Patch-Seq of synchronized and non-synchronized neurons in zebrafish dorsal pallium in response to CAS treatment
The goals of this study is to compare transcriptome profiles (RNA-seq) of synchronized and non-synchronized neurons in zebrafish dorsal pallium in response to CAS treatment.Tg(HuC:H2B-GCaMP6f) fish of 4-5 weeks old were were fixed in a recording chamber containing extracellular fluid, and neurons with synchronized or non-synchronized calcium activity after CAS treatment were identified by two-photon imaging and collected separately with glass pipette. Single cells were transferred to the lysis buffer solution. Total RNA was extracted using TRIzol (Invitrogen) and then purified on RNeasy columns (Qiagen). Total RNA quality was assessed on a bioanalyzer (Thermofisher). RNA-Seq libraries (N=10) for each group were prepared using the Illumina TruSeq Strand mRNA Prep Kit according to the manufacturer's instructions. RNA-Seq libraries were sequenced using Illumina NextSeq 500, generating 75 bp paired-end reads for each sample. RNA-Seq reads untrimmed. Differential expression analysis was performed using the CPM (counts per million) function in the Bioconductor package edgeR (v 3.14.0). Low expression genes were excluded to make a simple correction for gene counts. Genes with P-value (instead of adjusted P-value) < 0.05 were assigned as differentially expressed. We identified differentially expressed genes (DEGs) between synchronized and non-synchronized neurons in zebrafish dorsal pallium in response to CAS treatment. Synchronized neurons expressed much higher level of glutamate transporter genes (slc17a7a, slc17a6a), while non-synchronized neurons showed significantly higher expression of gad1b, suggesting that synchronized neurons are primarily glutamatergic, while non-synchronized neurons are mainly GABAergic. Overall design: To distinguish neurons with synchronized or non-synchronized calcium activity after CAS treatment, Tg(HuC:H2B-GCaMP6f) fish of 4-5 weeks old were fixed in a recording chamber containing extracellular fluid and subjected to two-photon imaging, and neurons were collected separately with glass pipette. RNA-Seq libraries (N=10) for each group were prepared using the Illumina TruSeq Strand mRNA Prep Kit according to the manufacturer's instructions.