Developmental and neurochemical features of cholinergic neurons in the murine cerebral cortex

The existence and role of intrinsic cholinergic cells in the cerebral cortex is controversial, because of their variable localization and morphology in different mammalian species. We have applied choline acetyltransferase (ChAT) immunocytochemistry to study the distribution of cholinergic neurons in the murine cerebral cortex, in the adult and during postnatal development. For more precise neurochemical identification of these neurons, the possible colocalization of ChAT with different markers of cortical neuronal populations has been analyzed by confocal microscopy. This method was also used to verify the relationship between cholinergic cells and cortical microvessels.

ChAT positive cells appeared at the end of the first postnatal week. Their density dramatically increased at the beginning of the second postnatal week, during which it remained higher than in perinatal and adult stages. In the adult neocortex, cholinergic neurons were particularly expressed in the somatosensory area, although their density was also significant in visual and auditory areas. ChAT positive cells tended to be scarce in other regions. They were mainly localized in the supragranular layers and displayed a fusiform/bipolar morphology. The colocalization of ChAT with pyramidal neuron markers was negligible. On the other hand, more than half of the cholinergic neurons contained calretinin, but none of them expressed parvalbumin or calbindin. However, only a fraction of the ChAT positive cells during development and very few in adulthood turned out to be GABAergic, as judged from expression of GABA and its biosynthetic enzymes GAD67/65. Consistently, ChAT showed no localization with interneurons expressing green fluorescent protein under control of the GAD67 promoter in the adult neocortex. Finally, the cortical cholinergic cells often showed close association with the microvessel walls, as identified with the gliovascular marker aquaporin 4, supporting previous hypotheses on the role of cholinergic cells in modulating the cortical microcirculation.

Our results show that the development of the intracortical cholinergic system accompanies the cortical rearrangements during the second postnatal week, a crucial stage for the establishment of cortical cytoarchitecture and for synaptogenesis. Although intrinsic ChAT positive cells usually expressed calretinin, they displayed a variable GABAergic phenotype depending on marker and on cortical developmental stage.