Temporal and region-dependent changes in muscarinic M4 receptors in the hippocampus and entorhinal cortex of adrenalectomized rats

Long-term adrenalectomy induces a dramatic loss of cells in the dentate gyrus and CA1-CA4 fields of the hippocampus resulting in an impairment of cognitive functions such as spatial learning, memory and exploratory behaviour. Muscarinic M₁ and M₄ receptor levels in the hippocampus and entorhinal cortex of adult male Wistar rats were examined 3, 14, 30, 90, and 150 days after adrenalectomy. Receptor levels in the entorhinal cortex and the hippocampus were determined by quantitative autoradiography using ¹²⁵I-M₁-toxin-1 and ¹²⁵I-M₄-toxin-1, M₁ and M₄ subtype selective antagonists, respectively. Moreover, the level of hippocampal M₁ and M₄ muscarinic receptors were evaluated 1 month after adrenalectomy by immunoblot analysis. Adrenalectomy induced apoptotic processes were examined by analysing apoptotic markers using Western blot analysis. No significant changes were observed in the level of muscarinic M₁ receptors in the entorhinal cortex, the dentate gyrus and in the different CA fields of the hippocampus of adrenalectomized (ADX) rats. However, M₄ receptors showed a significant decrease in the entorhinal cortex (at 3 days), dentate gyrus and CA4 (at 14 days), CA3 (at 30 days), and CA2 and CA1 (at 90 days) after adrenalectomy. Moreover, a decrease in the level of M₄ receptors was detected in ADX rats 1 month after adrenalectomy as compared with sham groups using M₄ specific antibody. Apoptotic markers such as PARP and p53 were significantly increased whereas Bcl-2 marker was decreased in ADX rat brain homogenates compared to controls. Our results show that M₁ and M₄ receptors are differentially affected by adrenalectomy and indicate that these subtypes have different functions in the hippocampus. Our data on time and region-dependent decreases in hippocampal M₄ receptors indicate that the M₄ receptor subtype is influenced by adrenal hormones and suggest that the M₄ receptor might be linked to memory function in the hippocampus.