Nicotinic acetylcholine receptors in a songbird brain

Abstract

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission and cell signaling, which contribute to learning, memory, and the execution of motor skills. Birdsong is a complex learned motor skill in songbirds. Although the existence of 15 nAChR subunits has been predicted in the avian genome, their expression patterns and potential contributions to song learning and production have not been comprehensively investigated. Here, we cloned all the 15 nAChR subunits (ChrnA1-10, B2-4, D, and G) from the zebra finch brain and investigated the mRNA expression patterns in the neural pathways responsible for the learning and production of birdsong during a critical period of song learning. Although there were no detectable hybridization signals for ChrnA1, A6, A9, and A10, the other 11 nAChR subunits were uniquely expressed in one or more major subdivisions in the song nuclei of the songbird brain. Of these 11 subunits, ChrnA3-5, A7, and B2 were differentially regulated in the song nuclei compared with the surrounding anatomically related regions. ChrnA5 was upregulated during the critical period of song learning in the lateral magnocellular nucleus of the anterior nidopallium. Furthermore, single-cell RNA sequencing revealed ChrnA7 and B2 to be the major subunits expressed in neurons of the vocal motor nuclei HVC and robust nucleus of the arcopallium, indicating the potential existence of ChrnA7-homomeric and ChrnB2-heteromeric nAChRs in limited cell populations. These results suggest that relatively limited types of nAChR subunits provide functional contributions to song learning and production in songbirds.

Genetically predisposed motor bias drives individuality in vocal learning

“…To this end, we conducted single-nuclei RNA sequencing (snRNA-seq) from the basal ganglia nucleus Area X and the cortical vocal output nucleus RA of F1 hybrids (n = 6 birds for each) at the subsong stage. In agreement with previous reports in zebra finches [53][54][55] , we identified the genetic signatures that matched multiple cell types reported for the song nuclei, such as Area X medium spiny neurons (MSNs), Area X pallidal-like neurons, RA glutamatergic projecting neurons, GABAergic neurons, astrocytes, microglia, oligodendrocytes, and oligo precursor cells (OPC) (Fig. 5A).…”

“…Subclusters in glutamatergic neurons in RA were identified based on a previous report 53,54 and in situ hybridization database, ZEBrA 87 ; SRD5A2 and SCUBE1 for the glutamatergic neurons in RA projecting to the nucleus of cranial nerve XII; CACNA1H, MGAT4C, and ADYAP1 for RA surrounding arcopallial neurons. Unknown clusters with no specific marker gene and several small clusters (less than 60 cells in each) were filtered out.…”

An underlying motor bias shapes individuality during vocal learning

“…In agreement with previous reports in ZFs [60][61][62], we identi ed marker genes representing speci c cell types reported for the song nuclei, such as Area X medium spiny neurons (MSNs), Area X pallidal-like neurons, RA glutamatergic projecting neurons, GABAergic neurons, astrocytes, microglia, oligodendrocytes, oligo precursor cells, and RA surrouding arcopallium glutamatergic neurons (Fig. 5A).…”

“…Subclusters in MSNs and pallidal-like neurons were identi ed with FOXP2, FOXP4, MTNR1A, and PENK based on a previous report [62]. Subclusters in glutamatergic neurons in RA were identi ed based on a previous report [60,61] and in situ hybridization database, ZEBrA [96]; SRD5A2 and SCUBE1 for the glutamatergic neurons in RA projecting to the nucleus of cranial nerve XII; CACNA1H, MGAT4C, and ADYAP1 for RA surrounding arcopallial neurons. Unknown clusters with no speci c marker gene and several small clusters (less than 60 cells in each) were ltered out.…”

Distinct patterns of gene expression in the medial preoptic area are related to gregarious singing behavior in European starlings (Sturnus vulgaris)

“…We also found multiple acetylcholine genes upregulated in high-singing starlings, including choline O-acetyltransferase (CHAT), a choline transporter (SLC5A7), and a muscarinic cholinergic receptor (CHRM4). Multiple immunolabeling studies have shown high concentrations of cholinergic proteins in the song control system in the zebra finch brain [57][58][59], and cholinergic stimulation of the HVC via carbachol modulates zebra finch song [60]. However, the literature regarding its role in brain regions involved in social behavior is limited.…”