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.

Keywords: acetylcholine; choline acetyltransferase; cholinergic locus; sensory neurons; splice variants; vesicular acetylcholine transporter.

Figure 1

Schematic representation of the VAChT gene and different rat splice variants. P indicates the different putative promoter sites. The arrows indicate the sites where the primers were designed.

Figure 2

Schematic representation of the cholinergic gene locus. The VAChT gene is expressed in the first intron of ChAT gene. P indicates the various putative promoter sites regulating ChAT splice variant expression. Some of these promoters are in common with the VAChT gene. The arrows indicate the sites where the primers were designed.

Figure 3

(A) Representative RT-PCR analysis of V1 VAChT splice variants at embryo E18, postnatal 2 and 15, and in adult DRG. HPRT was used as a housekeeping gene. The graph below reports the densitometric analysis of the bands normalized with respect to the HPRT housekeeping gene. The data are the mean of three independent experiments (*** p < 0.001); (B,C) Analysis by qRT-PCR of the R1 and R2 VAChT splice variants. The samples were compared with the respective E18 DRG sample. The data are the mean of three independent experiments performed in triplicate. (** p < 0.01; *** p < 0.001).

Figure 4

(A) Representative RT-PCR of the ChAT splice variants (R1, R2). HPRT was used as a housekeeping gene. The graphs indicate that the densitometric analysis of the bands normalized with respect to the HPRT housekeeping gene. The data are the mean of three independent experiments (* p < 0.001); (B–D) Analysis by qRT- PCR of the M, N1, and N2 ChAT splice variants, respectively. The data are the mean of three independent experiments performed in triplicate. All the samples were compared with the respective E18 DRG sample (** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 5

(A) ChAT activity was measured in DRG extracts at the E18, DRG 2dd; DRG 15dd; Adult DRGs. ChAT activity was reported as the total activity (pmol ACh tot/h) or specific activity (pmol ACh synthesized/mg of protein/h). All the samples were compared with the respective E18 DRG sample. Data are the mean ± SEM of at least 3 independent experiments performed in duplicate. (B) Representative Western blot analysis of the ChAT protein at E18, DRG 2dd; DRG 15dd; Adult DRGs. The graph (C) shows the densitometric analysis of the bands obtained after normalization with β-actin used as a protein reference. The data are the mean ± SEM of four independent experiments. All the samples were compared with the respective E18 DRG sample. (* p < 0.05; ** p < 0.01; **** p < 0.0001).