# Transcriptomic analysis of rat brain response to alternating current electrical stimulation: unveiling insights via single‐nucleus RNA sequencing

## TL;DR

This study uses single-nucleus RNA sequencing to show that electrical brain stimulation increases neurons and affects Rgs9 gene expression in rat brains.

## Contribution

The first single-nucleus RNA sequencing profiles of rat brain regions under intracranial alternating current stimulation.

## Key findings

- Unilateral iACS at 40 Hz increases neuron numbers and downregulates Rgs9 gene expression.
- Less than 0.1% of host gene expression in neurons is significantly altered by iACS.
- Rgs9 downregulation suggests enhanced neuronal sensitization to dopaminergic drugs.

## Abstract

Electrical brain stimulation (EBS) has gained popularity for laboratory and clinical applications. However, comprehensive characterization of cellular diversity and gene expression changes induced by EBS remains limited, particularly with respect to specific brain regions and stimulation sites. Here, we presented the initial single‐nucleus RNA sequencing profiles of rat cortex, hippocampus, and thalamus subjected to intracranial alternating current stimulation (iACS) at 40 Hz. The results demonstrated an increased number of neurons in all three regions in response to iACS. Interestingly, less than 0.1% of host gene expression in neurons was significantly altered by iACS. In addition, we identified Rgs9, a known negative regulator of dopaminergic signaling, as a unique downregulated gene in neurons. Unilateral iACS produced a more focused local effect in attenuating the proportion of Rgs9+ neurons in the ipsilateral compared to bilateral iACS treatment. The results suggested that unilateral iACS at 40 Hz was an efficient approach to increase the number of neurons and downregulate Rgs9 gene expression without affecting other cell types or genes in the brain. Our study presented the direct evidence that EBS could boost cerebral neurogenesis and enhance neuronal sensitization to dopaminergic drugs and agonists, through its downregulatory effect on Rgs9 in neurons.

We provide here the first single‐nucleus RNA sequencing profiles of the rat cortex, hippocampus, and thalamus under the intracranial alternating current stimulation (iACS). The findings would support as direct evidence that transcranial, intracranial, or deep brain iACS may increase neuronal number and sensitization to dopaminergic or dopaminergic agonists, improve analgesic effects, and prolong drug tolerance through its unique down‐regulatory effect on Rgs9 in neurons.