Patch-Seq of seven 5-HT neuronal subpopulations in zebrafish whole brain
The goals of this study is to compare transcriptome profiles (RNA-seq) of seven 5-HT neuronal subpopulations in zebrafish whole brain. Tg(tph2:GFP) fish at 7 dpf were fixed in a recording chamber containing extracellular solution. The somata of 5-HT neurons from seven subpopulations (PT, SR-R, SR-M, SR-C, IR-R, IR-C, MO) were visualized under a fluorescence microscopy, then targeted and collected separately using glass electrode. For each sample, 10-12 neurons were collected and transferred to lysis buffer solution. Total RNA was extracted for construction of cDNA libraries, involving fragmentation, PCR enrichment, N7 index linking, and purification. A total of 71 QuantSeq libraries (N=9-12 samples for each subpopulation) were prepared using the Lexogen's QuantSeq 3' mRNA-Seq Library Prep Kit for Illumina according to the manufacturer's instructions. Sequencing was carried out using Illumina NovaSeq 6000 with 150 bp paired-end reads. Gene expression level analysis was performed using R (Version 4.3.1) package Seurat (Version 5.0.2), and the mean gene expression matrix of all samples was obtained after processing by NormalizeData function. For marker gene analysis of each subpopulation, FindAllMarkers function in Seurat was used, with parameters set as min.pct=0.1 and logfc.threshold=0.25. Differential expression analysis revealed that each subpopulation expressed a set of featured genes, including specific markers for neurotransmitters, axon projection, neuron development, synapse, and ion channels, suggesting that seven 5-HT neuronal subpopulations display distinct genetic profiles.  We demonstrated that seven 5-HT neuronal subpopulations are molecularly discriminative by expressing distinct molecular markers. Overall design: To compare transcriptome profiles (RNA-seq) of seven 5-HT neuronal subpopulations in zebrafish whole brain, Tg(tph2:GFP) fish at 7 dpf were fixed in a recording chamber containing extracellular solution, and neurons were collected separately with glass pipette. A total of 71 QuantSeq libraries (N=9-12 samples for each subpopulation) were prepared using the Lexogen's QuantSeq 3' mRNA-Seq Library Prep Kit for Illumina according to the manufacturer's instructions.