Acetylcholine neurons become cholinergic during three time windows in the developing mouse brain

Abstract

Acetylcholine (ACh) neurons in the central nervous system are required for the coordination of neural network activity during higher brain functions, such as attention, learning, and memory, as well as locomotion. Disturbed cholinergic signaling has been described in many neurodevelopmental and neurodegenerative disorders. Furthermore, co-transmission of other signaling molecules, such as glutamate and GABA, with ACh has been associated with essential roles in brain function or disease. However, it is unknown when ACh neurons become cholinergic during development. Thus, understanding the timeline of how the cholinergic system develops and becomes active in the healthy brain is a crucial part of understanding brain development. To study this, we used transgenic mice to selectively label ACh neurons with tdTomato. We imaged serial sectioned brains and generated whole-brain reconstructions at different time points during pre- and postnatal development. We found three crucial time windows – two in the prenatal and one in the postnatal brain – during which most ACh neuron populations become cholinergic in the brain. We also found that cholinergic gene expression is initiated in cortical ACh interneurons, while the cerebral cortex is innervated by cholinergic projection neurons from the basal forebrain. Taken together, we show that ACh neuron populations are present and become cholinergic before postnatal day 12, which is the onset of major sensory processes, such as hearing and vision. We conclude that birth of ACh neurons and initiation of cholinergic gene expression are temporally separated during development but highly coordinated by brain anatomical structure.

Significance Statement Acetylcholine (ACh) neurons are required for higher brain functions and locomotion. Disturbed cholinergic signaling was observed in neurodevelopmental disorders and intellectual disability. While the role of ACh release in neural circuit function is well understood, it is unknown when ACh neurons become cholinergic. We labelled ACh neurons to investigate when ACh neurons become cholinergic in the developing brain and performed reconstructions of serial sectioned brains. Here, we show that ACh neuron populations become cholinergic during three time windows pre- and postnatally. ACh neurons become cholinergic following the caudorostral direction of brain formation. In cortex and hippocampus, activation of cholinergic gene expression in ACh interneurons coincides with cholinergic innervation from the basal forebrain. We highlight that brain ACh neurons are cholinergic before P12, the onset of major sensory functions, such as hearing and vision.