GSE134203
Genomic decoding of neuronal depolarization by stimulus-specific NPAS4 heterodimers, RNA sequencing
Cells regulate gene expression in response to salient external stimuli. In neurons, depolarization leads to the expression of inducible transcription factors (ITFs) that direct subsequent gene regulation. Depolarization encodes both a neuron's action potential (AP) output and synaptic inputs, via excitatory postsynaptic potentials (EPSPs). However, it is unclear if distinct types of electrical activity can be transformed by an ITF into distinct modes of genomic regulation. Here, we show that APs and EPSPs in mouse hippocampal neurons trigger two spatially segregated and molecularly distinct induction mechanisms that lead to the expression of the ITF NPAS4. These two pathways culminate with the formation of stimulus-specific NPAS4 heterodimers that exhibit distinct DNA binding patterns. Thus, NPAS4 differentially communicates increases in a neuron's spiking output and synaptic inputs to the nucleus, enabling gene regulation to be tailored to the type of depolarizing activity along the somato-dendritic axis of a neuron. Overall design: To augment neuronal activity, cells were incubated in the indicated drugs reconstituted in dimethyl sulfoxide (DMSO) or water, and diluted in maintenance media for 2 h (ChIP-seq and RNA-seq experiments). To silence neuronal activity for ChIP-seq and RNA-seq experiments, twenty seven DIV rat hippocampal neurons were incubated in (µM): 1 tetrodotoxin citrate (TTX), 10 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), and 10 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX) reconstituted in water and diluted in maintenance media for 24 h prior to harvesting on day of experiment. The next day, media was then replaced with the same (silenced, – Stim) or media with 50 µM PTX (stimulated, + Stim), media with PTX plus Nimodipine (Nim; 20 µM reconstituted in DMSO), or media with PTX plus CPP (10 µM, reconstituted in water), and incubated for 2 h at 37°C. Neurons were continuously maintained at 37°C and 5% CO2 until harvesting or fixation. Cells were lysed in TRI-reagent and RNA was extracted using a Direct-zol kit (Zymo Research Cat#R2051) with on-column DNase treatment, following the manufacturer's instructions. Strand-specific total RNA-seq libraries from ribosomal RNA-depleted RNA were prepared using the TruSeq kit Stranded Total RNA Library kit (Illumina) according to the manufacturer-supplied protocol. Library construction was performed by the IGM Genomics Center, University of California, San Diego, La Jolla, CA. Libraries were sequenced 76bp single-end on an Illumina NextSeq 500 instrument.