Mechanism of transcranial focused ultrasound regulating the neuronal activity of the retrosplenial cortex

Abstract

Non-invasive neuromodulation is crucial in fundamental research and clinical treatment. Among those non-invasive neuromodulations, transcranial focused ultrasound (tFUS) can penetrate the skull to focus energy on a specific brain region, temporarily affecting brain function. Compared with traditional neuromodulation methods, such as transcranial direct current stimulation and transcranial magnetic stimulation, tFUS has become a novel method for regulating neuronal activity by means of its non-invasion, reversibility, and accuracy. However, how tFUS regulates neuronal activity and cellular properties in response to tFUS remains unknown.

To investigate how tFUS regulates neuronal activity, I used behavioural tests, real-time fluorescence quantitative polymerase chain reaction, immunofluorescent staining, chemical genetics, and multi-channel in vivo recording to explore whether tFUS affected neuronal activity in the pain-related brain region, retrosplenial cortex (RSC). Moreover, tFUS significantly increased paw withdrawal threshold (PWT) and prolonged thermal withdrawal latency (TWL) both in naïve and neuropathic mice. tFUS also significantly increased mRNA and protein expression levels of early growth factor response 1 (Egr1). These tFUS-activated Egr1 cells were mainly neurons. Through multi-channel in vivo recording, it was found that the spike rate of pyramidal neurons and interneurons decreased remarkably under tFUS. A greater proportion of spike rate in pyramidal neurons and interneurons showed a decrease rather than an Subsequently, Egr1 positive cells activated by tFUS were specifically inhibited by the targeted recombination in the active population system (TRAP), and then the effect of tFUS on PWT and TWL was inhibited. These results suggested that Egr1 was an essential marker of tFUS responsive neurons in RSC. Subsequently, I performed transcriptome sequencing based on Egr1-GFP cells activated by tFUS. Transient receptor potential cation channel subfamily C member 4 (Trpc4) was selected according to the transcriptome sequencing data. Combined with calcium imaging, patch-clamp recording, and short hairpin ribonucleic acid (shRNA) interference, it was found that tFUS could activate Trpc4, which could be blocked by ML204, an inhibitor of Trpc4. In vivo, with specific inhibition of Trpc4 expression in RSC, the proportion of decreased neuronal activity induced by tFUS was significantly down-regulated. At the same time, the regulation of PWT and TWL by tFUS was also inhibited. The above results showed that Trpc4 was an important factor in regulating Egr1 response to tFUS, thus regulating RSC and further regulating the somatosensory threshold of mice.

To further explore the characteristics of tFUS responsive cells, I used single-cell RNA sequencing (scRNA-seq) to map the single-cell transcriptome expression of RSC. It was also identified tFUS-induced cell type-dependent transcriptome and functional changes in RSC. Subsequently, Egr1 was used as a marker to identify tFUS-activated cell types and populations. Then, it was found that Egr1 was highly expressed in neurons, endothelial cells (EC), and vascular smooth muscle cells (vSMC). These cells acted as tFUS-sensitive cells. Further analysis of cellular communication pathways between tFUS-sensitive cells and other cells revealed multiple signal pathways, which suggested that tFUS- sensitive cells received or transmitted information to other cell types, causing changes in the transcriptome.

In conclusion, this study found that Trpc4 was a key factor regulating Egr1 response to tFUS. In addition, it provided transcriptome expression atlas and cellular communication pathways of tFUS-sensitive cells by scRNA-seq. These results provided a basis for the cellular and molecular mechanisms of tFUS neuromodulation, which supported ultrasonic neuromodulation in basic neuroscience research and clinical applications.