The Absence of the Transient Receptor Potential Vanilloid 1 Directly Impacts on the Expression and Localization of the Endocannabinoid System in the Mouse Hippocampus

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective ligand-gated cation channel involved in synaptic transmission, plasticity, and brain pathology. In the hippocampal dentate gyrus, TRPV1 localizes to dendritic spines and dendrites postsynaptic to excitatory synapses in the molecular layer (ML). At these same synapses, the cannabinoid CB1 receptor (CB1R) activated by exogenous and endogenous cannabinoids localizes to the presynaptic terminals. Hence, as both receptors are activated by endogenous anandamide, co-localize, and mediate long-term depression of the excitatory synaptic transmission at the medial perforant path (MPP) excitatory synapses though by different mechanisms, it is plausible that they might be exerting a reciprocal influence from their opposite synaptic sites. In this anatomical scenario, we tested whether the absence of TRPV1 affects the endocannabinoid system. The results obtained using biochemical techniques and immunoelectron microscopy in a mouse with the genetic deletion of TRPV1 show that the expression and localization of components of the endocannabinoid system, included CB1R, change upon the constitutive absence of TRPV1. Thus, the expression of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) drastically increased in TRPV1−/− whole homogenates. Furthermore, CB1R and MAGL decreased and the cannabinoid receptor interacting protein 1a (CRIP1a) increased in TRPV1−/− synaptosomes. Also, CB1R positive excitatory terminals increased, the number of excitatory terminals decreased, and CB1R particles dropped significantly in inhibitory terminals in the dentate ML of TRPV1−/− mice. In the outer 2/3 ML of the TRPV1−/− mutants, the proportion of CB1R particles decreased in dendrites, and increased in excitatory terminals and astrocytes. In the inner 1/3 ML, the proportion of labeling increased in excitatory terminals, neuronal mitochondria, and dendrites. Altogether, these observations indicate the existence of compensatory changes in the endocannabinoid system upon TRPV1 removal, and endorse the importance of the potential functional adaptations derived from the lack of TRPV1 in the mouse brain.

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective ligand-gated cation channel involved in synaptic transmission, plasticity, and brain pathology. In the hippocampal dentate gyrus, TRPV1 localizes to dendritic spines and dendrites postsynaptic to excitatory synapses in the molecular layer (ML). At these same synapses, the cannabinoid CB 1 receptor (CB 1 R) activated by exogenous and endogenous cannabinoids localizes to the presynaptic terminals. Hence, as both receptors are activated by endogenous anandamide, co-localize, and mediate long-term depression of the excitatory synaptic transmission at the medial perforant path (MPP) excitatory synapses though by different mechanisms, it is plausible that they might be exerting a reciprocal influence from their opposite synaptic sites. In this anatomical scenario, we tested whether the absence of TRPV1 affects the endocannabinoid system. The results obtained using biochemical techniques and immunoelectron microscopy in a mouse with the genetic deletion of TRPV1 show that the expression and localization of components of the endocannabinoid system, included CB 1 R, change upon the constitutive absence of TRPV1. Thus, the expression of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) drastically increased in TRPV1 −/− whole homogenates. Furthermore, CB 1 R and MAGL decreased and the cannabinoid receptor interacting protein 1a (CRIP1a) increased in TRPV1 −/− synaptosomes. Also, CB 1 R positive excitatory terminals increased, the number of excitatory terminals decreased, and CB 1 R particles dropped significantly in inhibitory terminals in the dentate ML of TRPV1 −/− mice. In the outer 2/3 ML of the TRPV1 −/− mutants, the proportion of CB 1 R particles decreased in dendrites, and increased in excitatory terminals and astrocytes.
The use of immunoelectron microscopy has revealed localizations from where TRPV1 regulates neural activity. Thus, TRPV1 localized presynaptically to excitatory synaptic terminals in the CA1 hippocampus facilitates AEA-mediated glutamate release (Bialecki et al., 2020). Also, TRPV1 localized postsynaptically to both, inhibitory synapses in the inner 1/3 of the dentate ML (Canduela et al., 2015) regulates GABAergic synaptic transmission (Chavez et al., 2014), and to excitatory synapses in the outer 2/3 of the ML  where AEA triggers TRPV1-dependent and CB 1 R-independent longterm depression of the excitatory synaptic transmission (eLTD) at the MPP synapses (Chávez et al., 2010). Interestingly, at these same excitatory synapses, MPP stimulation (10 Hz, 10 min) triggered a group I metabotropic glutamate receptor-dependent and CB 1 R-mediated eLTD that required intracellular calcium and 2-AG synthesis . So, the question raises as whether both receptors would influence each other by acting from opposite loci of the same synapse. In this sense, the existence of interactions between both the endocannabinoid and endovanilloid system has been suggested (Starowicz et al., 2007). AEA increase reduces 2-AG effect on presynaptic CB1Rs through postsynaptic TRPV1 resulting in short-term plasticity regulation (Maccarrone et al., 2008;Musella et al., 2010;Lee et al., 2015). Genetic deletion of endocannabinoid system components leads to compensatory changes in TRPV1; for instance, TRPV1 expression decreases in dentate gyrus and increases in the cerebellar granule cell layer in mice lacking CB 1 R (Cristino et al., 2006). Also, CB 1 R and TRPV1 expressed in the same cell mediate opposite effects on intracellular calcium levels (Szallasi and Di Marzo, 2000), glutamate release (Marinelli et al., 2003) or excitatory and inhibitory neurotransmission (Tahmasebi et al., 2015). Altogether, these previous findings endorse reciprocal and complex bidirectional interactions between CB 1 R and TRPV1 expressed at the same synapse (Zádor and Wollemann, 2015).
We sought in this study compensatory mechanisms in the endocannabinoid system in a mouse model carrying the genetic deletion of TRPV1 (TRPV1 −/− ), which might eventually have an impact on neural information processing. Hence, biochemical and immunohistochemical tools were used to explore the overall expression patterns of some key endocannabinoid system components (DAGL, MAGL, NAPE-PLD, FAAH). Also, immunoelectron microscopy was applied to assess CB 1 R rearrangements in the TRPV1 −/− hippocampal dentate gyrus where both receptors are involved in mechanistically distinct eLTD at the MPP synapses (Chávez et al., 2010;Peñasco et al., 2019). The results show that the expression and localization of some components of the endocannabinoid system change upon the constitutive absence of TRPV1.

Animal Procedures
All protocols were approved by the Committee of Ethics for Animal Welfare of the University of the Basque Country (CEEA/M20/2015/105; CEIAB/M30/2015/106) and were in accordance to the European Communities Council Directive of 22nd September 2010 (2010/63/EU) and Spanish regulations (Real Decreto 53/2013, BOE 08-02-2013. All efforts were made to minimize pain and suffering and to reduce the number of animals used. Eight week-old-male TRPV1 −/− mice and their wild type (WT) littermates (TRPV1+/+) were used (n = 18 each). The TRPV1 −/− mice (C57BL/6 J background; Caterina et al., 2000) were derived from heterozygous breeding pairs generated by crossing of B6.129X1-Trpv1 tm1Jul /J mice (The Jackson Laboratory, Bar Harbor, ME) with C57BL/6 j mice (Janvier Labs) at the General Animal Unit Service of the University of the Basque Country (UPV/EHU). The mice used were genotyped in the Genomics and Proteomics Unit of the University of the Basque Country (UPV/EHU).
Mice were housed in pairs or groups of maximum three littermates in standard Plexiglas cages (17 × 14.3 × 36.3 cm) and before experiments were conducted, they were allowed to acclimate to the environment for at least 1 week. They were maintained at standard conditions with food and tap water ad libitum throughout all experiments in a room with constant temperature (22 • C), and kept in a 12:12 h light/dark cycle with lights off at 9:00 p.m.

Tissue Preservation
The TRPV1 −/− and WT mice were deeply anesthetized by intraperitoneal administration of a mixture of ketamine/xilacine (80/10 mg/kg body weight). They were transcardially perfused at room temperature (RT) with phosphate buffered saline (0.1 M PBS, pH 7.4) for 20 s, followed by the iced-cooled fixative solution made up of 4% formaldehyde (freshly depolymerized from paraformaldehyde), 0.2% picric acid, and 0.1% glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 10-15 min. Then, brains were carefully removed from the skull and post-fixed in the fixative solution for 1 week at 4 • C followed by their storage in 1:10 fixative solution diluted in 0.1 M PB with 0.025% sodium azide at 4 • C until use.

CB 1 R Immunoelectron Microscopy
The procedure has already been described in detail elsewhere . Briefly, hippocampal sections were preincubated in a blocking solution of 10% HNS, 0.1% sodium azide, and 0.02% saponine prepared in TBS (pH 7.4) for 30 min at RT. Then hippocampal sections were incubated with a goat anti-CB 1 R antibody (2 µg/ml, #CB1-Go-Af450, Frontier Science Co.; RRID: AB_257130) in 10% HNS/TBS containing 0.1% sodium azide and 0.004% saponine on a shaker for 2 days at 4 • C. After several washes in 1% HNS/TBS, tissue sections were incubated with a 1.4 nm gold-labeled rabbit anti-goat IgG (Fab' fragment, 1:100, Nanoprobes Inc., Yaphank, NY, USA Cat#2004; RRID: AB_2631182) in 1% HNS/TBS with 0.004% saponine on a shaker for 4 h at RT. Thereafter, hippocampal sections were washed in 1% HNS/TBS overnight at 4 • C, postfixed in 1% glutaraldehyde in TBS for 10 min and washed in double-distilled water. Then, gold particles were silver-intensified with a HQ Silver kit (Nanoprobes Inc., Yaphank, NY, USA; Cat#2012) for ∼12 min in the dark and washed in 0.1 M PB. Stained sections were osmicated (1% OsO4 (v/v) in 0.1 M PB, 20 min; Electron Microscopy Sciences; Cat#19150), dehydrated in graded alcohols to propylene oxide and plastic-embedded in Epon resin 812. Ultrathin sections (50 nm-thick) were collected on nickel mesh grids, stained with 2.5% lead citrate for 20 min and examined in an electron microscope (Philips EM208S). Tissue preparations were photographed by using a digital camera (Digital Morada Camera, Olympus) coupled to the electron microscope. Adjustments in contrast and brightness were made to the figures using Adobe Photoshop (CS3, Adobe Systems; RRID: SCR_014199).

Double CB 1 R and Glial Fibrillary Acidic Protein (GFAP) Immunoelectron Microscopy
Co-labeling experiments were performed as described . The first steps were shared with the single pre-embedding immunogold method. Then, the hippocampal sections were simultaneously incubated with the goat anti-CB 1 R antibody (2 µg/ml, #CB1-Go-Af450, Frontier Science Co.; RRID: AB_257130) and a mouse anti-GFAP antibody (20 ng/ml; G3893; Sigma-Aldrich, mouse monoclonal; RRID: AB_257130) in 10% HNS/TBS with 0.1% sodium azide and 0.004% saponin on a shaker for 2 days at 4 • C. After several washes in 1% HNS/TBS, tissue sections were incubated with both 1.4 nm goldlabeled rabbit anti-goat IgG (Fab' fragment, 1:100, Nanoprobes Inc., Yaphank, NY, USA) for the localization of CB 1 R and a biotinylated horse anti-mouse IgG (1:200 Vector Labs, Cat#BA-2000; RRID: AB_2313581) for the localization of GFAP, diluted in 1% HNS/TBS with 0.004% saponin on a shaker for 4 h at RT. Then, sections were incubated in avidin-biotin peroxidase complex (ABC) prepared in 1% HNS/TBS for 1.5 h at RT. They were subsequently washed in 1% HNS/TBS overnight at 4 • C and postfixed in 1% glutaraldehyde in TBS for 10 min at RT. Following several washes in double-distilled water, gold particles were silver intensified with an HQ Silver kit (Nanoprobes Inc., Cat#2012) for ∼12 min in the dark and washed in 0.1 M PB, pH 7.4. Then, the tissue was incubated in 0.05% DAB (Sigma-Aldrich, Cat#D5637; RRID: AB_2336819) and 0.01% hydrogen peroxide prepared in 0.1 M PB for 3 min. Labeled sections were osmicated (1% osmium tetroxide, Electron Microscopy Sciences, Cat#19150) in 0.1 M PB, pH 7.4, 20 min, dehydrated in graded alcohols to propylene oxide, and plastic-embedded in Epon resin 812. Ultrathin sections (50 nm-thick) were collected on nickel mesh grids, counterstained with 2.5% lead citrate for 20 min and examined with an electron microscope (JEOL JEM 1400 Plus). Tissue samples were imaged using a digital camera (sCMOS). Figures were created with Adobe Photoshop (CS3, Adobe Systems; RRID: SCR_014199).

Semi-quantification Analysis
Hippocampal sections from TRPV1 −/− (n = 3) and WT mice (n = 3) were visualized under a light microscope in order to select portions of the inner 1/3 (hilar mossy cell axon terminal synapses) and outer 2/3 of the dentate ML (perforant path synapses) with good, reproducible immunolabeling and wellpreserved ultrastructure. All electron micrographs were taken at ×22,000 magnification and showed similar labeling intensity, indicating that the selected areas were at the same depth. Furthermore, only ultrathin sections within the first 1.5 µm from the surface of the tissue block were examined to avoid false negatives. Metal particles placed on membranes were counted.
Positive labeling was considered if at least one immunoparticle was over the membrane or within ∼30 nm of it. Image-J (FIJI) (NIH, USA; RRID: SCR_003070) was used to measure the membrane length. Sampling was carefully and accurately carried out in the same way for all the animals studied, and experimenters were blinded to the subject during CB 1 R quantification.
Synaptic terminals were identified by ultrastructural features. Thus, asymmetric excitatory synapses showed typical presynaptic terminals containing abundant clear and spherical synaptic vesicles, and thick postsynaptic densities mostly on dendritic spines. Inhibitory synapses had presynaptic terminals with pleomorphic synaptic vesicles forming symmetric contacts with postsynaptic dendrites. Astrocytes were identified by GFAP immunoreaction product inside their cell bodies and processes. The analysis was done over 1,910 synapses, 4,574 mitochondria and 549 astrocytic profiles in TRPV1 −/− ; 2,177 synapses, 5,565 mitochondria and 413 astrocytic profiles in WT.
Image-J (FIJI) (NIH, USA; RRID: SCR_003070) was used to measure the following parameters: percentage of CB 1 Rpositive terminals, mitochondria, and astrocytic profiles; density of CB 1 R particles in terminal and astrocytic membranes (particles/µm membrane); terminal perimeter; number of terminals, mitochondria and astrocytic profiles; and proportion of CB 1 R particles in each compartment vs. total CB 1 R labeling. All values were shown as mean ± S.E.M. using a statistical software package (GraphPad Prism 5; GraphPad Software; RRID: SCR_002798). The normality test (Kolmogorov-Smirnov) was always applied before running statistical tests. Sample uniformity was assessed by one-way ANOVA or Kruskal-Wallis multiple comparison test. Data from each group (n = 3) were pooled since no significant differences were detected among mice (p > 0.05). Finally, data were analyzed by parametric and non-parametric tests (Unpaired t-test or Man-Whitney test). Values of p < 0.05 were considered statistically significant.

Hippocampal Membrane Preparation
Hippocampal sections from TRPV1 −/− and WT were thawed in ice-cold 20 mM Tris-HCl, pH 7.4, containing 1 mM EGTA (Tris/EGTA buffer), and then homogenized in 20 times the volume of the same hypotonic buffer using a glass homogenizer. Cell debris was discarded by centrifugation at 1,000 g (10 min, 4 • C) and then membranes were obtained by centrifugation at 40,000 g (30 min, 4 • C). Finally, the pellet was re-suspended and re-centrifuged under the same conditions. Membranes were aliquoted in microcentrifuge tubes, centrifuged again (40,000 g, 30 min, 4 • C) and the pellets were stored at −75 • C prior to use. Protein content was determined using the Bio-Rad dye reagent with bovine γ-globulin as a standard.

Western Blotting of Hippocampal Synaptosomes
Hippocampal synaptosomes were prepared as previously described (Garro et al., 2001). TRPV1 −/− and WT mice were anesthetized with isoflurane and decapitated; brains were removed and placed on ice-cold 0.32 M sucrose, pH 7.4, containing 80 mM Na2HPO4 and 20 mM NaH2PO4 (sucrose phosphate buffer) with protease inhibitors (Iodoacetamide 50 µM, PMSF 1 mM). The hippocampal tissue was minced and homogenized in 10 volumes of sucrose/phosphate buffer using a motor-driven Potter Teflon glass homogenizer (motor speed 800 rpm; 10 up and down strokes; mortar cooled in an ice-water mixture throughout). The homogenate was centrifuged at 1,000 × g for 10 min and obtained pellet (P1) was re-suspended and pelleted. The supernatants (S1 + S1') were pelleted at 15,000 × g (P2) and re-suspended in the homogenization buffer to a final volume of 16 ml. This P2 fraction is a mixture of myelin fragments, synaptosomes and free mitochondria. The suspension was layered directly onto tubes containing 8 ml 1.2 M sucrose phosphate buffer, and centrifuged at 180,000 × g for 20 min. The material retained at the gradient interface (synaptosome + myelin + microsome) was carefully collected with a Pasteur-pipette and diluted with ice-cold 0.32 M sucrose/phosphate buffer to a final volume of 16 ml. The diluted suspension was then layered onto 8 ml of 0.8 M sucrose phosphate buffer, and centrifuged as described above. The obtained pellet was re-suspended in ice-cold phosphate buffer, pH 7.5 and aliquoted in microcentrifuge tubes. Aliquots were then centrifuged at 40,000 × g for 30 min, the supernatants were aspirated and the pellets corresponding to the nerve terminal membranes were stored at −80 • C. Protein content was determined using the Bio-Rad dye reagent with bovine γ-globulin as standard.
For western blotting, hippocampal synaptosome fractions were boiled in urea-denaturing buffer [20 mM Tris-HCl, pH 8.0, 12% glycerol, 12% Urea, 5% dithiothreitol, 2% sodium dodecyl sulfate (SDS), 0.01% bromophenol blue] for 5 min. Denaturized proteins were resolved by electrophoresis on SDS-polyacrylamide (SDS-PAGE) gels and transferred to nitrocellulose or PVDF membranes at 30 V constant voltage overnight at 4 • C. Blots were blocked in 5% non-fat dry milk/phosphate buffered saline containing 0.5% BSA and 0.1% Tween for 1 h, and incubated with the antibodies overnight at 4 • C. Blots were washed and incubated with specific HRP conjugated secondary antibodies diluted in blocking buffer for 1.5 h at RT. Immunoreactive bands were incubated with the ECL system according to the manufacturer instructions. In these experiments, differences between the relative expressions of proteins were analyzed by regression line slopes comparison method by a statistical software package (GraphPad Prism, GraphPad Software Inc, San Diego, USA).

Cannabinoid Immunohistochemistry in TRPV1 −/− Hippocampus
The patterns of CB 1 R and the main enzymes for synthesis and degradation of 2-AG and AEA were studied in TRPV1 −/− (Figure 1). An increase in MAGL, FAAH, and NAPE-PLD, and a slight decrease in DAGLα immunoreactivity was observed in TRPV1 −/− vs. WT. Though MAGL and FAAH immunostainings were faint in both TRPV1 −/− and WT hippocampus, MAGL immunoreactivity increased more than FAAH and NAPE-PLD, especially in the hilus and CA3 stratum lucidum. Noticeably, CB 1 R staining increased overall TRPV1 −/− hippocampus, but particularly stronger was in CA3 stratum radiatum, CA1 pyramidal cell layer and dentate ML. In the latter, CB 1 R immunoreactivity was more intense in a fiber meshwork in the inner 1/3 of the layer and weaker but yet more conspicuous than in WT, in fibrous profiles distributed in the outer 2/3 ML (Figure 1).

DISCUSSION
The main finding of this study was that the constitutive deletion of the TRPV1 gene impacts on the expression and localization of some elements of the ECS. Thus, the increase in CB 1 R, FAAH, MAGL and NAPE-PLD immunoreactivity in the TRPV1 −/− hippocampus indicates the existence of compensatory changes. However, CB 1 R did not change significantly and only FAAH and MAGL increased when measured in western blots of whole hippocampal homogenates. Certainly, this is a powerful technique to detect low protein levels (Garro et al., 2001), but subtle changes in protein expression can be better achieved by homogenate fractioning into P2 extracts and raw synaptosomes (Garro et al., 2001). Thus, MAGL increased in whole TRPV1 −/− hippocampal homogenates, but a significant MAGL decrease with no changes in FAAH stood out in TRPV1 −/− synaptosomes. One plausible explanation for this discrepancy would be that MAGL expression in astrocytes (Uchigashima et al., 2011) increases in the absence of TRPV1, but decreases the MAGL pool localized in presynaptic terminals (Gulyas et al., 2004;Uchigashima et al., 2011). Finally, 2-AG dysregulation would be expected to occur in TRPV1 −/− as DAGLα expression did not change in the absence of TRPV1. FAAH mostly localizes to intracellular organelle membranes but is also on somatic and dendritic membranes (Gulyas et al., 2004), while NAPE-PLD is very highly localized in hippocampal granule cell axons (Egertová et al., 2008). Hence, the FAAH increase in whole homogenates with no obvious changes in NAPE-PLD in TRPV1 −/− synaptosomes would drop AEA in TRPV1 −/− . However, NAPE-PLD immunoreactivity increased in the dentate hilar region and CA3 stratum lucidum, indicating that AEA might be augmented in certain subcellular compartments, e.g., granule cell axons. Interestingly, CB 1 R expression did not suffer any variation in P2 fractions from TRPV1 −/− but significantly decreased in synaptosomal extracts. To circumvent the limiting factors of raw synaptosomes unable to discriminate between presynaptic and postsynaptic compartments, we investigated the CB 1 R localization in TRPV1 −/− by high resolution immunoelectron microscopy. We focused on the dentate ML because its outer 2/3 correspond to the termination zone of the glutamatergic entorhino-dentate pathway (Grandes and Streit, 1991) which transmits spatial information through the medial perforant path (Fyhn et al., 2004) and non-spatial information via the lateral perforant path (Burwell, 2000). The inner 1/3 ML receives the glutamatergic mossy cell commissural/associational axons which innervate the dentate granule cells involved in the signaling of environmental and context information (Scharfman and Myers, 2012). Our anatomical data confirmed the biochemical results. Thus, similar changes were found in the inner 1/3 and outer 2/3 ML but with slight differences, that might be reflecting that distinct TRPV1 expression patterns trigger specific compensatory effects. For instance, the relative increase in CB 1 R positive excitatory terminals in TRPV1 −/− was more pronounced in the inner 1/3 than outer 2/3 ML. However, taking into account that the CB 1 R density in excitatory terminals did not change throughout the entire ML, the reduction in the number of CB 1 R negative excitatory terminals might likely be explaining the increase seen in CB 1 R positive excitatory terminals. Thus, TRPV1 deletion modifies unevenly the total number of excitatory terminals boosting the proportion of excitatory terminals equipped with CB 1 R, which is in line with the reduced glutamatergic innervation observed in TRPV1 −/− hippocampus (Hurtado-Zavala et al., 2017). We observed in TRPV1 −/− a decrease in the proportion of CB 1 R particles located in inhibitory terminals in both the inner 1/3 (34.5% ↓) and outer 2/3 ML (15.5% ↓) as well as an increase in CB 1 R labeling in excitatory terminals in the inner 1/3 (83.80% ↑) and outer 2/3 (73.50% ↑) (see below). Furthermore, considering the total number of CB 1 R particles counted, the particles localized in inhibitory terminals decreased ∼53% in the inner 1/3 and ∼21% in the outer 2/3 ML in TRPV1 −/− . Nevertheless, the differences in inhibitory terminals in TRPV1 −/− relative to WT were minimal because only a small reduction in CB 1 R density was found to be significant in the outer 2/3 ML. Bearing in mind that the majority of CB 1 R particles are localized to GABAergic terminals in the hippocampus (Kano et al., 2009;Gutiérrez-Rodríguez et al., 2017, 2018Bonilla-Del Río et al., 2019), it would be plausible that an impairment in receptor renewal could be more pronounced in inhibitory than excitatory terminals and therefore easier to be detected. This would also lead to guess that if the overall CB 1 R expression at excitatory terminals remains unchanged, the decrease in CB 1 R density at inhibitory terminals could indeed be responsible for the CB 1 R fall in synaptosomal fractions. Actually, the CRIP1a increase observed in TRPV1 −/− might also be playing role as its overexpression interferes with CB 1 R activity and receptor downregulation (Smith et al., 2015).
About 75% of the total TRPV1 positive granule cell dendritic spines and 56% of the dendrites were in the outer 2/3, and the rest in the inner 1/3 ML . Furthermore, about 30% of the inhibitory synapses in the inner 1/3 ML have TRPV1 mostly localized to postsynaptic dendritic membranes (Canduela et al., 2015). In the absence of TRPV1, CB 1 R may exert a major regulatory effect on the excitatory transmission with important functional consequences in the dentate ML. In this sense, we have recently shown that CB 1 R immunolabeling decreases by 34% in excitatory terminals and the proportion of CB 1 R immunopositive excitatory boutons decreases by 35% FIGURE 6 | CB 1 R localization in the inner 1/3 ML of WT and TRPV1 −/− mice. Pre-embedding immunogold method for electron microscopy. CB 1 R immunoparticles (arrows) are localized to inhibitory terminals (ter, red arrows, red shading), excitatory terminals (ter, green arrows, green shading), and mitochondrial outer membranes (m, purple arrows, purple shading) in WT (A,C) and TRPV1 −/− (B,D). CB 1 R particles are also on membranes of GFAP positive astrocytic processes (as, yellow arrows, yellow shading) in WT (E) and TRPV1 −/− (F). Combined pre-embedding immunoperoxidase and immunogold method. sp, dendritic spine; den, dendrite; GC, granule cell. Scale bars: 0.5 µm.
in the middle 1/3 ML of the adult mouse subjected to ethanol intake during adolescence (binge drinking model). These deficits in glutamatergic CB 1 Rs were associated with the loss of eCB-eLTD at the MPP-granule cell synapses and an impairment of recognition memory (Peñasco et al., 2020). TRPV1 changes upon the loss or absence of CB 1 R remain to be investigated.
CB 1 Rs are also in mitochondria where they regulate cellular respiration, energy production, and memory formation in the  (J) Number of CB 1 R particles found in membranes in 20 µm 2 . Mann-Whitney U-test or Student's t-test. p < 0.05*; p < 0.01**; p < 0.001***; p < 0.0001****. All data are represented as mean ± S.E.M. hippocampus (Hebert-Chatelain et al., 2016). The proportion of mitochondrial CB 1 R in TRPV1 −/− was maintained regardless TRPV1 is expressed in mitochondrial membranes (Miyake et al., 2015). However, despite that the number of mitochondrial profiles was kept in the inner 1/3 ML, a significant reduction was observed in the outer 2/3 ML, suggesting that TRPV1 could have a direct implication in mitochondrial dynamics. The differences observed between both ML zones may be due to differences in their neuronal composition and distinct effects of TRPV1 absence. Thus, strong TRPV1-mediated mitochondrial calciuminflux causes cytotoxicity and cell death in HEK 293 cells and dorsal root ganglion neurons (Stueber et al., 2017), while TRPV1 knockdown improves mitochondrial function and apoptosis inhibition in primary cardiomyocytes (Sun et al., 2014). The CB 1 R is also localized to astrocytes (Navarrete and Araque, 2010;Metna-Laurent and Marsicano, 2015;Gutiérrez-Rodríguez et al., 2017). Calcium rise linked to TRPV1 activation drives cytoskeletal rearrangements, microtubule disassembly, and filament reorganization leading to astrocyte migration (Goswami et al., 2007;Morales-Lázaro et al., 2013). However, TRPV1 antagonism has an opposite effect (Ho et al., 2014). We did not detect differences in astrocytic parameters in TRPV1 −/− , thus astrocytic disturbance does not seem to happen in the absence of TRPV1. TRPV1 deletion could have triggered compensatory mechanisms in other receptors/channels that would replace its function. Furthermore, there were not changes in the proportion of CB 1 R positive astrocytic profiles in TRPV1 −/− ML. However, a significant reduction in CB 1 R density in astrocytes was observed in TRPV1 −/− outer 2/3 ML resembling the CB 1 R density decrease in astrocytes revealed in a binge-drinking model of ethanol intake . Astrocytes participate in inflammatory responses through the release of pro-inflammatory molecules (Farina et al., 2007) that can be soothing by astroglial CB 1 R-mediated mechanisms (Metna-Laurent and Marsicano, 2015). Hence, because of the reduced CB 1 R density in astrocytes, it is reasonable to expect an impairment of an anti-inflammatory response in TRPV1 −/− . Furthermore, the decrease in astrocytic CB 1 R density could also have functional consequences in synaptic transmission and plasticity, as astrocytes may not be effective in detecting the endocannabinoids produced on demand by neural activity, compromising gliotransmitter availability elicited by cannabinoids at the synapses (Araque et al., 2014).
Altogether, the lack of TRPV1 causes changes in the ECS that might be affecting synaptic transmission and plasticity, and eventually behavior.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by Committee of Ethics for Animal Welfare of the University of the Basque Country (CEEA/M20/2015/105; CEIAB/M30/2015/106).