washing solution for 1 hour at room temperature. 8. Several rinses with1% horse serum and 0.5% triton X-100 in Tris-Hydrogen Chloride buffered saline. Last rinses were done with 0.1M phosphate buffer and 0.5% triton X-100. 9. Incubation with 0.05% diaminobenzidine as a chromogen in 0.1 M phosphate buffer containing 0.5% triton-X100 and 0.01% hydrogen peroxide, for 5 minutes at room temperature. 10. Several washes in 0.1 M phosphate buffer with 0.5% triton-X100. 11. Mounting on gelatinized slides. 12. Dehydration in graded alcohols (50°, 70°, 96°, 100°) for 5 minutes each.

PREEMBEDDING SILVER-INTENSIFIED IMMUNOGOLD METHOD FOR ELECTRON MICROSCOPY

The following protocol was applied:

1. Coronal hippocampal vibrosections were cut at 50 µm and collected in 0.1 M phosphate buffer (pH 7.4) at room temperature. 2. Preincubation of the sections in a blocking solution of 10% bovine serum albumin, 0.1% sodium azide, and 0.02% saponin prepared in Tris-

PREEMBEDDING DOUBLE LABELING OF SILVER-INTENSIFIED IMMUNOGOLD AND IMMUNOPEROXIDASE METHOD FOR ELECTRON MICROSCOPY

This is the protocol followed:

1. Coronal hippocampal vibrosections were cut at 50 µm and collected in 0.1 M phosphate buffer (pH 7.4) at room temperature. 2. Preincubation in a blocking solution of 10% bovine serum albumin, 0.1% sodium azide, and 0.02% saponin prepared in Tris-Hydrogen Chloride buffered saline 1X, pH 7.4 for 30 minutes at room temperature. 3. Incubation with the primary goat polyclonal anti-CB1 receptor antibody

(1:100) and the primary mouse monoclonal anti-GFAP (1:1000) or the primary rabbit polyclonal hrGFP (1:500) prepared in the blocking solution in 10% Bovine Serum Albumin / Tris-Hydrogen Chloride buffered saline containing 0.1% sodium azide and 0.004% saponin on a shaker for 2 days at 4°C. 4. Several washes in 1% Bovine Serum Albumin / Tris-Hydrogen Chloride buffered saline. 5. Incubation with the corresponding biotinylated secondary antibody (1:200) in 1% Bovine Serum Albumin / Tris-Hydrogen Chloride buffered saline with 0.004% saponin on a shaker for 3 hours at room temperature. 6. Several washes in 1% Bovine Serum Albumin / Tris-Hydrogen Chloride buffered saline overnight on a shaker at 4°C. 7. Incubation in a secondary 1.4 nm gold-labeled rabbit anti-goat Immunoglobulin-G (Fab' fragment, 1:100, Nanoprobes Inc., Yaphank, NY, USA) in 1% Bovine Serum Albumin / Tris-Hydrogen Chloride buffered saline with 0.004% saponin on a shaker for 3 hours at room temperature.

SEMI-QUANTIFICATION OF THE CB1 RECEPTOR IMMUNOGOLD STAINING

With the aim of maximizing the standard conditions, the pre-embedding immunogold method was systematically applied simultaneously to all the sections collected from the all different animals (n=3 of each condition) in each of the three replicated experiments done for each mouse performed.

Immunogold-labeled hippocampal sections were then visualized in the light microscope in order to select portions of the CA1 and the dentate molecular layer with good and reproducible CB1 receptor immunolabeling. Then the semithin sections were cut with an ultra-microtome and just the first 5 ultrathin sections (60 nm thick) were collected onto two grids. Moreover, to further standardize the conditions between the immunolabeled sections obtained from the different mice, only the first 1.5 µm from the section surface was photographed. All electron micrographs were taken at 18,000x using a Digital Morada Camera from Olympus. Sampling was always performed accurately in the same way for all the animals studied.

The analyzed excitatory and inhibitory synapses were identified by their characteristic ultrastructural features. Hence, excitatory synapses were distinguished by their asymmetric synapses with thick postsynaptic densities and presynaptic terminals containing abundant, clear and spherical synaptic vesicles. Inhibitory synapses were identified by their symmetric synaptic contacts and terminal boutons with pleomorphic synaptic vesicles.

To determine the proportion of the labeled CB1 receptor terminals, positive labeling was considered if at least one immunoparticle was within approximately 30 nm from the plasmalemma of the synaptic terminal. Metal particles at these synaptic membranes were visualized and counted. Immunopositive astrocytic processes were considered positive if the precipitates of DAB immunoreaction product for hrGFP or GFAP, were inside the astrocytic elements. Image-J software was used to measure the membrane length and distance to synapses. Percentages of CB1 receptor positive profiles, density (particles/µm membrane) and proportion of CB1 immunoparticles in terminals versus total CB1 receptor expression in cellular membranes were analyzed and displayed as mean ± Standard Error of the Mean using a statistical software package (GraphPad Prism, GraphPad Software Inc, San Diego, USA). The normality test (Kolmogorov-Smirnov normality test) was applied before running statistical tests and subsequently data were analyzed using nonparametric tests (Man-Whitney U test when k=2 or Kruskal-Wallis test when k>2). A potential variability between mice of the same mutant line was analyzed statistically. Since there were no differences between them, all data from each line were pooled.

SEMI-QUANTIFICATION OF THE DISTANCE FROM ASTROCYTIC CB 1 RECEPTORS TO THE NEAREST SYNAPSE.

Immunogold and immunoperoxidase hippocampal sections were visualized in the light microscope in order to select portions of the CA1 and the dentate molecular layer with good and reproducible immunolabeling. The first 5 ultrathin sections (60 nm thick) were collected onto two grids. Moreover, to further standardize the conditions between the immunolabeled sections obtained from the different mice, only the first 1.5 µm from the section surface was photographed. Once again, all electron micrographs were taken at 18,000x using a Digital Morada Camera from Olympus. Sampling was always accurately done in the same way for all the animals studied.

Positive astrocytic processes were considered by the presence of immunoprecipitation of DAB in the cytoplasm (see above). Moreover, to determine the proportion of labeled CB1 receptor astrocytic processes, positive labeling was considered if at least one immunoparticle was within approximately 30 nm from the plasmalemma.

Afterward, the nearby synapses surrounding the immunopositive astrocytic process were identified, the distances were measured using the Image-J software, and the nearest synapse to the astrocytic CB1 receptor immunogold particle was selected. The data from all the nearest synapses to the astrocytic CB1 receptor particles were tabulated, analyzed and displayed as mean ± S.E.M. using a statistical software package (GraphPad Prism, GraphPad Software Inc, San Diego, USA).

CB 1 RECEPTOR DISTRIBUTION IN THE BRAIN OF GLU-CB 1 -RS AND GABA-CB 1 -RS MICE. LIGHT MICROSCOPY

The pattern of CB1 receptor immunostaining was first analyzed in the Glu-CB 1 -RS and GABA-CB 1 -RS brains by light microscopy (Figs. 11 and 12). As expected, a much weaker and diffuse CB1 receptor pattern was observed in Glu-CB 1 -RS than in GABA-CB 1 -RS, in accordance with the distribution of CB1 receptor in glutamatergic and GABAergic cells, respectively (Figs. 11 and 12). Thus, noticeable CB1 receptor staining was observed in the striatum, cortex, olfactory tubercle and amygdala of Glu-CB 1 -RS (Fig. 11). In GABA-CB 1 -RS, strong CB1 receptor immunoreactivity was detected in the cortex, anterior olfactory nucleus, piriform cortex, globus pallidus, entopeduncular nucleus, amygdala and the substantia nigra (Fig. 12). The striatum exhibited moderate to strong immunostaining (Fig. 12). Altogether, the overall CB1 receptor pattern in Glu-CB 1 -RS and GABA-CB 1 -RS matches the known receptor distribution in the brain.

CB 1 RECEPTOR DISTRIBUTION IN HIPPOCAMPUS OF GLU-CB 1 -RS AND GABA-CB 1 -RS MICE. LIGHT MICROSCOPY

We particularly focused on the pattern of the CB1 receptor immunostaining in the hippocampus (Fig. 13). In CB 1 -WT, a distinct intensity pattern was observed throughout the layers (Fig. 13a). Thus, the strongest CB1 receptor immunostaining was revealed in the stratum pyramidale and at the limit between the strata radiatum and lacunosum-moleculare. Also, a dense neuropil labeling was seen throughout the strata radiatum and oriens. In the dentate gyrus, the most prominent CB1 receptor staining localized in the innermost third of the stratum moleculare whereas a remarkable but weaker staining was found in the outer 2/3 of the layer. Finally, immunoreactive fibrous processes surrounding the granule cell bodies were visible in the stratum granulosum (Fig. 13a).

In the Glu-CB 1 -RS hippocampus, a faint CB1 receptor immunoreactivity was observed throughout the strata oriens and radiatum of the hippocampal Ammon´s horn (Cornu ammonis, CA) (Fig. 13b). Remarkably, a strong band of CB1 receptor immunostaining appeared in the innermost third of the dentate molecular layer which corresponds to the zone of the synapses of the commissural/associational system (Fig. 13b) [START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF][START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF]. In GABA-CB 1 -RS, heavy CB1 receptor immunoreactivity was distributed throughout the CA stratum radiatum and the dentate molecular layer (Fig. 13c). However, a stronger CB1 receptor staining was found in the pyramidal cell layer and at the limit between strata radiatum and lacunosum-moleculare of the CA1, as well as in the molecular innermost third and the infragranular zone of the dentate gyrus (Fig. 13c). Importantly, the CB1 receptor immunostaining was absence in the CB 1 -KO hippocampus, indicating the specificity of the CB1 receptor antibody used (Fig. 13d).

SUBCELLULAR LOCALIZATION OF THE CB 1 RECEPTOR IN HIPPOCAMPUS OF GLU-CB 1 -RS AND GABA-CB 1 -RS. HIGH RESOLUTION ELECTRON MICROSCOPY

To study the localization of CB1 receptor in detail, a preembedding immunogold method for electron microscopy was used. Hence, hippocampal sections of CB 1 -WT, CB 1 -KO, Glu-CB 1 -RS, GABA-CB 1 -RS, STOP-CB 1 , Glu-CB 1 -KO and GABA CB 1 -KO mice were used to investigate the cellular and subcellular localization of the receptor in the CA1 stratum radiatum (Fig. 14) and in the inner third of the dentate molecular layer (Fig. 15). As expected in CB 1 -WT, presynaptic inhibitory terminal membranes forming symmetric synapses were decorated with a high density of CB1 receptor immunoparticles, whereas a much lower labeling was observed in excitatory terminals making asymmetric synapses. CB1 receptor immunoparticles were located at a distance from the active zones of the symmetric and asymmetric synapses formed with postsynaptic dendrites and dendritic spines, respectively (Figs. 14a and15a). Noticeably, the CB1 receptor pattern virtually disappeared in CB 1 -KO meaning that the CB1 receptor antibody used was highly specific (Figs. 14b and15b).

In Glu-CB 1 -RS, the CB1 receptor was re-expressed in excitatory synapses identified by their typical ultrastructural features, namely, axon boutons with abundant, clear and spherical synaptic vesicles forming asymmetric synapses with postsynaptic dendritic spines, whereas CB1 receptor immunolabeling was absent in symmetric synapses (Figs. 14 d and15d). CB1 receptor was also re-expressed in synaptic terminals of the commissural/associational system in the inner third of the dentate molecular layer that contained abundant tightly packed synaptic vesicles and forming asymmetric synapses with granule cell dendrites and dendritic spines (Fig. 15d) [START_REF] Ribak | The development, ultrastructure and synaptic connections of the mossy cells of the dentate gyrus[END_REF][START_REF] Li | The hippocampal CA3 network: An in vivo intracellular labeling study[END_REF][START_REF] Blasco-Ibáñez | Distribution, ultrastructure, and connectivity of calretinin-immunoreactive mossy cells of the mouse dentate gyrus[END_REF][START_REF] Scharfman | The CA3 "backprojection" to the dentate gyrus[END_REF]. By contrast, in Glu-CB 1 -KO CB1 receptor immunonegative excitatory synapses but numerous CB1 receptor immunoparticles accumulated in terminals with pleomorphic synaptic vesicles and symmetric synapses with dendrites were observed (Figs. 14e and15e).

In the case of GABA-CB 1 -RS, rich CB1 receptor immunolabeling was restricted to presynaptic terminal plasma membranes making symmetric synapses with dendrites; no immunoparticles were found at excitatory synapses (Figs. 14f and15 f). On the other hand, only presynaptic boutons forming asymmetric synapses with spines were CB1 receptor immunopositive in GABA-CB 1 -KO (Figs. 14g and15g).

No immunolabeling was observed in excitatory or inhibitory synapses of STOP-CB 1 hippocampus (Figs. 14c and15c). In addition, CB1 receptor immunoparticles were also observed in mitochondrial membranes of CB 1 -WT and the conditional mutants, but not in CB 1 -KO (Figs. 14b and15b)

SUBCELLULAR LOCALIZATION OF THE CB 1 RECEPTOR IN HIPPOCAMPUS OF GFAP-CB 1 -RS AND GFAPhrGFP-CB 1 -WT. HIGH RESOLUTION ELECTRON MICROSCOPY

For the study in detail of the localization of CB1 receptors in astrocytes of the hippocampus, a preembedding immunogold and immunoperoxidase method for electron microscopy were applied. In order to investigate the cellular and subcellular localization of the receptor in the CA1 stratum radiatum (Fig. 16) and in the the dentate molecular layer (Fig. 17), hippocampal sections of CB 1 -WT, CB 1 -KO, GFAP-CB 1 -RS, STOP-CB 1 and GFAP-CB 1 -KO mice were used. Besides, CB 1 -WT and CB 1 -KO mice expressing hrGFP in astrocytes (GFAPhrGFP-CB 1 -WT and GFAPhrGFP-CB 1 -KO respectively) were also analyzed.

As expected, CB1 receptor immunoparticles were distributed in astrocytic processes membranes and terminal plasmalemma of CB 1 -WT (Figs. 16a and17a) and of GFAPhrGFP-CB 1 -WT (Figs 16f,16g,17f and 17g). However, in GFAP-CB 1 -RS, CB1 receptor immunoparticles appeared only in astrocytic processes but not in synaptic terminals (Figs. 16c and17c). Conversely in GFAP-CB 1 -KO (Figs. 16d and17d), CB1 receptor labeling disappeared from astrocytes membranes whereas the pattern of the CB1 receptor in synaptic terminals remained unchangeable relative to CB 1 -WT (Figs. 16a and17a). Importantly, there was not CB1 receptor immunolabeling in synaptic terminals nor in astrocytic processes of STOP-CB 1 hippocampus (Figs. 16e and17e). Furthermore, the CB1 receptor staining pattern disappeared in CB 1 -KO (Figs. 16b and17b) and in GFAPhrGFP-CB 1 -KO (Figs. 16h and17h) hence demonstrating the great specificity of the anti-CB1 receptor antibody used.

STATISTICS OF THE CB 1 RECEPTOR IN EXCITATORY TERMINALS OF THE GLU-CB 1 -RS AND GABA-CB 1 -RS HIPPOCAMPUS

The proportion of CB1 receptor immunopositive excitatory synaptic terminals in the CA1 stratum radiatum of CB 1 -WT (24.29% ± 1.09), Glu-CB 1 -RS (21.89% ± 1.21) and GABA-CB 1 -KO (21.37% ± 1.13) was not significantly different (Fig. 18a). However, the percentage of positive terminals greatly decreased in Glu-CB 1 -KO (2.50% ± 0.76) and virtually disappeared in STOP-CB 1 , GABA-CB 1 -RS and CB 1 -KO (Fig. 18a). Furthermore, no statistical differences were found in CB1 receptor density (particles/µm) between excitatory synaptic terminals of CB 1 -WT (0.45 ± 0.01), Glu-CB 1 -RS (0.45 ± 0.02) and GABA-CB 1 -KO (0.46 ± 0.02) (Fig. 18b).

Finally, the proportion of CB1 receptor gold particles in asymmetric terminals versus total CB1 receptor expression in plasmalemmal structures was analyzed in CA1 stratum radiatum. 88.78% ± 1.96 of the total CB1 receptor immunoparticles were located in excitatory terminals of Glu-CB 1 -RS, while 12.35% ± 1.04 and 27.30% ± 3.34 were found in CB 1 -WT and GABA-CB 1 -KO respectively. Only residual CB1 receptor immunoparticles were observed in STOP-CB 1 , GABA-CB 1 -RS, Glu-CB 1 -KO and CB 1 -KO (Fig. 18c).

The analysis of the inner third of the dentate molecular layer showed comparable percentages of CB1 receptor immunopositive excitatory synaptic terminals in CB 1 -WT (55.98% ± 2.51), Glu-CB 1 -RS (53.19% ± 2.89) and GABA-CB 1 -KO (52.12% ± 3.00) (Fig. 19a). The proportion decreased in Glu-CB 1 -KO (5.48% ± 1.70) and practically disappeared in STOP-CB 1 , GABA-CB 1 -RS and CB 1 -KO (Fig. 19a). CB1 receptor immunoparticle density, expressed as the number of particles per µm of cell perimeter, was statistically similar in CB 1 -WT (0.39 ± 0.02), Glu-CB 1 -RS (0.39 ± 0.02) and GABA-CB 1 -KO (0.39 ± 0.02) (Fig. 19b). Furthermore, the proportion of CB1 receptor immunogold particles in glutamatergic terminals versus total CB1 receptor expression in plasmalemma was 12.80% ± 1.39 in CB 1 -WT, 88.64% ± 1.71 in Glu-CB 1 -RS, 29.22% ± 3.65 in GABA-CB 1 -KO and only residual CB1 receptor immunoparticles were found in excitatory terminals of GABA-CB 1 -RS, Glu-CB 1 -KO, STOP-CB 1 and CB 1 -KO mice (Fig. 19c). Altogether, these results indicate that the subcellular CB1 receptor distribution and expression levels at excitatory synapses were preserved in the Glu-CB 1 -RS and GABA-CB 1 -KO mutants relative to the wild type, remaining residual at the same synapses of Glu-CB 1 -KO and GABA-CB 1 -RS mice.

STATISTICS OF THE CB 1 RECEPTOR IN INHIBITORY TERMINALS OF THE GLU-CB 1 -RS AND GABA-CB 1 -RS HIPPOCAMPUS

As to the inhibitory synapses, the amount of CB1 receptor immunopositive terminals in CB 1 -WT (78.50% ± 2.44), GABA-CB 1 -RS (77.92% ± 2.63) and Glu-CB 1 -KO (79.63% ± 2.35) was maintained in the CA1 stratum radiatum (Fig. 20a). However, this proportion drastically decreased in STOP-CB 1 , GABA-CB 1 -KO and Glu-CB 1 -RS and virtually disappeared in CB 1 -KO (Fig. 20a). There were not statistically significant differences in CB1 receptor immunoparticle density between inhibitory synaptic terminals of CB 1 -WT (4.57 ± 0.13), GABA-CB 1 -RS (4.33 ± 0.11) and Glu-CB 1 -KO (4.50 ± 0.15) (Fig. 20b). Furthermore, 44.06% ± 2.67 of the total immunoparticles in CB 1 -WT, 97.10% ± 0.46 in GABA-CB 1 -RS and 54.87% ± 3.94 in Glu-CB 1 -KO were located in inhibitory terminals. Just residual particles were observed in STOP-CB 1 , Glu-CB 1 -RS, GABA-CB 1 -KO and CB 1 -KO mice (Fig. 20c).

The proportion of CB1 receptor immunopositive inhibitory synaptic terminals in the inner third of the dentate molecular layer was found to be similar between CB 1 -WT (84.27% ± 2.38), GABA-CB 1 -RS (85.07% ± 1.76) and Glu-CB 1 -KO (88.02% ± 1.93) (Fig. 21a). These values almost disappeared in STOP-CB 1 , GABA-CB 1 -KO, Glu-CB 1 -RS and in CB 1 -KO (Fig. 21a). No statistical differences in labeling density were found between inhibitory terminals of CB 1 -WT (7.54 ± 0.16), GABA-CB 1 -RS (7.47 ± 0.14) and Glu-CB 1 -KO (7.69 ± 0.21) (Fig. 21b). Moreover, 45.38% ± 3.18 of the total immunoparticles in the inner third of the dentate molecular layer were located in GABAergic terminals of CB 1 -WT, 93.87% ± 0.93 in GABA-CB 1 -RS and 59.45% ± 4.00 in Glu-CB 1 -KO, whereas only residual particles were found in inhibitory terminals of STOP-CB 1 , Glu-CB 1 -RS, GABA-CB 1 -KO and CB 1 -KO mice (Fig. 21c). Hence, the subcellular distribution and expression levels of CB1 receptors at inhibitory synapses were kept in the GABA-CB 1 -RS and Glu-CB 1 -KO mutants relative to the wild type, remaining residual at the same synapses of STOP-CB 1 , GABA-CB 1 -KO and Glu-CB 1 -RS mice.

STATISTICS OF THE CB 1 RECEPTOR IN ASTROCYTIC PROCESSES OF THE GFAP-CB 1 -RS AND GFAPhrGFP-CB 1 -WT HIPPOCAMPUS

The analysis of the percentage of CB1 receptor immunopositive astrocytic processes (Fig. 22a) did not show statistical differences between CB 1 -WT (42.06% ± 3.56) and GFAP-CB 1 -RS (37.12% ± 3.79) in the CA1 stratum radiatum. In GFAPhrGFP-CB 1 -WT, the proportion of astrocytic elements immunolabeled for CB1 receptors increased to 59.91% ± 3.29 being the difference statistically significant when compared with CB 1 -WT. Conversely, only scarce CB1 receptor immunolabeling was found in astrocytes of GFAP-CB 1 -KO, STOP-CB 1 , CB 1 -KO and in GFAPhrGFP-CB 1 -KO mice.

Additionally, CB1 receptor immunoparticle density on astrocytic membranes (particles/µm) was analyzed (Fig. 22b). Our data showed statistically similar densities in CB 1 -WT (0.135 ± 0.019) and GFAP-CB 1 -RS (0.128 ± 0.020), whereas a great statistically significant increase was found in GFAPhrGFP-CB 1 -WT (0.384 ± 0.039). Nevertheless, only residual unspecific particles were found in GFAP-CB 1 -KO (0.005 ± 0.003), in STOP-CB 1 (0.005 ± 0.003), in CB 1 -KO (0.001 ± 0.001) and in GFAPhrGFP-CB 1 -KO (0.004 ± 0.002).

Finally, the proportion of CB1 immunogold particles in astrocytic processes versus total CB1 expression in plasmalemma was analyzed in CA1 stratum radiatum (Fig. 22c). 95.45% ± 1.82 of the total CB1 immunoparticles were located in astrocytic processes of GFAP-CB 1 -RS, while 23.08% ± 1.99 of the total immunogold particles were in the astrocytic elements of CB 1 -WT. This proportion reached 33.71% ± 2.75 in GFAPhrGFP-CB 1 -WT. As expected, only residual CB1 receptor immunoparticles were located in astrocytes of GFAP-CB 1 -KO, STOP-CB 1 , GFAPhrGFP-CB 1 -KO and CB 1 -KO mice. Furthermore, 75.13% ± 4.06 of the total CB1 immunoparticles in plasmalemma were in terminal membranes of GFAP-CB 1 -KO, while 65.52% ± 2.44 were in CB 1 -WT, and 56.32% ± 2.73 in GFAPhrGFP-CB 1 -WT (Fig. 22d). As expected, only residual CB1 receptor immunoparticles were located in astrocytic processes of GFAP-CB 1 -RS, STOP-CB 1 , GFAPhrGFP-CB 1 -KO and CB 1 -KO mice (Fig. 22d).

The analysis of the hippocampal dentate molecular layer showed comparable percentages of CB1 receptor immunopositive astrocytic processes between CB 1 -WT (44.67% ± 3.85) and GFAP-CB 1 -RS (39.84% ± 3.50), whereas the proportion significantly increased to 59.99% ± 3.37 in GFAPhrGFP-CB 1 -WT. The proportion decreased in GFAP-CB 1 -KO, STOP-CB 1 , GFAPhrGFP-CB 1 -KO and CB 1 -KO mice (Fig. 23a).

Besides, no statistical differences were found between the CB1 receptor density (particles/µm) on astrocytic processes of CB 1 -WT (0.112 ± 0.011) and GFAP-CB 1 -RS (0.138 ± 0.016) mice but there was when compared with GFAPhrGFP-CB 1 -WT (0.334 ± 0.033) (Fig. 23b). However, only residual particles were observed in GFAP-CB 1 -KO (0.006 ± 0.003), STOP-CB 1 (0.006 ± 0.003), GFAPhrGFP-CB1-KO (0.002 ± 0.002) and CB1-KO mice (0.004 ± 0.002) (Fig. 23b). Furthermore, we examined the proportion of CB1 receptor labeling in astrocytic processes versus total CB1 receptor expression (Fig. 23c). The values were 22.40% ± 2.28 in CB 1 -WT, 95.61% ± 1.56 in GFAP-CB 1 -RS and 32.57% ± 2.09 in GFAPhrGFP-CB 1 -WT. Only non-specific CB1 receptor immunoparticles were found in astrocytic processes of GFAP-CB 1 -KO, STOP-CB 1 , and GFAPhrGFP-CB 1 -KO and CB1-KO mice (Fig. 23c).

Finally, CB1 gold labeling in terminal membranes versus total CB1 expression was also studied. 76.17% ± 4.70 of the total CB1 immunoparticles were located in terminals of GFAP-CB 1 -KO, 64.27% ± 2.88 in CB 1 -WT and 57.17% ± 2.19 in GFAPhrGFP-CB 1 -WT (Fig. 23d). Just residual particles were in astrocytic processes of GFAP-CB 1 -RS, STOP-CB 1 , GFAPhrGFP-CB 1 -KO and CB 1 -KO mice (Fig. 23d).

STATISTICAL ANALYSES OF THE DISTANCE FROM ASTROCYTIC CB 1 RECEPTORS TO THE NEAREST SYNAPSE

Taking advantage of the better detection of astrocytes achieved with the hr-GFP protein in the electron microscope, the distance between the astrocytic CB1 receptor immunoparticles and the midpoint of the nearest synapse surrounded by the immunopositive astrocytic element was measured in GFAPhrGFP-CB 1 -WT.

In CA1 (Fig 24a), 50.76% ± 4.10 of the total synapses surrounded by astrocytic processes were at a distance between 400-800 nm away from the nearest astrocytic immunoparticle. Of those, 40.89% ± 2.57 were excitatory synapses whereas 9.86% ± 1.97 were inhibitory synapses. 20.60% ± 6.1 of the synapses were found to be located in a range of 0 nm to 400 nm from the astrocytic CB1 receptor to the nearest synapse. Of these, 17.52% ± 5.63 were excitatory synapses and only 3.08% ± 0.49 were inhibitory synapses. 18.46% ± 2.53 of the synapses were found to be located in a range of 800 nm to 1200 nm of which 13,96% ± 1,60 were excitatory and 4.50% ± 0.95 were inhibitory synapses. Only 10.18% ± 7.81 synapses were found at more than 1200 nm from the astrocytic CB1 receptor metal particle, being 9.62% ± 8.01 of them excitatory and only 0.56% ± 0.56 were inhibitory synapses.

In the dentate molecular layer (Fig 24b), 57.25% ± 3.19 of the synapses surrounded by astrocytic processes were at 400-800 nm from the astrocytic CB1 receptor particle, 46.67% ± 2.17 of them were excitatory synapses and the other 10.59% ± 2.18 were inhibitory synapses. 19.83% ± 0.58 of the synapses were in a range of 0 nm to 400 nm from the astrocytic CB1 receptor metal particle of which 16.67% ± 1.31 were excitatory and 3.16% ± 0.82 inhibitory synapses. Finally, 16.42% ± 4.37 of the total synapses were found between 800 nm and 1200 nm; 13.22% ± 3.19 being excitatory and 3.21% ± 2.25 inhibitory synapses. Only 6.48% ± 2.31 synapses were found at more than 1200 nm from the astrocytic CB1 receptor. 5.16% ± 2.72 of them were excitatory and 1.32% ± 0.66 inhibitory synapses. An immunoperoxidase method for light microscopy was used for CB1 receptor immunostaining detection in the GABA-CB1-RS mouse brain. Representative rostro-caudal coronal brain sections (left to right and top to bottom). Strong CB1 receptor immunostaining is found in AON, Pir, PFCx, Cx, Str, GP, EP, Amyg, SN and hippocampus.

AON, Anterior olfactory nucleus; Pir, Piriform cortex; PFCx, Prefrontal cortex; Cx, Cortex; Str, Striatum; GP, Globus pallidus; Amyg, Amygdala; Hip, Hippocampus; EP, Entopeduncular nucleus; SN, Substantia nigra; a to d: Bregma 2.80 to 1.70 mm; e to h: Bregma 1.54 to 1.18 mm; i to l: Bregma 0.74 to -0.94 mm; m to p: Bregma -1.22 to -1.58 mm; q to t: Bregma -2.46 to -2.92. An immunoperoxidase method for light microscopy was used. In the CB1-WT hippocampus a stronger immunolabeling is observed in Py and at the limit Rad and LMol than throughout the Or and Rad. In the dentate gyrus, the densest immunolabeling is found in the inner third of MoDG whereas less staining, although intense, was found in the outer parts of this layer. Fibrous processes surrounding the granule cell bodies are observed in GrDG (a). Detailed magnification of the Glu-CB1-RS hippocampus: noticeable CB1 receptor neuropil labeling is detected in Or and Rad. A dense band of immunostaining is observed in the inner third of MoDG (b). Detailed magnification of the GABA-CB1-RS hippocampus: a denser labeling area is seen in Py and also at the limit between Rad and LMol. MoDG is heavily CB1 receptor immunoreactive exhibiting a conspicuous band at its inner third (c). Detailed magnification of the CB1-KO hippocampus: immunostaining completely disappears in all the CB1-KO layers demonstrating that the CB1 antibody is highly specific (d). In CB1-WT, immunoparticles are localized on excitatory (asymmetric synapses) and inhibitory (symmetric synapses) terminal membranes making synaptic contacts with dendritic spines or dendrites, respectively (a).

No CB1 receptor immunolabeling is detected in CB1-KO, indicating that the CB1 receptor antibody used is specific (b). In STOP-CB1, no CB1 receptor labeling is observed in excitatory or inhibitory terminals (c). In Glu-CB1-RS, CB1 receptor immunoparticles are at asymmetric but not symmetric synapses (d). On the contrary, CB1 receptor immunolabeling is distributed on inhibitory presynaptic terminal membranes and not on membranes of excitatory boutons in Glu-CB1-KO (e). Similarly, many CB1 receptor immunoparticles are confined to symmetric synapses in GABA-CB1-RS (f). However, synaptic CB1 receptor immunoparticles are observed on excitatory presynaptic terminal membranes and not in inhibitory terminals of GABA-CB1-KO (g). Note the presence of CB1 receptor labeling in mitochondria (a, d-g).

Black arrowheads: excitatory synapses; white arrowheads: inhibitory synapses; black arrows: CB1 receptor immunoparticles. ter, terminal; den, dendrite; sp, dendritic spine. Scale bars: 0.5 µm. In CB1-WT, scarce CB1 receptor immunoparticles are localized on presynaptic terminal membranes making excitatory synapses (asymmetric) with dendritic spines while numerous metal particles are at inhibitory terminals (a). The CB1 receptor labeling pattern is neither observed in CB1-KO (b) nor in STOP-CB1 mice (c). In Glu-CB1-RS, CB1 receptor gold particles are only seen on presynaptic mossy cell axon boutons and not on presynaptic inhibitory terminal membranes (d). However, in Glu-CB1-KO, excitatory mossy cell terminals are immunonegative but inhibitory boutons contain abundant CB1 receptor labeling (e). In GABA-CB1-RS, numerous CB1 receptor immunoparticles are only on presynaptic inhibitory axon terminals but not at excitatory synapses (f). In contrast, immunolabeling is not observed in GABAergic terminals but it is present in excitatory boutons of GABA-CB1-KO (g). Observe CB1 receptor immunoparticles on mitochondria (a, d-g).

Black arrowheads: excitatory synapses; white arrowheads: inhibitory synapses; black arrows: CB1 receptor immunoparticles. ter, terminal; den, dendrite; sp, dendritic spine. Scale bars: 0.5 µm. In CB1-WT and in GFAPhrGFP-CB1-WT mice, CB1 receptor immunoparticles are localized in membranes of astrocytic processes. CB1 receptor immunoparticles are also observed on terminal membranes (a, f, g). CB1 receptor immunolabeling is neither detected in CB1-KO nor in GFAPhrGFP-CB1-KO, indicating that the CB1 receptor antibody used is highly specific (b, h). CB1 receptor immunoparticles appear in astrocytic processes but not in terminals of GFAP-CB1-RS (c). In GFAP-CB1-KO, CB1 receptors disappear from membranes of astrocytic processes whereas they remain in synaptic terminals (d). There is not CB1 receptor immunoparticles in STOP-CB1 mice (e).

Black arrowheads: excitatory synapses; white arrowheads: inhibitory synapses; black arrows: CB1 receptor immunoparticles; white arrows: astrocytic CB1 receptor immunoparticles. as: astrocytic processes; ter: terminal; den: dendrite; sp: dendritic spine. Scale bars: 0.5 µm. In CB1-WT and GFAPhrGFP-CB1-WT mice, CB1 receptor is localized on presynaptic terminal membranes and on membranes of astrocytic processes (a, f, g). The CB1 receptor labeling pattern disappears in CB1-KO and in GFAPhrGFP-CB1-KO showing the specificity of the antibody used (b, h). In GFAP-CB1-RS, CB1 receptor immunogold particles are on astrocytic processes and not on neuronal terminal membranes (c). However, in GFAP-CB1-KO, astrocytic processes are CB1 receptor immunonegative but inhibitory and excitatory terminals contain the receptor (d). There is no CB1 receptor immunolabeling in STOP-CB1 (e).

Black arrowheads: excitatory synapses; white arrowheads: inhibitory synapses; black arrows: CB1 receptor immunoparticles; white arrows: astrocytic CB1 receptor immunoparticles. as: astrocytic processes; ter: terminal; den: dendrite; sp: dendritic spine. Scale bars: 0.5 µm. Analysis of the distance from the astrocytic CB1 receptors and the midpoint of the nearest synapse surrounded by the astrocytic processes in the GFAPhrGFP-CB1-WT mice. In CA1, of the total analyzed synapses (n= 159) 50.76% ± 4.10 are localized at a distance between 400-800 nm from the astrocytic CB1 receptor immunoparticle. 40.89% ± 2.57 of them are excitatory and 9.86% ± 1.97 are inhibitory synapses. 20.60% ± 6.12 of the synapses are in a range of 0-400 nm from the astrocytic CB1 receptor immunoparticle. Of those, 17.52% ± 5.63 are excitatory synapses and 3.08% ± 0.49 are inhibitory synapses. 18.46% ± 2.53 of the total synapses are between 800 nm and 1200 nm; 13,96% ± 1,60 are excitatory and 4.50% ± 0.95 are inhibitory synapses. Only 10.18% ± 7.81 of the synapses are found at more than 1200 nm from the astrocytic CB1 receptor. Of those, 9.62% ± 8.01 are excitatory and only 0.56% ± 0.56 are inhibitory synapses (a). In the dentate molecular layer, 57.25% ± 3.19 of the total analyzed synapses (n=166) are placed at a distance between 400-800 nm from the astrocytic CB1 receptor particle; 46.67% ± 2.17 of them are excitatory and 10.59% ± 2.18 are inhibitory synapses. 19.83% ± 0.58 of the synapses are in a range of 0-400 nm; 16.67% ± 1.31 of them are excitatory and 3.16% ± 0.82 are inhibitory synapses. 16.42% ± 4.37 of the total synapses are located between 800-1200 nm. Of these synapses, 13.22% ± 3.19 are excitatory and 3.21% ± 2.52 are inhibitory. Only 6.48% ± 2.31 of the synapses are observed at more than 1200 nm from the astrocytic CB1 receptor metal particle; 5.16% ± 2.72 being excitatory and 1.32% ± 0.66 inhibitory synapses (b). Data are expressed as mean ± Standard Error of the Mean of three different animals.

THE IMPORTANCE OF CB 1 RECEPTOR MUTANTS IN THE STUDY OF CB 1 RECEPTOR

The CB1 receptor is expressed in different brain cell types populations [START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF][START_REF] Tsou | Cannabinoid CB1 receptors are localized primarily on cholecystokinin-containing GABAergic interneurons in the rat hippocampal formation[END_REF]Nyíri et al., 2005b;[START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF][START_REF] Häring | Identification of the cannabinoid receptor type 1 in serotonergic cells of raphe nuclei in mice[END_REF][START_REF] Scavone | Characterization of cannabinoid-1 receptors in the locus coeruleus: Relationship with mu-opioid receptors[END_REF][START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF][START_REF] Metna-Laurent | Rising stars: Modulation of brain functions by astroglial type-1 cannabinoid receptors[END_REF]. Moreover, a wide variety of intracellular effects such as modulation of kinases, ion channels and transcription factors are triggered by the activation of CB1 receptors [START_REF] Bosier | Functionally selective cannabinoid receptor signaling: therapeutic implications and opportunities[END_REF][START_REF] Pertwee | Endocannabinoids and Their Pharmacological Actions[END_REF]. One of the most known and important result of these intracellular events is the retrograde inhibition of transmitter release [START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF]. Consequently, the activation of CB1 receptors modulates the release of several neurotransmitters, such as: glutamate, GABA, glycine, acetylcholine, norepinephrine, dopamine, serotonin and cholecystokinin [START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF].

Anatomically, the CB1 receptor is also widely distributed in the brain with a preferential localization in motor, limbic, reward and cortical regions [START_REF] Matsuda | Localization of cannabinoid receptor mRNA in rat brain[END_REF][START_REF] Tsou | Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system[END_REF]. Its high concentration in certain brain areas is an advantage for studying the functional role of the receptor in the neural circuits where it is abundantly localized [START_REF] Katona | Presynaptically Located CB1 Cannabinoid Receptors Regulate GABA Release from Axon Terminals of Specific Hippocampal Interneurons[END_REF][START_REF] Kawamura | The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum[END_REF][START_REF] Ludányi | Downregulation of the CB1 cannabinoid receptor and related molecular elements of the endocannabinoid system in epileptic human hippocampus[END_REF][START_REF] Marsicano | Anatomical Distribution of Receptors, Ligands and Enzymes in the Brain and in the Spinal Cord: Circuitries and Neurochemistry[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] De-May | Cell type-specific regulation of inhibition via cannabinoid type 1 receptors in rat neocortex[END_REF][START_REF] Steindel | Neuron-type specific cannabinoid-mediated G protein signaling in mouse hippocampus[END_REF][START_REF] Hu | Distribution of the Endocannabinoid System in the Central Nervous System[END_REF].

However, CB1 receptor density is not uniform through the regions expressing the receptor which makes extremely difficult to identify low CB1 receptor expression in cell types and/or in subcellular compartments of wild-type brains [START_REF] Busquets-Garcia | Dissecting the cannabinergic control of behavior: The where matters[END_REF].

The hippocampus is one of the brain structures with the highest CB1 receptor immunoreactivity [START_REF] Herkenham | Cannabinoid receptor localization in brain[END_REF][START_REF] Mailleux | Distribution of neuronal cannabinoid receptor in the adult rat brain: A comparative receptor binding radioautography and in situ hybridization histochemistry[END_REF][START_REF] Matsuda | Localization of cannabinoid receptor mRNA in rat brain[END_REF][START_REF] Tsou | Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system[END_REF][START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF][START_REF] Egertová | Localisation of cannabinoid receptors in the rat brain using antibodies to the intracellular C-terminal tail of CB[END_REF][START_REF] Katona | Molecular composition of the endocannabinoid system at glutamatergic synapses[END_REF][START_REF] Kawamura | The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum[END_REF][START_REF] Ludányi | Downregulation of the CB1 cannabinoid receptor and related molecular elements of the endocannabinoid system in epileptic human hippocampus[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] Steindel | Neuron-type specific cannabinoid-mediated G protein signaling in mouse hippocampus[END_REF][START_REF] Hu | Distribution of the Endocannabinoid System in the Central Nervous System[END_REF] and where CB1 receptors have shown to play key functional roles [START_REF] Campbell | delta 9-Tetrahydrocannabinol differentially affects sensory-evoked potentials in the rat dentate gyrus[END_REF][START_REF] Heyser | Effects of delta-9tetrahydrocannabinol on delayed match to sample performance in rats: alterations in short-term memory associated with changes in task specific firing of hippocampal cells[END_REF][START_REF] Stella | A second endogenous cannabinoid that modulates long-term potentiation[END_REF][START_REF] Hampson | Cannabinoids, hippocampal function and memory[END_REF]Katona et al., 2000;[START_REF] Carlson | Endocannabinoids facilitate the induction of LTP in the hippocampus[END_REF]Chevaleyre andCastillo, 2003, 2004;[START_REF] Vanderbyl | Transgene expression after stable transfer of a mammalian artificial chromosome into human hematopoietic cells[END_REF][START_REF] Robbe | Cannabinoids reveal importance of spike timing coordination in hippocampal function[END_REF][START_REF] Akirav | The role of cannabinoids in modulating emotional and nonemotional memory processes in the hippocampus[END_REF][START_REF] Puighermanal | Cellular and intracellular mechanisms involved in the cognitive impairment of cannabinoids[END_REF][START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF][START_REF] Basavarajappa | CB1 receptor-mediated signaling underlies the hippocampal synaptic, learning, and memory deficits following treatment with JWH-081, a new component of spice/K2 preparations[END_REF].

The development and use of cell type specific CB1-receptor-knockout mice, lacking the CB1 receptor in specific brain cell populations served to identify low CB1 receptor expression in cellular, subcellular or intracellular compartments as well as to understand its physiological functions in those compartments [START_REF] Marsicano | CB1 cannabinoid receptors and on-demand defense against excitotoxicity[END_REF][START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF][START_REF] Monory | Genetic dissection of behavioural and autonomic effects of Delta(9)-tetrahydrocannabinol in mice[END_REF][START_REF] Puighermanal | Cannabinoid modulation of hippocampal long-term memory is mediated by mTOR signaling[END_REF][START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF][START_REF] Bénard | Mitochondrial CB₁ receptors regulate neuronal energy metabolism[END_REF][START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF][START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Steindel | Neuron-type specific cannabinoid-mediated G protein signaling in mouse hippocampus[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF][START_REF] Busquets-Garcia | Dissecting the cannabinergic control of behavior: The where matters[END_REF][START_REF] Martín-García | Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors[END_REF][START_REF] Oliveira Da Cruz | Astroglial type-1 cannabinoid receptor (CB1): A new player in the tripartite synapse[END_REF]. For instance, in the GABA-CB 1 -KO brain, a drastic decrease of the CB1 receptor immunoreactivity was observed throughout the hippocampus but remaining a noticeable immunoreactive band in the inner third of the dentate molecular layer [START_REF] Martín-García | Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors[END_REF]. On the other hand, just a very faint decrease of the CB1 receptor immunostaining was noticed in the Glu-CB 1 -KO hippocampus [START_REF] Martín-García | Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors[END_REF]. Yet, there was not a full disappearance of the CB1 receptor immunoreactivity in either condition as it occurred in CB 1 -KO. The reason is because the CB1 receptor localization is mostly, but not exclusively, restricted to neuronal membrane compartments, as CB1 receptors are also distributed in astrocytic processes [START_REF] Bosier | Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes[END_REF] and mitochondrial membranes of the hippocampus [START_REF] Bénard | Mitochondrial CB₁ receptors regulate neuronal energy metabolism[END_REF]Hebert-Chatelain et al., 2014a;b). The light microscopic observations previously described by our group [START_REF] Martín-García | Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors[END_REF] correlate well with the present findings in the electron microscope of the subcellular CB1 receptor localization in hippocampal glutamatergic and GABAergic axon terminals of GABA-CB 1 -KO and Glu-CB 1 -KO, respectively. This work has also shown that CB1 receptor immunolabeling was abolished in excitatory terminals of Glu-CB 1 -KO while the proportion and density of CB1 receptors in GABA-CB 1 -KO were maintained in the glutamatergic excitatory terminals of CA1 stratum radiatum [START_REF] Takahashi | The CB1 cannabinoid receptor mediates glutamatergic synaptic suppression in the hippocampus[END_REF][START_REF] Takács | Extrinsic and local glutamatergic inputs of the rat hippocampal CA1 area differentially innervate pyramidal cells and interneurons[END_REF][START_REF] Witter | The Mouse Nervous System[END_REF]. On the contrary, CB1 receptor metal particles were not observed in the hippocampal inhibitory synaptic terminals of GABA-CB 1 -KO though CB1 receptor expression and distribution in CA1 and dentate inhibitory boutons were undistinguishable from CB 1 -WT.

Moreover, the using of GFAP-CB 1 -KO mice have already demonstrated that the activation of CB1 receptors in CA1 astrocytes mediates the impairment of working memory elicited by acute cannabinoids through the modulation of hippocampal long term depression [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF]. The results obtained on the proportion of CB1 receptor immunopositive CA1 hippocampal astrocytes in in this thesis is similar to the value reported in our previous work [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF]. Furthermore, the expression levels of CB1 receptor immunoparticles in terminals of GFAP-CB 1 -KO mice was comparable to CB 1 -WT indicating that GFAP-CB 1 -KO are good tools for studies based on the loss of function of astrocytic CB1 [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF].

However, conditional mutant mice have limitations as a biological compensation derived from the CB1 receptor deletion could occur. In addition, they do not allow establishing a link between the anatomical CB1 receptor localization in some nerve cells and a certain physiological task [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF].

CB 1 RECEPTOR EXPRESSION IN SPECIFIC BRAIN CELL TYPES OF RESCUE MUTANTS

The CB1 receptor expression protein restoration in phenotype-specific brain cell populations can provide great insights into the sufficiency of the CB1 receptor for specific brain functions and behaviors. Thus, it is crucial to ensure that the rescue of CB1 receptors in particular cell types of CB1 receptor-null mutants correlates with the endogenous CB1 receptor expression and localization in the wild-type mouse brain. This thesis work has analyzed in detail the anatomical distribution pattern of the CB1 receptor in the mutant mice.

CB 1 RECEPTOR EXPRESSION IN SPECIFIC NEURONAL CELL TYPES OF THE RESCUE MUTANT MICE

The cellular CB1 receptor staining described here for the Glu-CB 1 -RS and GABA-CB 1 -RS mutants by a preembedding immunoperoxidase method for light microscopy reflects the distribution of CB1 receptors throughout the brain [START_REF] Herkenham | Cannabinoid receptor localization in brain[END_REF][START_REF] Tsou | Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system[END_REF][START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF][START_REF] Egertová | Localisation of cannabinoid receptors in the rat brain using antibodies to the intracellular C-terminal tail of CB[END_REF][START_REF] Mackie | Distribution of cannabinoid receptors in the central and peripheral nervous system[END_REF][START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF][START_REF] Monory | Genetic dissection of behavioural and autonomic effects of Delta(9)-tetrahydrocannabinol in mice[END_REF][START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF][START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Martín-García | Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors[END_REF]. CB1 receptor immunoreactivity was much fainter in Glu-CB 1 -RS than in GABA-CB 1 -RS, as expected for the much lower CB1 receptor expression in glutamatergic [START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF][START_REF] Katona | Molecular composition of the endocannabinoid system at glutamatergic synapses[END_REF][START_REF] Kawamura | The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum[END_REF][START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF][START_REF] Monory | Genetic dissection of behavioural and autonomic effects of Delta(9)-tetrahydrocannabinol in mice[END_REF][START_REF] Martín-García | Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors[END_REF][START_REF] Lu | An Introduction to the Endogenous Cannabinoid System[END_REF] than in GABAergic neurons [START_REF] Katona | Presynaptically Located CB1 Cannabinoid Receptors Regulate GABA Release from Axon Terminals of Specific Hippocampal Interneurons[END_REF][START_REF] Hájos | Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations[END_REF]Nyíri et al., 2005a;[START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF][START_REF] Takács | Quantitative ultrastructural analysis of basket and axo-axonic cell terminals in the mouse hippocampus[END_REF][START_REF] Lu | An Introduction to the Endogenous Cannabinoid System[END_REF]. Subcellularly, CB1 receptors were localized in glutamatergic terminals of the CA1 stratum radiatum and the inner third of the dentate molecular layer of the Glu-CB 1 -RS, the CB 1 -WT and the GABA-CB 1 -KO hippocampus. Furthermore, no significant differences in the proportion of immunopositive excitatory synaptic terminals and density of CB1 receptors were detected between Glu-CB 1 -RS, CB 1 -WT or GABA-CB 1 -KO [START_REF] Gutiérrez-Rodríguez | Anatomical characterization of the cannabinoid cb 1 receptor in cell type-specific mutant mouse rescue models[END_REF]. This pattern corresponds well with the distribution and expression of CB1 receptors in the intrahippocampal excitatory pathways [START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF][START_REF] Katona | Molecular composition of the endocannabinoid system at glutamatergic synapses[END_REF][START_REF] Kawamura | The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum[END_REF][START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF][START_REF] Uchigashima | Molecular and morphological configuration for 2arachidonoylglycerol-mediated retrograde signaling at mossy cell-granule cell synapses in the dentate gyrus[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF]. Very importantly, the inhibitory presynaptic profiles in the Glu-CB 1 -RS hippocampus were virtually devoid of CB1 immunoparticles. Also, the labeling was almost nil in the CB 1 -KO, indicating that the CB1 receptor antibody used in this study was highly specific. Previous electron microscopy studies described that more than 80% of excitatory synapses are CB1 receptor immunopositive in the inner third of the dentate molecular layer, compared to only 30-50% in other hippocampal layers [START_REF] Katona | Molecular composition of the endocannabinoid system at glutamatergic synapses[END_REF][START_REF] Uchigashima | Molecular and morphological configuration for 2arachidonoylglycerol-mediated retrograde signaling at mossy cell-granule cell synapses in the dentate gyrus[END_REF]. Although these values were somehow higher than those obtained in CB 1 -WT and Glu-CB 1 -RS in our study, it is plausible to assume that this is probably due to the different CB1 receptor antibodies used and/or the immunocytochemical protocols applied [START_REF] Katona | Molecular composition of the endocannabinoid system at glutamatergic synapses[END_REF][START_REF] Uchigashima | Molecular and morphological configuration for 2arachidonoylglycerol-mediated retrograde signaling at mossy cell-granule cell synapses in the dentate gyrus[END_REF]. Besides, in these studies only anatomical features of the synapses were used to distinguish between excitatory and inhibitory synapses. In our study, the CB1 receptor localization was determined not only based on anatomical features but also using genetic tools (Glu-CB 1 -RS mice and GABA-CB 1 -RS mice) probably making a difference.

CB1 receptors in GABA-CB 1 -RS were densely localized in axon terminals of presumably cholecystokinin-containing interneurons as well as of calbindin D28k positive interneurons innervating the proximal portions of the pyramidal cell dendrites in the CA1 stratum radiatum and the dentate molecular layer [START_REF] Katona | Presynaptically Located CB1 Cannabinoid Receptors Regulate GABA Release from Axon Terminals of Specific Hippocampal Interneurons[END_REF][START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF][START_REF] Hájos | Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations[END_REF]Nyíri et al., 2005;[START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF][START_REF] Katona | Multiple functions of endocannabinoid signaling in the brain[END_REF][START_REF] Lu | An Introduction to the Endogenous Cannabinoid System[END_REF][START_REF] Takács | Quantitative ultrastructural analysis of basket and axo-axonic cell terminals in the mouse hippocampus[END_REF]. Significantly, the excitatory presynaptic profiles in the GABA-CB 1 -RS hippocampus were virtually lacking CB1 immunoparticles. Furthermore, the proportion of the CB1 receptor immunopositive inhibitory synaptic terminals was similar in GABA-CB 1 -RS, Glu-CB 1 -KO and CB 1 -WT [START_REF] Gutiérrez-Rodríguez | Anatomical characterization of the cannabinoid cb 1 receptor in cell type-specific mutant mouse rescue models[END_REF], and the localization coincides with the CB1 receptor distribution pattern described in rodent interneurons [START_REF] Katona | Presynaptically Located CB1 Cannabinoid Receptors Regulate GABA Release from Axon Terminals of Specific Hippocampal Interneurons[END_REF][START_REF] Hájos | Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations[END_REF]Nyíri et al., 2005a) and human hippocampus [START_REF] Katona | GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus[END_REF].

Another parameter studied here was the density of immunogold particles in the CB1 receptor positive excitatory and inhibitory terminals. The molecular and microscopic analysis of the hippocampal CB1 receptors in Glu-CB 1 -RS and GABA-CB 1 -KO, on the one hand, and GABA-CB 1 -RS and Glu-CB 1 -KO, on the other, confirmed that the CB1 receptor is present at much higher density in hippocampal GABAergic than glutamatergic cells [START_REF] Katona | Molecular composition of the endocannabinoid system at glutamatergic synapses[END_REF][START_REF] Kawamura | The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum[END_REF][START_REF] Gutiérrez-Rodríguez | Anatomical characterization of the cannabinoid cb 1 receptor in cell type-specific mutant mouse rescue models[END_REF]. The study by [START_REF] Kawamura | The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum[END_REF] revealed a CB1 receptor density of 5.75 particles/µm in inhibitory terminals and only 0.29 particles/µm in excitatory terminals of the CA1 stratum radiatum. In the same study, inhibitory terminals in the inner third of the dentate molecular layer had 7.14 particles/µm in contrast to 0.62 particles/µm found in excitatory terminals [START_REF] Kawamura | The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum[END_REF]. The CB1 receptor densities observed in the CB1 receptor positive excitatory and inhibitory terminals of the mutant mice are in line with these previous hippocampal data obtained in CB 1 -WT mice, though the differences observed could be attributed to the diverse antibodies and methods used, as mentioned before. To mention that higher receptor abundancy in inhibitory synapses does not directly correlate with downstream signaling activation, as the glutamatergic CB1 receptor is more efficiently coupled to G protein signaling than the GABAergic CB1 receptor [START_REF] Steindel | Neuron-type specific cannabinoid-mediated G protein signaling in mouse hippocampus[END_REF].

CB 1 RECEPTOR EXPRESSION IN ASTROCYTES OF RESCUE MUTANT MICE

In this study, the subcellular CB1 receptor labeling was also described in rescue mice where the CB1 receptor expression has been specifically restored in astrocytes of GFAP-CB 1 -RS mice. The low levels of expression of CB1 receptors in astrocytes can only be accurately detected using high resolution immunocytochemical techniques for electron microscopy. Hence, a preembedding immunogold and immunoperoxidase methods applied to hippocampal sections had previously shown in our laboratory to be an excellent methodological strategy for the localization of CB1 in astrocytes [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF][START_REF] Bosier | Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes[END_REF]. Our present results have shown the proportion of the CB1 receptor positive astrocytic processes and the density of the receptor in the astrocytic elements (particles /µm) were not statistically significant different in CB 1 -WT and GFAP-CB 1 -RS. Moreover, the percentage of the CB1 receptor immunopositive CA1 astrocytes obtained here is similar to the previous values reported by our group [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF]. The analysis of the CB1 particles localized in astrocytic processes or terminals versus total CB1 receptor particles in plasmalemmal structures of the GFAP-CB 1 -RS hippocampus showed that almost all the labeling was in astrocytic processes and just only residual particles were in terminals confirming the high specify of the astrocytic CB1 receptor genetic rescue approach carried in the mutant mice. Consequently, these rescue mice emerge as excellent models to study the contribution to brain functions of the CB1 receptors in astrocytes. Specifically, a more comprehensive characterization of the functional consequences of the eCB signaling through the astrocytic CB1 receptor could be achieve in the tripartite synapses using these rescue mice.

Importantly, the CB1 receptor expression virtually disappeared in the STOP-CB 1 mice corroborating previous findings published with these mutants [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] De Salas-Quiroga | Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons[END_REF].

To sum up, the genetic rescue mouse models of the Glu-CB 1 -RS expressed CB1 receptors only in dorsal telencephalic glutamatergic neurons [START_REF] Ruehle | Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions[END_REF][START_REF] Soria-Gómez | The endocannabinoid system controls food intake via olfactory processes[END_REF][START_REF] De Salas-Quiroga | Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons[END_REF][START_REF] Gutiérrez-Rodríguez | Anatomical characterization of the cannabinoid cb 1 receptor in cell type-specific mutant mouse rescue models[END_REF], the GABA-CB 1 -RS only in GABAergic neurons (de [START_REF] De Salas-Quiroga | Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons[END_REF][START_REF] Gutiérrez-Rodríguez | Anatomical characterization of the cannabinoid cb 1 receptor in cell type-specific mutant mouse rescue models[END_REF] and the GFAP-CB 1 -RS exclusively in astrocytic processes. Furthermore, these results may suggest that the regulation of the CB1 receptor expression in glutamatergic and GABAergic cells may be independent one from another. Moreover, astrocytic CB1 receptor expression may be also independent from the CB1 receptor in neurons. However, the absence of a crosstalk in the expression of CB1 receptor between these type of cells cannot be proved with the present experimental design, since the analysis of only one point in the mouse's life time is insufficient to assess the regulation pathway. Therefore, further analysis would be required in order to corroborate this assumption. The demonstration in this thesis work that the hippocampus of the rescue Glu-CB 1 -RS, GABA-CB 1 -RS and GFAP-CB 1 -RS mutant mice maintain the normal anatomical distribution and expression levels of CB1 receptors in the cell types with restored receptors, proves the great potential of these mutants for the study of the CB1 receptor function in specific brain cell populations. In fact, the rescue strategies have the advantage of the reestablishment and visualization of existing CB1 receptors levels in glutamatergic neurons, GABAergic neurons and astrocytes more accurately, without the interference of additional cells expressing CB1 receptors. This is especially important for the anatomical and functional investigation of the endocannabinoid system in cell types or brain areas with sparse CB1 receptors. Finally, the comprehension of the exact expression and distribution of the CB1 receptor will not only help to better understand the receptor architecture of the brain, but will also improve the conceptual framework for a more specific pharmacological intervention against complex brain diseases in regions where balanced CB1 receptors are crucial for brain function.

CB 1 RECEPTOR EXPRESSION IN GFAPhrGFP-CB 1 -WT MUTANT MICE

Additionally, mutant mice target to express hrGFP into astroglial cells were used in this thesis. In this animal model, the percentage and density (particles/µm) of astrocytic elements expressing CB1 receptor were higher and more statistically significant when compared with the GFAP-CB 1 -RS mutants using GFAP as a marker for astrocytes. These results suggest that the expression of CB1 receptors on astrocytes could actually be higher than in previous observations reported using GFAP as a marker for the identification of the astrocytes [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF][START_REF] Bosier | Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes[END_REF]. A plausible explanation could be based on the different molecular nature of the GFAP and hr-GFP proteins. GFAP is a cytoskeletal protein which is assembled in intermediate filament packet [START_REF] Inagaki | Glial Fibrillary Acidic Protein: Dynamic Property and Regulation by Phosphorylation[END_REF][START_REF] Eng | Glial Fibrillary Acidic Protein: GFAP-Thirty-One Years (1969-2000)[END_REF][START_REF] Hol | Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system[END_REF], then GFAP immunostaining only shows the main radial processes of the astrocyte. However, hr-GFP is a diffusible protein which fills all the cytoplasmic regions, including the fine processes of astrocytes that are normally lacking GFAP [START_REF] Nolte | GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue[END_REF], hence, a better detection of astrocytic processes can be accomplished.

TRIPARTITE SYNAPSE: ANATOMICAL INTERPLAY BETWEEN CB 1 RECEPTORS IN ASTROCYTES AND THE NEARBY SYNAPSES

Taking advantage of the better detection of CB1 receptors in astrocytes of the GFAPhrGFP-CB 1 -WT mouse, we also examined the distance between the astrocytic CB1 receptor and the nearest synapse in order to put the CB1 receptors in astrocytes in the anatomical context of the functional tripartite synapse (Navarrete andAraque, 2008, 2010;[START_REF] Navarrete | Astrocyte calcium signal and gliotransmission in human brain tissue[END_REF][START_REF] Navarrete | Astrocytes in endocannabinoid signaling[END_REF][START_REF] Gómez-Gonzalo | Endocannabinoids Induce Lateral Long-Term Potentiation of Transmitter Release by Stimulation of Gliotransmission[END_REF]. It was demonstrated that the endocannabinoids coming from a postsynaptic neuron activate astrocytic CB1 receptors leading to an increase in the intracellular calcium that triggers a release of astrocytic glutamate, which stimulates a neurotransmitter release in heteroneuronal synapses (Navarrete andAraque, 2008, 2010;[START_REF] Navarrete | Astrocyte calcium signal and gliotransmission in human brain tissue[END_REF][START_REF] Navarrete | Astrocytes in endocannabinoid signaling[END_REF][START_REF] Gómez-Gonzalo | Endocannabinoids Induce Lateral Long-Term Potentiation of Transmitter Release by Stimulation of Gliotransmission[END_REF].

Glial cells constitute the most abundant cellular population in the central nervous system. Amongst glial cell types, astrocytes are excellent players in brain information processing [START_REF] Volterra | Astrocytes, from brain glue to communication elements: the revolution continues[END_REF], due to the bidirectional communication established with neurons [START_REF] Araque | Dynamic Signaling Between Astrocytes and Neurons[END_REF][START_REF] Bezzi | Astrocytes: Powering Memory[END_REF] through intricate morphological and biochemical interactions. The morphology of astrocytes is particularly complex, suggesting that their structures have important functional roles in brain functions. In rodents, astrocytes are distributed covering non-overlapping domains in the cerebral cortex, where they contact tens of thousands of synapses [START_REF] Halassa | Synaptic islands defined by the territory of a single astrocyte[END_REF]. Astrocytes are in close apposition to the synaptic structures, forming tripartite synapses, and play important roles in maintaining and regulating synaptic physiology [START_REF] Perez-Alvarez | Structural and functional plasticity of astrocyte processes and dendritic spine interactions[END_REF]. Functionally, endocannabinoids, through CB1, promote astroglial differentiation and mediate neuron-astrocyte communication regulating synaptic transmission [START_REF] Aguado | The endocannabinoid system promotes astroglial differentiation by acting on neural progenitor cells[END_REF]Navarrete andAraque, 2008, 2010). Furthermore, our laboratories [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF] showed that the impairment of spatial working memory and in vivo long-term depression at hippocampal CA3-CA1 synapses, induced by an acute exposure of exogenous cannabinoids, is fully abolished in conditional mutant mice lacking CB1 receptors in brain astroglial cells but is conserved in mice lacking CB1 receptors in glutamatergic or GABAergic neurons. Blockade of neuronal glutamate N-methyl-D-aspartate receptors (NMDAR) and of synaptic trafficking of glutamate α-amino-3-hydroxy-5-methylisoxazole propionic acid receptors (AMPAR) also abolished cannabinoid effects on spatial working memory and long-term depression induction and expression [START_REF] Han | Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD[END_REF].

In addition, CB1 activation in astrocytes is involved in energy supply to the brain. Our findings have revealed that genetic and pharmacological manipulations of the CB1 receptor expression and activity in cultured cortical and hypothalamic astrocytes demonstrated that cannabinoid signaling controls the levels of leptin receptors expression [START_REF] Bosier | Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes[END_REF]. Lack of CB1 receptors also markedly impaired leptin-mediated activation of signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) in astrocytes. In particular, CB1 deletion determined a basal over activation of STAT5, thereby leading to the downregulation of leptin receptors expression, and leptin failed to regulate STAT5dependent glycogen storage in the absence of CB1 receptors in astrocytes [START_REF] Bosier | Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes[END_REF].

Our present results showed that the most common distance between astrocytic CB1 receptors and the nearest synapses is in a rank of 400-800 nm. Moreover, most of those synapses were found to be excitatory. Thus, it is tempting to suggest that this distance would represent the distance travelled by the endocannabinoid molecules from the postsynaptic neuron to the CB1 receptor localized in the astrocyte. According to this interpretation, once inside the astrocyte, the endocannabinoid would activate the astrocytic CB1 receptor, hence triggering the increase of calcium and stimulate the release of neurotransmitter that modulates synaptic transmission and plasticity. Finally, the fixed anatomical distribution pattern of the astrocytic CB1 receptors relative to the nearest synapse both in the CA1 stratum radiatum and in the dentate molecular layer suggests the existence of a molecular architecture underlying the functional activity of the astrocytic CB1 receptors at the tripartite synapse.

The conclusions of the Thesis work are the following:

1. The CB1 receptor in glutamatergic neurons of the Glu-CB 1 -RS mouse hippocampus maintains the same levels of expression and localization of the CB 1 -WT mouse hippocampus. 2. The CB1 receptor in GABAergic neurons of the GABA-CB 1 -RS mouse hippocampus maintains the same levels of expression and localization of the CB 1 -WT mouse hippocampus. 3. The CB1 receptor in astrocytes of the GFAP-CB 1 -RS mouse hippocampus maintains the same levels of expression and localization of the CB 1 -WT mouse hippocampus. 4. The detection of CB1 receptors in astrocytes of the GFAPhrGFP-CB 1 -WT mouse hippocampus is significantly higher than in CB 1 -WT mouse hippocampus with GFAP as an astrocytic marker. 5. The most common distance between the astrocytic CB1 receptors and the nearest synapses in the hippocampus, is between 400-800 nm. 6. The majority of the CA1 and dentate molecular layer synapses surrounded by CB1 receptor immunopositive astrocytes in the 400-800 nm range are of excitatory nature. 7. The CB1 receptor rescue mutant mice characterized in this Doctoral Thesis have proven: 1) to express CB1 receptors in specific brain cell types; 2) the re-expression is limited to the particular neuronal populations or to astrocytes; 3) the endogenous levels of CB1 receptors are maintained in the brain cell types re-expressing the receptor. 8. Altogether, the studied CB1 receptor rescue mutant mice are excellent tools for functional and translational investigations on the role of the CB1 receptors in the normal and diseased brain.

-2-AG: 2-arachidonoylglycerol.

-AA: Arachidonic acid.

-ABHD12: α/β-hydrolase domain containing 12.

-ABHD6: α/β-hydrolase domain containing 6.

-AC: Adenylate cyclase.

-AEA: N-arachidonoylethanolamine or anandamide.

-AMPAR: α-amino-3-hydroxy-5-methyl-isoxazole propionic acid receptor.

-Amyg: Amygdala.

-AON: Anterior olfactory nucleus.

-as: Astrocyte.

-Astro: Astrocytic.

-CA: Cornu ammonis or Ammon´s horn.

-CA1: Region 1 of Cornu Ammonis.

-CA2: Region 2 of Cornu Ammonis.

-CA3: Region 3 of Cornu Ammonis.

-Cb: Cerebellar cortex.

-CB1: Cannabionid type I receptor -CB 1 -KO: Cannabinoid type-1 receptor knock-out mouse.

-CB 1 -WT: Cannabinoid type-1 receptor wild type mouse.

-CB 2 : Cannabionid type 2 receptor.

-CNS: Central Nervous System.

-CPu: Caudate putamen.

-Cx: Cortex.

-DAB: Diaminobezidine.

-den: Dendrite.

-DG: Dentate gyrus.

-DG: Diacylglycerol.

-DGL: Diacylglycerol lipase.

-DSE: Depolarization-induced supression of excitation.

-DSI: Depolarization-induced suppression of inhibition.

-eCB-LTD: Endocannabinoid-mediated long-term depression.

-eCBs: Endocannabinoids.

-eCB-STD: Endocannabinoid-mediated short-term depression.

-EP: Entopeduncular nucleus.

-EXCIT: Excitatory.

-FAAH: Fatty acid amide hydrolase.

-GABA: Gamma-Aminobutyric acid.

-GABA-CB 1 -KO: GABAergic neurons cannabinoid type-1 receptor knock-out mouse. -GABA-CB 1 -RS: GABAergic neurons cannabinoid type-1 receptor rescue mouse -GFAP: Glial Fibrillary Acidic Protein.

-GFAP-CB 1 -KO: Astrocyte cannabinoid type-1 receptor knock-out mouse -GFAP-CB 1 -RS: Astrocyte cannabinoid type-1 receptor rescue mouse -GFAPhrGFP-CB 1 -KO: CB 1 -KO mouse that express hrGFP in astrocytes.

-GFAPhrGFP-CB 1 -WT: CB 1 -WT mouse that express hrGFP in astrocytes.

-GFP: Green Fluorescence Protein.

-Glu-CB 1 -KO: Dorsal telencephalic glutamatergic neurons cannabinoid type-1 receptor knock-out mouse. -Glu-CB 1 -RS: Dorsal telencephalic neurons cannabinoid type-1 receptor rescue mouse.

-GP: Globus pallidus.

-GPCRs: G protein coupled receptors.

-GrDG: Granular dentate gyrus.

-Hi: Hippocampus.

-INHIB: Inhibitory.

-LEA: Lateral entorhinal cortex.

-LMol: Lacunosum moleculare, -LTD: Long-term depression.

-LTP: Long-term potentiation.

-M1: Primary motor cortex.

-MAGL: Monoacylglicerol lipase.

-MEA: Medial Entorhinal Cortex.

-mGluR: Metabotropic glutamate receptors.

-Mid: Midbrain.

-MO: Medulla oblongata.

-MoDG: Molecular dentate gyrus.

-NAc: Nucleus accumbens.

-NAPE: N-arachidonoyl phosphatidylethanolamine.

-NAPE-PLD: N-acyl phosphatidylethanolamine especific phospholipase D.

-NAT: N-acyltransferase.

-NMDAR: N-Methyl-D-aspartate receptor.

-NOS: Nitric oxide synthase.

-OEA: Oleoylethanolamine.

Figure 1 .

 1 Figure 1. Chemical structures of the two major endocannabinoids. N-arachidonylethanolamide (AEA; "anandamide") and 2-arachidonoylglycerol (2-AG).

Figure 2 .

 2 Figure 2. Major pathways of anandamide and 2-AG synthesis and degradation. Modified from Lee et al., 2015.

Figure 3 .

 3 Figure 3. Three-dimensional representation of CB1 receptor and CB2 receptor structure. Modified from Ramos et al., 2011.

Figure 4 .

 4 Figure 4. Distribution of CB1 receptors in the central nervous system of adult mice.Primary somatosensory cortex (S1), primary motor cortex (M1), primary visual cortex (V1), hippocampus (Hi), dentate gyrus (DG), cerebellar cortex (Cb), midbrain (Mid), caudate putamen (CPu), thalamus (Th), anterior olfactory nucleus (AON), nucleus accumbens (NAc), ventral pallidum (VP), substantia nigra pars reticulata (SNR), pons (Po), medulla oblongata (MO). Modified from[START_REF] Kano | Endocannabinoid-mediated control of synaptic transmission[END_REF]

Figure 5 .

 5 Figure 5. Schematic representation of rescued CB1 receptor allele after excision of the stop cassette mediated by Cre recombinase.

Figure 6 .

 6 Figure 6. Signaling mechanisms underlying synaptic regulation by endocannabinoids through stimulation of astrocytes. Endocannabinoids are released by a postsynaptic neuron acting on presynaptic CB1 receptors. As a response, a DSE is triggered in homoneuronal synapses. In addition, the released endocannabinoids also are able to activate astrocytic CB1 receptors. The activation of astrocytic CB1 receptors increases the intracellular calcium and triggers the release of glutamate by the astrocyte, which stimulates the release of neurotransmitter in hetero-neuronal synapses. Modified from Navarrete et al., 2014.

Figure 7 .

 7 Figure 7. Representation of the hippocampal formation and the parahippocampal region in the rat brain. Dentate gyrus, DG (dark brown); Region 3 of Cornu Ammonis, CA3 (medium brown); Region 2 of Cornu Ammonis CA2 (not indicated); Region 1 of Cornu Ammonis, CA1 (orange). The subiculum, Sub (yellow); presubiculum, PrS (medium blue) and parasubiculum (PaS; dark blue). The entorhinal cortex, which has a lateral, LEA (dark green) and a medial, MEA (light green) areas. The perirhinal cortex (consisting of Brodmann areas: A35 (pink) and A36 (purple). Stratum oriens, Or; Pyramidal cell hippocampus, Py; Stratum radiatum, Rad; stratum lucidum, SLu; Lacunosum moleculare, LMol; Molecular dentate gyrus, MoDG and Granular dentate gyrus, GrDG. Modified from van Strien et al., 2009.

  Furthermore, a few neuronal cell types are also present in the molecular layer. The first type of neurons is located in the innermost area, shows a triangular or CA1

Figure 8 .

 8 Figure 8. Representation of a sagittal section of the mouse hippocampal formation. Laminar structure of the hippocampus. DG, Dentate gyrus; CA1, Region 1 of Cornu Ammonis; CA2, Region 2 of Cornu Ammonis; CA3, Region 3 of Cornu Ammonis; Or, Stratum oriens; Py, Stratum pyramidale; Rad, Stratum radiatum; SLu, stratum lucidum; LMol, Stratum lacunosum moleculare; MoDG, Molecular dentate gyrus; GrDG, Granular dentate gyrus; PoDG, Polymorph dentate gyrus. Modified from Franklin and Paxinos, 2008.

13 .

 13 Clearing in xylol (3 times of 5 min). 14. Coverslipped with DPX. 15. Tissue sections were observed and photographed with a Zeiss Axiocam light microscope coupled to a Zeiss AxioCam HRc Camera.

Figure 9 .

 9 Figure 9. Graphical representation of the Immunoperoxidase method for light microscopy.

Figure 10 .

 10 Figure 10. Graphical representation of the Immunoperoxidase method for light microscopy.

Figure 11 .Figure 12 .

 1112 Figure 11. CB1 receptor immunostaining in the Glu-CB1-RS mouse brain.An immunoperoxidase method for light microscopy was used for CB1 receptor immunostaining detection in the Glu-CB1-RS mouse brain. Representative rostro-caudal coronal brain sections stained with the CB1 antibody (left to right and top to bottom). Observe a diffuse CB1 receptor staining throughout but somehow more detectable in OT, Cx, Str, Amyg and hippocampus. PFCx, Prefrontal cortex; OT, Olfactory tubercle; Cx, Cortex; Str, Striatum; Amyg, Amygdala; Hip, Hippocampus; a to d: Bregma 2.34 to 1.34 mm; e to h: Bregma 0.98 to 0.14 mm; i to l: Bregma -0.46 to -1.70 mm; m to p: Bregma -2.46 to -2.80 mm.

Figure 13 .

 13 Figure 13. CB1 receptor immunostaining in the hippocampus of CB1-WT, Glu-CB1-RS and GABA-CB1-RS and CB1-KO mice.

Figure 14 .

 14 Figure 14. Subcellular CB1 receptor localization in synaptic terminals of the CA1 stratum radiatum. Preembedding immunogold method for electron microscopy.

Figure 15 .

 15 Figure 15. Subcellular CB1 receptor localization in synaptic terminals of the dentate inner 1/3 molecular layer. Preembedding immunogold method for electron microscopy.

Figure 16 .

 16 Figure 16. Subcellular CB1 receptor localization in astrocytic processes of the hippocampal CA1 stratum radiatum. Combined preembedding immunogold and immunoperoxidase methods for electron microscopy.

Figure 17 .

 17 Figure 17. Subcellular CB1 receptor localization in astrocytic processes of the dentate molecular layer. Combined preembedding immunogold and immunoperoxidase methods for electron microscopy.

Figure 18 .

 18 Figure 18. Statistics of the CB1 receptor immunopositive glutamatergic terminals in the CA1 stratum radiatum. No significant differences are found between CB1-WT (24.29% ± 1.09), Glu-CB1-RS (21.89% ± 1.21) and GABA-CB1-KO (21.37% ± 1.13). Just residual excitatory terminals with metal particles were found in Glu-CB1-KO (2.50% ± 0.76), GABA-CB1-RS (1.92% ± 0.66) STOP-CB1 (2.20% ± 1.54) and CB1-KO (1.68% ± 0.62) (a). Graphical representation of the CB1 receptor density (particles/µm) in CB1 receptor positive excitatory terminals. No statistical differences are obtained between CB1-WT (0.45 ± 0.01), Glu-CB1-RS (0.45 ± 0.02) and GABA-CB1-KO (0.46 ± 0.02) (b). Proportion of CB1 receptor in glutamatergic terminals normalized to the total CB1 receptor signal in plasmalemma of cellular structures: 12.35% ± 1.04 of the total immunoparticles are located in glutamatergic terminals of CB1-WT and 27.30% ± 3.34 of GABA-CB1-KO. In Glu-CB1-RS the proportion increases to 88.78% ± 1.96 and only residual CB1 receptor immunoparticles are found in excitatory terminals of GABA-CB1-RS (1.10% ± 0.30), Glu-CB1-KO (3.39% ± 0.96), STOP-CB1 (3.03% ± 1.52) and CB1-KO (2.65% ± 1.45) (c). Data are expressed as mean ± Standard Error of the Mean of three different animals. Data were analyzed by means of Kruskal-Wallis Test and the Dunn's Multiple Comparison Post-hoc test. *** indicate statistically significant differences with p < 0.001. EXCIT, excitatory; PART, particles; TER, terminals.

Figure 19 .Figure 20 .

 1920 Figure 19. Statistics of the CB1 receptor distribution in glutamatergic terminals of the dentate inner 1/3 molecular layer. The percentage of CB1 receptor immunopositive excitatory synaptic terminals is statistically similar between CB1-WT (55.98% ± 2.51), Glu-CB1-RS (53.19% ± 2.89) and GABA-CB1-KO (52.12% ± 3.00). The terminal labeling drastically decreases in Glu-CB1-KO (5.48% ± 1.70) and practically disappears in GABA-CB1-RS (3.19% ± 1.08), STOP-CB1 (1.69% ± 1.36) and CB1-KO (3.41% ± 1.36) (a). Graphical representation of the CB1 receptor density (particles/µm) in CB1 receptor positive excitatory terminals. Density in CB1-WT (0.39 ±0.02), Glu-CB1-RS (0.39 ± 0.02) and GABA-CB1-KO (0.39 ± 0.02) is not statistically different (b). Proportion of CB1 receptors in glutamatergic terminals normalized to the total CB1 receptor signal in plasmalemma of cellular structures: 12.80% ± 1.39 of the total CB1 receptor immunoparticles in CB1-WT, 29.22% ± 3.65 in GABA-CB1-KO and 88.64% ± 1.71 in Glu-CB1-RS are localized in excitatory terminals. Only scarce CB1 receptor immunoparticles are in excitatory terminals of GABA-CB1-RS (1.78% ± 0.45), Glu-CB1-KO (0.65% ± 0.27), STOP-CB1 (1.54% ± 1.10) and CB1-KO (1.87% ± 1.14) (c). Data are expressed as mean ± Standard Error of the Mean of three different animals. Data were analyzed by means of Kruskal-Wallis Test and the Dunn's Multiple Comparison Post-hoc test. *** indicate statistically significant differences with p < 0. 001. EXCIT, excitatory; PART, particles; TER, terminals.

Figure 21 .Figure 22 .Figure 23 .Figure 24 .

 21222324 Figure 21. Statistics of the CB1 receptor distribution in GABAergic terminals of the dentate inner 1/3 molecular layer. Percentages of CB1 receptor immunopositive inhibitory synaptic terminals do not defer statistically between CB1-WT (84.27% ± 2.38), GABA-CB1-RS (85.07% ± 1.76) and Glu-CB1-KO (88.02% ± 1.93). Only residual particles are detected in scarce terminals of GABA-CB1-KO (3.82% ± 1.68), Glu-CB1-RS (2.30% ± 1.32), STOP-CB1 (1.28% ± 0.90) and CB1-KO (2.84% ± 1.48) (a). Graphical representation of the CB1 receptor density (particles/µm) in CB1 receptor positive inhibitory terminals. Density in CB1-WT (7.54 ± 0.16), GABA-CB1-RS (7.47 ± 0.14) and Glu-CB1-KO (7.69 ± 0.21) is not statistically different (b). Proportion of CB1 receptor immunoparticles in GABAergic terminals normalized to the total CB1 signal in plasmalemma of cellular structures: 45.38% ± 3.18 in CB1-WT, 59.45% ± 4.00 in Glu-CB1-KO and 93.87% ± 0.93 in GABA-CB1-RS are distributed in GABAergic terminals. Rare CB1 receptor immunoparticles are in inhibitory terminals of Glu-CB1-RS (0.60% ± 0.43), GABA-CB1-KO (0.53% ± 0.53), STOP-CB1 (0.86% ± 0.86) and CB1-KO (0.93% ± 0.93) (c). Data are expressed as mean ± Standard Error of the Mean of three different animals. Data were analyzed by means of Kruskal-Wallis Test and the Dunn's Multiple Comparison Post-hoc test. *** indicate statistically significant differences with p < 0.001. INHIB, inhibitory; PART, particles; TER, terminals.

  nigra pars reticulata. -sp: Dendritic spine. -STAT: Signal transducer and activator of transcription. -STOP-CB 1 : mouse carrying a loxP-flanked stop cassette inserted in the 5'UTR upstream of the CB1 receptor translational start codon. -Str: Striatum. -Sub: The subiculum. -TER: Terminals. -Th: Thalamus.

Table 1 . Details of the antibodies used in this Thesis.

 1

	ANTIBODY			MANUFACTURER	HOST	CONCENTRATION
	Polyclonal Anti-Cannabinoid Receptor Type-1 (CB1)	Frontier Institute	Goat	1:100
	Monoclonal		Anti-Glial
	Fibrillary	Acidic	Protein	Sigma-Aldrich	Mouse	1:1000
	(GFAP)
	Polyclonal humanized Renilla
	reniformis		Green	Stratagene	Rabbit	1:500
	Fluorescence Protein (hrGFP)

RESULTS