Layer specific distribution of muscarinic receptor M2 in the the sensorimotor areas of rat brain

Abstract

Current understanding of attentional mechanisms within the tactile modality is not fully known. We have recently studied muscarinic receptor-mediated responses of vibrotactile neurons which vary according to cortical depth due to the differences in the local connectivity in the cortex. The aim of this study is to characterize specific muscarinic receptor subtype (M2) by immunofluorescence technique for understanding the role of these receptors within the associated cortex. In particular, differences between barrel field (S1BF), motor cortex (M1) and hindlimb area (S1HL) of the rat SI were investigated. Coronal sections (50 m) from 7 Wistar Albino rats were obtained for each area. Mouse monoclonal anti-mAChR2 was used as primary antibody and goat anti-IgG1 with Alexa Fluor 594 was used as secondary antibody. Ethidium bromide was used on additional sections to determine layer thicknesses and total number of cells within a layer. Statistical analyses were performed on three dependent variables: Average number of M2 receptor complexes (M2RC) in a layer (N), average number of M2RC normalized with layer thickness (D), and average number of M2RC per total number of cells in a layer (C). 2-way ANOVA showed significant main effects of layer on N, D, and C (p<0.001, p=0.002, p=0.053, respectively). Area and layer interaction was only observed for N (p<0.001). On the other hand, area did not have a main effect for any dependent variable. The number of M2RC was highest in layer V, VI, IV for M1, S1BF and S1HL, respectively. Additionally, when the average number M2RC were normalized according to thickness, highest M2 density were observed in layer II and III for all areas. In other layers, M2 density was similar for S1HL and S1BF, but lower than M1. There was no general difference among the three cortical area regarding the number and density of M2RC which is consistent with the cholinergic innervation in the sensory-motor areas. However, the distribution of M2RC varied within each area.