Dual recombinase fate mapping reveals a transient cholinergic phenotype in multiple populations of developing glutamatergic neurons

Abstract

Cholinergic transmission shapes the maturation of glutamatergic circuits, yet the developmental sources of acetylcholine have not been systematically explored. Here, we have used Cre‐recombinase‐mediated genetic labeling to identify and map both mature and developing CNS neurons that express choline acetyltransferase (ChAT). Correction of a significant problem with a widely used ChatCre transgenic line ensures that this map does not contain expression artifacts. ChatCre marks all known cholinergic systems in the adult brain, but also identifies several brain areas not usually regarded as cholinergic, including specific thalamic and hypothalamic neurons, the subiculum, the lateral parabrachial nucleus, the cuneate/gracilis nuclei, and the pontocerebellar system. This ChatCre fate map suggests transient developmental expression of a cholinergic phenotype in areas important for cognition, motor control, and respiration. We therefore examined expression of ChAT and the vesicular acetylcholine transporter in the embryonic and early postnatal brain to determine the developmental timing of this transient cholinergic phenotype, and found that it mirrored the establishment of relevant glutamatergic projection pathways. We then used an intersectional genetic strategy combining ChatCre with Vglut2Flp to show that these neurons adopt a glutamatergic fate in the adult brain. The transient cholinergic phenotype of these glutamatergic neurons suggests a homosynaptic source of acetylcholine for the maturation of developing glutamatergic synapses. These findings thus define critical windows during which specific glutamatergic circuits may be vulnerable to disruption by nicotine in utero, and suggest new mechanisms for pediatric disorders associated with maternal smoking, such as sudden infant death syndrome.

Motor neurons involved in fine motor control are labeled by tracingAtoh1-lineage neurons in the spinal cord

“…Therefore, we pursued a second intersectional cross using Chat IRES-Cre and Cdx2::FLPo crossed to the intersectional Ai65 tdTomato reporter (hereafter called Chat Cdx2 neurons). Chat IRES-Cre/+ labels all cholinergic cells and cells with transient Chat expression (Rossi et al, 2011; Nasirova et al, 2020) while the Cdx2::FLPo labels all cells caudal to the mid-cervical area of the spinal cord (Bourane et al, 2015). We found that in the spinal cord, labeling of MNs was much more robust with 92% of the MNs being labeled (n=1) (Fig.…”

“…The lack of robust MN labeling in the Chat Hoxa4 mice might be due to the dorsal-enriched expression of Hoxa4::Cre that would miss ventrally generated neuronal populations (Yuengert et al, 2015). In contrast, the Chat Cdx2 mice labeled almost all the MNs (92%), but also labeled many other neurons with potentially ascending projections in the spinal cord likely due to transient Chat expression (Nasirova et al, 2020). In both mice, other cholinergic neurons in the spinal cord (SPN, V 0c ) were labeled, although these should not have any ascending projections (Zagoraiou et al, 2009; Deuchars and Lall, 2015).…”

“…The projections to the facial nucleus were not cholinergic (Fig. 5J’’) and are likely evidence of transient cholinergic expression in these neurons that become glutamatergic in adulthood (Nasirova et al, 2020). The projections to folia III are of unknown neurotransmitter status as antibodies for VACHT do not work well in the cerebellar granule cell layer and these TOM + projections in Chat Hoxa4 mice did not express VGLUT2.…”

“…As for other ascending projections in either the Chat Cdx2 or Chat Hoxa4 mouse, the TOM + processes in IRt are not as prominent in the Chat Cdx2 mouse as in the Chat Hoxa4 mouse indicating that these cholinergic IRt processes in the Chat Hoxa4 mice might be from cells labeled between the caudal medulla and mid-cervical spinal cord. This would suggest that the cholinergic innervation of IRt neurons involved in respiration might come from the cervical spinal cord area (Anderson et al, 2016; Nasirova et al, 2020). In the Chat Hoxa4 mice, we found areas of the medulla that were previously reported to contain cholinergic projections in Chat IRES-Cre/+ mice (facial nucleus (7N), spinal trigeminal nucleus (Sp5), ambiguus nucleus (Amb), and paraflocculus (PFl))(Nasirova et al, 2020) and were able to isolate these projections as originating from cell bodies caudal to the lower medulla.…”

“…This would suggest that the cholinergic innervation of IRt neurons involved in respiration might come from the cervical spinal cord area (Anderson et al, 2016; Nasirova et al, 2020). In the Chat Hoxa4 mice, we found areas of the medulla that were previously reported to contain cholinergic projections in Chat IRES-Cre/+ mice (facial nucleus (7N), spinal trigeminal nucleus (Sp5), ambiguus nucleus (Amb), and paraflocculus (PFl))(Nasirova et al, 2020) and were able to isolate these projections as originating from cell bodies caudal to the lower medulla. Furthermore, to get a three-dimensional view of MN axonal trajectories that could be missed in thin cryosections, we cleared the spinal cord and hindbrain of a Chat Hoxa4 mouse.…”