Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons

Abstract

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a “cholinergic locus”, and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

The Mechanisms Mediated by α7 Acetylcholine Nicotinic Receptors May Contribute to Peripheral Nerve Regeneration

“…The present data reinforce previous results obtained by our research group, reaffirming that acetylcholine, probably released in extra-synaptic regions of motor and sensory axons [38], may significantly contribute to Schwann cell development and plasticity through the activation of different cholinergic receptor types. In this study, we assessed the expression of the α7 nAChR in Schwann cells after peripheral nerve injury and proved that the selective activation of this receptor subtype may be relevant to the establishment of the microenvironment favorable to improving nerve regeneration.…”

“…Several studies were conducted in the peripheral nervous system where the cholinergic receptors have been identified in SCs, revealing that glial cells are able to respond to cholinergic stimuli [11,12,37]. Interestingly, ACh is present in sciatic nerves and can be potentially released in extrasynaptic regions both by motor and sensory axons [38], suggesting its involvement in axon-glia crosstalk. However, when isolated by the axons and cultured in vitro, SCs are able to synthesize and release ACh that may be useful to contribute to the balance between the proliferative and differentiative phase [39].…”

The Efficiency of Direct Maturation: the Comparison of Two hiPSC Differentiation Approaches into Motor Neurons

“…In contrast, ChAT gene expression levels were decreased in both approaches, which may be due to posttranscriptional regulation in process of maturation [55]. These findings were also observed in rats by Corsetti et al [53]. As ChAT is not expressed in the upper MNs, this may suggest successful NPC differentiation into mature and functional lower MNs [52,56].…”

“…ChAT is crucial for the synthesis of the neurotransmitter acetylcholine and is selectively expressed in MNs of the spinal cord [3]. Several studies found that ChAT-positive neurons possess electrophysiological activity [27,46,[52][53][54]. On protein level, ChAT was detected in both approaches by flow cytometry and immunohistochemistry.…”