Inter‐ and intra‐specific differences in muscarinic acetylcholine receptor expression in the neural pathways for vocal learning in songbirds

Abstract

Acetylcholine receptors (AChRs) abound in the central nervous system of vertebrates. Muscarinic AChRs (mAChRs), a functional subclass of AChRs, mediate neuronal responses via intracellular signal transduction. They also play roles in sensorimotor coordination and motor skill learning by enhancing cortical plasticity. Learned birdsong is a complex motor skill acquired through sensorimotor coordination during a critical period. However, the functions of AChRs in the neural circuits for vocal learning and production remain largely unexplored. Here, we report the unique expression of mAChRs subunits (chrm2-5) in the song nuclei of zebra finches. The expression of excitatory subunits (chrm3 and chrm5) was downregulated in the song nuclei compared with the surrounding brain regions. In contrast, the expression of inhibitory mAChRs (chrm2 and chrm4) was upregulated in the premotor song nucleus HVC relative to the surrounding nidopallium. Chrm4 showed developmentally different expression in HVC during the critical period. Compared with chrm4, individual differences in chrm2 expression emerged in HVC early in the critical period. These individual differences in chrm2 expression persisted despite testosterone administration or auditory deprivation, which altered the timing of song stabilization. Instead, the variability in chrm2 expression in HVC correlated with parental genetics. In addition, chrm2 expression in HVC exhibited species differences and individual variability among songbird species. These results suggest that mAChRs play an underappreciated role in the development of species and individual differences in song patterns by modulating the excitability of HVC neurons, providing a potential insight into the gating of auditory responses in HVC neurons.

Nicotinic acetylcholine receptors in a songbird brain

“…When the initial cloning failed, we used putative mRNA sequences predicted by the NCBI for primer design. Partial DNA fragment of each targeted nAChR subunit was amplified by PCR (Ex Taq polymerase, Takara Bio, Shiga, Japan) using the synthesized cDNA template and cloned into pGEM‐T easy plasmids (Promega) based on a previous method (Asogwa et al., 2018; Wada et al., 2004). The cloned cDNA fragments were verified by DNA sequencing.…”

“…The brain tissues were sectioned at 12 μm thickness on the sagittal plane and mounted on silane‐coated slides. The radioisotope in situ hybridization and quantification of expressed mRNA levels were conducted according to previous studies (Asogwa et al., 2018). Using specific T7 or SP6 RNA polymerases (Roche, Basel, Switzerland) to the inserted sense‐/antisense direction of nAChR subunits, 35 S‐labeled riboprobes were synthesized from the T7 or SP6 promoter sites of pGEM‐T.…”

“…The radioisotope in situ hybridization and quantification of expressed mRNA levels were conducted according to previous studies (Asogwa et al, 2018). Using specific T7 or SP6 RNA polymerases (Roche, Basel, Switzerland) to the inserted sense-/antisense direction of nAChR subunits, 35 S-labeled riboprobes were synthesized from the T7 or SP6 promoter sites of pGEM-T. Fresh frozen brain sections were fixed in 3% paraformaldehyde/1× phosphate-buffered saline (PBS, pH 7.0), washed three times in 1× PBS, acetylated, washed three times in 2× SSPE, dehydrated in ascending ethanol concentrations (50%, 70%, 90%, and 100%), and then air-dried.…”

“…These findings suggest the expression of AChRs in the song nuclei that mediates cholinergic functions during song learning and production. Our previous investigation revealed developmental regulation and inter-and intraspecific differences in the expression of muscarinic AChR subunits (mAChRs) in the song pathways (Asogwa et al, 2018). In contrast to mAChRs, only a limited number of nAChR subunits have been reported to be expressed in the songbird brain (Lovell et al, 2008(Lovell et al, , 2018Watson et al, 1988).…”

Transcriptional regulatory divergence underpinning species-specific learned vocalization in songbirds

“…The importance of these song nuclei in determining species-specific song traits suggests an underlying causative role of species differences in the structure and activity of these regions. Consistent with this, a variety of genes, including transcription factors and neuromodulator receptors, are differentially expressed in these song nuclei between species, even in a laboratory-controlled environment [25–27]. However, a key gap in our knowledge is how species-specific patterns of gene expression in these regions arise via regulatory differences between species.…”