In Vivo Thoracic Dorsal Root Ganglia (DRG) Calcium Imaging and ECG Recording for Studying Peripheral Nerve Stimulation

Summary

This study presents a surgical manipulation to expose the T-DRG in anesthetized mice for in vivo calcium imaging, along with synchronous ECG recordings. This method represents a cutting-edge tool for studying peripheral electric nerve stimulation and thoracic visceral organ inputs, as well as their interactions at the primary sensory level.

Abstract

The dorsal root ganglia (DRG), housing primary sensory neurons, transmit somatosensory and visceral afferent inputs to the dorsal horn of the spinal cord. They play a pivotal role in both physiological and pathological states, including neuropathic and visceral pain. In vivo calcium imaging of DRG enables real-time observation of calcium transients in single units or neuron ensembles. Accumulating evidence indicates that DRG neuronal activities induced by somatic stimulation significantly affect autonomic and visceral functions. While lumbar DRG calcium imaging has been extensively studied, thoracic segment DRG calcium imaging has been less explored due to surgical exposure and stereotaxic fixation challenges. Here, we utilized in vivo calcium imaging at the thoracic1 dorsal root ganglion (T1-DRG) to investigate changes in neuronal activity resulting from somatic stimulations of the forelimb. This approach is crucial for understanding the somato-cardiac reflex triggered by peripheral nerve stimulations (PENS), such as acupuncture. Notably, synchronization of cardiac function was observed and measured by electrocardiogram (ECG), with T-DRG neuronal activities, potentially establishing a novel paradigm for somato-visceral reflex in the thoracic segments.

Introduction

Dorsal root ganglia (DRG) neurons process afferent sensory information from both somatic and visceral receptors. Regulation of cardiac function involves not only primary sensory afferents from viscera but also somatosensory neurons within the same thoracic DRG segment (T-DRG). Recently published research in 'Circulation' has indicated that T-DRG plays a role in cardiac function regulation. Blocking Piezo1/IL-6 in T-DRG inhibited IL-6/STAT3 inflammatory signaling, thereby attenuating ventricular remodeling post-myocardial infarction (MI)¹. Additionally, Cui et al.² found in rats with MI that sympathetic sprouting ....

Protocol

All procedures followed the guidelines of the National Institutes of Health for the care and use of laboratory animals and were approved by the Animal Care and Use Ethics Committee of the Institute of Acupuncture & Moxibustion, China Academy of Chinese Medical Sciences. For in vivo calcium imaging of DRG neurons, adult Pirt-GCaMP6s mice (20-25 g, both sexes) were used. These mice were generated by crossing Pirt-Cre mice with Rosa26-loxP-STOP-loxP-GCaMP6s mice (see Table of Materials). They were housed in the animal facility of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, under controlled conditi....

Results

Following the described protocol, the T1-DRG of transgenic Pirt-GCaMP6s mice were exposed to various somatic stimuli. The aim of this experiment was to observe changes in the number and type of neurons and cardiac function induced by different stimuli.

As depicted in Figure 2A, under baseline conditions, most neurons in the T1-DRG did not exhibit GFP fluorescence. This baseline fluorescence could be influenced by two factors: the GCaMP expression level a.......

Discussion

In this study, a method for calcium imaging of the thoracic segment T1 DRG is described, which has significant value for studying the afferent transmission of cardiopulmonary visceral sensory neurons and somato-visceral communication. Additionally, a general approach is presented for monitoring calcium activity in DRG neurons and changes in cardiac function simultaneously, enabling correlation analysis of neural activity and cardiac responses.

Calcium imaging techniques, using Ca²⁺ .......

Materials

Name	Company	Catalog Number	Comments
Acupuncture Needle	ZhongYanTianHe	0.25/13s
Anesthesia System	Kent Scientific	SomnoSuite
Animal Bio Amp	ADInstruments	NSW
Confocal Microscope	Leica	STELLARIS 8
DC Temperature Controller	FHC	40-90-8D
DC Temperature Controller Heating Pad	FHC	40-90-2-05
Fiji	National Institute of Health	N/A
Fine Forceps	RWD	F11028-13
Fine Ophthalmic Forceps	Jinzhong	JD1060
Gelatin Sponges	Coltene	274-007
Intubation Cannula	Harward Apparatus	73-2737
Isoflurane	RWD	R510
LabChart Professional Software	ADInstruments	Version 8.0
LAS X	Leica	N/A
Pirt-cre mice	Johns Hopkins University	N/A
Retractor	Fine Science Tools	16G212
Rosa-GCaMP6s  mice (AI96)	Jax Laboratory	28866
Spinal Clamp	N/A	N/A	Custom made
Spring Scissors	Jinzhong	YBC040
Stimulator	AMPI	Master-8
Tribromoethanol	Sigma	T48402
Wireless Biological Acquisition System	Kardiotek Biomedical Technologie	KLB-1