Utilizing transgenic rats with eGFP‐tagged serotonergic (5‐HT) neurons to assess differential gene expression associated with age‐dependent changes in cellular CO 2 /H + chemosensitivity (1092.7)

The ventilatory response to hypercapnia in rodents increases throughout postnatal development, which parallels the increases in cellular CO2 sensitivity of serotonergic (5‐HT) neurons in primary cultures. Herein we begin to test the hypothesis that specific changes in gene expression in 5‐HT neurons during development contribute to the changes in cellular and/or organismal CO2/pH sensitivity. To begin to test this, acutely dissociated medullary raphe neurons obtained from adult (P56) transgenic rats (n=2) that express enhanced green fluorescent protein (eGFP) in cells expressing Pet‐1 (ePet‐1:eGFP rats) were sorted into pools (n= 5000 cells) using Fluorescent‐Assisted Cell Sorting (FACS). Total RNA was then extracted, and RNA quality and abundance measured before quantifying transcript abundance (Δ ct) with qRT‐PCR of 5‐HT neuron‐specific (slc6a4 (SERT); slc18a2 ( VMAT2); Tph2, and Trh) or non‐5‐HT specific raphe (Gad2 and Hprt) genes. We found high expression of 5‐HT neuron‐specific genes in the eGFP+ cell pools, including TRH (∆ct 6.94), TPH (∆ct 6.95), SERT (∆ct 3.18), and VMAT2 (∆ct 2.27), with little or no detectable expression of these genes in eGFP‐ cell pools. Expression levels of Gad2 and the housekeeping gene Hprt were comparable in both eGFP+ and eGFP‐ cells. These data indicate effective sorting of 5‐HT and non‐5‐HT raphe neurons in adult transgenic rats, and allow for further studies utilizing RNA sequencing to identify, measure and compare age‐related differences in 5‐HT neuron and non‐5‐HT neuron raphe transcriptomes. Grant Funding Source : NIH HL097033