Summary

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

Published: August 16, 2024
doi:

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 and sympatho-sensory coupling occurred in T1-5 DRGs and upper limb skin, contributing to cardiac-related referred pain. Somatic stimulation in the referred pain area increased sympathetic discharge and regulated cardiac function. However, due to technical limitations, changes in T-DRG from cardiac and somatic inputs pre- and post-MI or somatic stimulation were observed only after animal sacrifice. Therefore, observing neural activities within T-DRG is crucial for understanding its intricate relationship with visceral function alterations.

In recent years, advancements in sensitive genetically encoded calcium indicators (GECIs)3, along with confocal microscopy and multi-photon imaging technology, have enabled scientists not only to describe neuronal activity and diameter, but also to combine genetic labeling techniques such as Pirt4 and neural tracing to observe specific neurons during imaging5. This integrated approach aids in uncovering deeper scientific principles. However, until recently, methods for studying in vivo calcium imaging of DRG have been predominantly limited to lumbar segments6.

The T-DRG in mice are relatively small and circular, located anterior and medial to the intervertebral foramen, with a total of 13 pairs. Due to the physiological curvature of the thoracic vertebrae, the space between adjacent thoracic vertebral segments, especially T1-5, is very narrow, increasing the difficulty of exposure. Fixation presents another challenge for stable calcium imaging of DRG in a single plane. Each side of the T-DRG is adjacent to two rib surfaces, and each rib surface of the vertebra is attached to the corresponding rib, further complicating the already cramped space. Building upon early calcium imaging methods for lumbar DRG7, this research team has developed an in vivo calcium imaging method for T-DRG using a custom-made spinal clamp. Additionally, numerous studies have demonstrated that peripheral electric nerve stimulation, such as acupuncture, can induce DRG neural activity and regulate visceral function8,9,10. To better understand the relationship between DRG and acupuncture-mediated cardiac function regulation, synchronous recording of cardiac function during DRG calcium imaging has been implemented, offering novel research insights into how somatic stimulation-induced neuronal activity in T-DRG influences visceral function regulation.

This unique research details the exposure and fixation of mouse T-DRG, ensuring stable calcium imaging alongside cardiac function recording. This provides a cutting-edge scientific tool for studying the peripheral mechanisms of thoracic visceral functional changes and further exploring visceral-somatic afferent inputs.

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 w…

Representative 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+

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

This study was funded by the National Natural Science Foundation of China (No. 82174518, 82074561, 82105029).

Materials

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

Referencias

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Li, X., Liu, Y., Liu, K., Du, L., Lv, T., Zhu, B., Gao, X. In Vivo Thoracic Dorsal Root Ganglia (DRG) Calcium Imaging and ECG Recording for Studying Peripheral Nerve Stimulation. J. Vis. Exp. (210), e67283, doi:10.3791/67283 (2024).

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