Alterations in the immunoreactivity for muscarinic acetylcholine receptors and colocalized PKCγ in mouse hippocampus induced by spatial discrimination learning
Authors: Van der Zee Eddy A. , Compaan Josje C. , Bohus Béla , Luiten Paul G. M. 
Published: May 2005

This study describes changes in the immunoreactivity for muscarinic acetylcholine receptors (mAChRs) in the hippocampus of mice in relation to spatial discrimination behavior, employing the monoclonal antibody M35 raised against purified bovine mAChR protein. Performance in a hole board in which the animals learned the pattern of 4 baited holes out of 16 holes served as the measure of spatial discrimination learning and memory. Twenty‐six adult male house mice were used, divided into four groups. Three groups served as various controls: group N (naive; blank controls); group H (habituated; animals were introduced to the hole board with all holes baited for 5 consecutive days), and group P (pseudotrained; the animals were admitted to the hole board for 13 consecutive days with all holes baited). The T group (trained) was subjected to the hole board for 5 consecutive habituation days with all holes baited (similar to the H and P groups), followed by 8 successive training days with only four holes baited in a fixed pattern. During the 8 training days, the T group gradually acquired a pattern to visit the baited holes, whereas the P group continued visiting holes in a random fashion. The mice were killed 24 h after the last behavioral session.

All principal cells in the cornu ammonis (CA) and dentate gyrus (DG) of the habituated animals revealed increased levels of mAChR immunoreactivity (mAChR‐ir) over the naive mice. A minor increase in mAChR‐ir was found in the apical dendrites of the CA1 pyramidal cells. Pseudotraining resulted in a CA1‐CA2 region with a low level of mAChR‐ir, resembling naive animals, whereas the trained mice showed a further increase in mAChR‐ir in the CA1‐CA2 pyramidal cell bodies and apical dendrites. Optical density measures of the mAChR‐ir in the CA1 region revealed a significant (P < 0.05) increase in the pyramidal cell bodies of the H and T group over the N and P group, and a significant (P < 0.05) increase in the apical dendrites of the T group over all other groups. In contrast to the CA1‐CA2 region, both pseudotrained and trained mice revealed high mAChR staining in the CA3‐CA4 region and the DG. These results indicate that prolonged exposure to the hole board is sufficient for an enhanced mAChR‐ir in the CA3‐CA4 and DG, whereas the increase in CA1‐CA2 pyramidal cells is a training‐specific feature related to spatial orientation. Nonpyramidal neurons within the CA1‐CA2 region with enhanced mAChR‐ir in the pyramidal cells, however, revealed a decreased level of mAChR‐ir. The opposing effect of pyramidal and nonpyramidal cells suggests a shift in the excitability of the hippocampal microcircuitry.

Previously we demonstrated an increase and redistribution of hippocampal protein kinase C γ‐immunoreactivity (PKC γ‐ir) induced by hole board learning in mice (Van der Zee et al., 1992, J Neurosci 12:4808–4815). Immunofluorescence double‐labeling experiments conducted in the present study in naive and trained animals revealed that the principal cells and DG interneurons co‐express mAChRs and PKCγ, and that the immunoreactivity for both markers increased in relation to spatial orientation within these neurons. The mAChR‐positive nonpyramidal cells of the CA1‐CA2 region were devoid of PKCγ and revealed an opposite training‐induced effect. These results suggest that the postsynaptic changes in mAChR‐ and PKCγ‐ir reflect functional alterations of the hippocampal formation induced by spatial learning. © 1995 Wiley‐Liss, Inc.