Localization of the Ca2+-binding proteins, cannabinoid CB1 receptor (CB1R) and fatty acid amide hydrolase (FAAH) in the swine claustrum

Introduction: Elevation of intracellular Ca2+ is associated to the retrograde suppression of the synaptic transmission mediated by cannabinoid CB1R. Also, in cultured trigeminal ganglion neurons, the endocannabinoid anandamide directly caused Ca2+ influx in a dose-dependent manner, which then triggered an increase of intracellular Ca2+ concentration. Moreover, the endocannabinoid anandamide degrading enzyme FAAH is a postsynaptic enzyme associated with membranes organelles known to store Ca2+ (e.g. mitochondria, smooth endoplasmic reticulum) localized in somata and dendrites. Thus we investigated here the immunohistochemical localization of CB1 receptor, FAAH and the Ca2+-binding proteins Parvalbumin (PV), Calbindin (CB) and Calretinin (CR) in the pig claustrum. Materials and Methods: Claustrum samples, obtained from four healthy slaughtered animals (females, 10 months old), were promptly formalin fixed for paraffin embedding. Commercially available antibodies were used to reveal CB1R, FAAH, PV, CB and CR presence by mean of indirect immunohistochemistry using the anti-peroxidase method. Results: Immunoreactive somata and nerve fibers were recorded for PV, CR and CB throughout the pig claustrum. The CB1R antibody labeled nerve fibers, whereas the anti-FAAH antibody stained cell bodies. All the structures identified in the stained sections revealed that FAAH-immunoreactive (ir) neuronal somata and dendrites were surrounded by CB1R-ir fibers. Conclusion: CB1R and FAAH complementary immunostaining and their cellular localization reported here provide the first anatomical evidence for existence of the endocannabinoid system in the swine claustrum. The observed populations of Ca2+-binding proteins positive interneurons lead to the hypothesis that the endocannabinoid system could play a role in the so-called depolarization- induced suppression of inhibition. Further investigations including colocalization studies may help clarifying how interneurons are involved in the claustrum endocannabinoid system of pigs.