In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

Summary

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

Abstract

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

Introduction

Acupuncture, an integral part of Traditional Chinese medicine, has gained global recognition primarily for its effectiveness in pain management, including the alleviation of chronic visceral pain¹. Over the past decades, our knowledge of the central nervous mechanisms underlying acupuncture analgesia has undergone considerable growth^1,2. However, little attention has been paid to exploring the functional roles of dorsal root ganglia (DRG) neurons in inducing the analgesic effect of acupuncture in visceral nociception. Visceral nociception and acupuncture analgesic studies are potentiall....

Protocol

This animal protocol was approved by the Animal Care and Use Ethics Committees of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, and complied with the National Institutes of Health Guide for the Care and Use of Experimental Animals to ensure minimal animal use and discomfort. Pirt-cre mice were kindly donated by Dr. Xinzhong Dong from Johns Hopkins University (Baltimore, MD). Rosa26-loxP-STOP-loxP-GCaMP6s mice were obtained from a commercial source (see Table of Materials.......

Discussion

It is believed that acupuncture analgesia is modulated by integrative processes in the DRG, involving an interplay between afferent impulses from pain regions and impulses from acupoints. Here, we describe an elaborate procedure for L6 DRG imaging. The advantages of imaging are manifold, including remarkable spatial resolution, the possibility for high-efficiency imaging of large areas of neurons simultaneously, and the ability to monitor specific cellular subtypes and subcellular domains using gene-targeting probes

Materials

Name	Company	Catalog Number	Comments
Anesthesia System	Kent Scientific	SomnoSuite
Confocal Microscope	Leica	STELLARIS 8
DC Temperature Controller	FHC	40-90-8D
DC Temperature Controller Heating Pad	FHC	40-90-2-05
Fiji software	National Institute of Health	N/A
Fine Scissors	Fine Science Tools	14558-11
Friedman-Pearson Rongeurs	Fine Science Tools	16220-14
Gelatin Sponges	Coltene	274-007
Graefe Forceps	Roboz	RS-5137
Han’s Acupoint Nerve Stimulator	Jason Scientific	HANS-200A
Intubation Cannula	Harward Apparatus	73-2737
Isoflurane	RWD	R510
LAS X	Leica	N/A
Pirt-cre mice	Johns Hopkins University	N/A
Rosa-GCaMP6s  mice (AI96)	Jax Laboratory	28866
Spinal Adaptor	N/A	N/A	Custom made
Spring Scissors	Fine Science Tools	15023-10
Tribromoethanol	Sigma	T48402
Vannas Spring Scissors	Fine Science Tools	15019-10