Neurochemical heterogeneity of ChAT-immunoreactive neurons in the basal forebrain cholinergic nuclei and striatum in reference to CGRP, CCK, and calcium-binding proteins

Cholinergic neurons in the basal forebrain cholinergic nuclei (BFCN) and neostriatum (CPu) play key roles in learning, attention, and motor control. The loss of cholinergic neurons causes major neurodegenerative diseases such as Alzheimer’s disease. This study aimed to elucidate the molecular diversity of choline acetyltransferase immunoreactive (ChAT-ir) neurons in these brain regions. We performed immunohistochemistry to determine the co-expression of ChAT-ir neurons with two neuropeptides, calcitonin gene-related peptide (CGRP) and cholecystokinin (CCK), as well as three calcium-binding proteins, such as calbindin, calretinin, and parvalbumin, in the adult mouse brain. The results showed that ChAT, calbindin, CGRP and CCK were strongly expressed in the BFCN, including medial septal nucleus (MS), nucleus of vertical limb and horizontal limb of the diagonal band of Broca (VDB and HDB), substantia innominata basal part (SIB), and in the caudate putamen (CPu). CGRP and CCK showed a high immunoreactive co-expression with ChAT, especially in the HDB and CPu. Calbindin immunoreactivity was widely present and coincided with ChAT in the VDB, HDB, and CPu. However, calretinin immunoreactivity showed a selective co-expression with ChAT in the VDB, SIB, and CPu. Although parvalbumin immunoreactivity was observed throughout the BFCN and CPu, but there was no co-expression between ChAT and parvalbumin. The neurochemical diversity of ChAT-ir neurons in the BFCN and neostriatum suggests the specialized functions of cholinergic neurons across different circuits, especially by modulating CGRP, CCK, or calbindin. These results could provide new insight into cholinergic modulation throughout the BFCN and striatum.