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.
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.
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.
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 conditions (room temperature: 22-24 °C, humidity: 50%-60%, 12-h light-dark cycle), with ad libitum access to food and water. The animals underwent one week of acclimatization before experimentation. During experiments, animals were anesthetized, maintained at a warm temperature, and monitored for pain response to ensure adequate anesthesia depth. After the experiments, animals were euthanized under deep anesthesia (following institutionally approved protocols). The details of the reagents and the equipment used are listed in the Table of Materials.
1. Preoperative preparation
2. Tracheotomy
3. Exposure of thoracic vertebrae
4. Exposure of T1-DRG
5. Thoracic vertebrae fixation and ECG detection
6. Hardware and software setup for imaging
7. Somatic stimuli and imaging
8. Data analysis and processing
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 and potential damage to the DRG during surgery. Somatic stimulation resulted in a rapid and transient increase in GCaMP fluorescence, along with an increase in the number and intensity of GFP, as illustrated in Figure 2B. Similar changes were observed when PENS-PC6 was applied, as shown in Figure 2C. Figure 2D highlights selected and numbered cells circled using imaging software in Figure 2C.
As mentioned earlier, changes in fluorescence intensity exceeding 130% of the F0 threshold level are considered positive responses. The heat map and line chart in Figure 2E,F display the responses of all neurons circled in the imaging software to PENS-PC6 stimulation. Figure 2G presents a histogram of the different diameters of neurons responsive to PENS-PC6, while Figure 2H illustrates a schematic diagram of the increase in heart rate with PENS-PC6.
Figure 1: Surgical procedure of the T1-DRG. (A) Intubation of trachea in mouse. (B) Thoracic vertebrae after removing surrounding tissues. (C) Exposed T1-DRG. (D) Customized-stage, this stage can be tilted at different angles, and the spinal clamp with universal beads is installed. (E) Local fixation of spinal clamp. (F) ECG monitor connection. (G) Calcium imaging using confocal microscopy. (H) Experimental setup for calcium imaging of T1-DRG. Please click here to view a larger version of this figure.
Figure 2: Representative images and analysis of in vivo calcium imaging of the T1-DRG neurons. Representative images in (A–C) show T1-DRG neurons at Baseline, Brush, and PENS stimulation. (D) The cells marked and numbered within a single T1-DRG after tracing with imaging software. Scale bars: 100 µm. (E,F) Heat map and line graph depicting the changes in fluorescence intensity of traced cells under PENS-PC6 stimulation. (G) Description of the number of responsive cells to different sizes of PENS-PC6 stimulation. Error bars represent mean ± SEM (N = 1). (H) The increase in heart rate during PENS stimulation of PC6. Please click here to view a larger version of this figure.
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+ sensitive dyes or genetically encoded Ca2+ indicators to image intracellular Ca2+ as an indirect measure of action potential firing, enable real-time observation of individual neurons or neuronal clusters' responses to various stimuli. Currently, most calcium imaging studies on DRG have been limited to the lumbar segments12. Compared to the lumbar DRG, the thoracic DRG are smaller, situated close to the ribs and thoracic vertebrae, and poses an increased risk of pneumothorax during surgical procedures. Therefore, two major issues must be overcome to conduct calcium imaging in T-DRG.
The first issue is the exposure of T-DRG. Using T1-DRG as an example, T1-DRG is located below the vertebral arch of T1. When bone is removed from the lamina arcus vertebrae and articular process, it is prone to bleeding and requires careful handling. As calcium imaging requires tracking changes in calcium ion activity in neurons before and after stimulation, it is necessary to ensure that the DRG are stably fixed in place. However, due to the physiological curvature of the cervical and thoracic vertebrae, the space between the T1-T3 vertebrae is narrow, making the fixation of the thoracic DRG the second critical issue to address.
The spinal clamp intended for the lumbar segments was modified by grinding the front end of the alligator clips with sandpaper to a thickness of 1 mm for fixation7. Meanwhile, the muscles and connective tissues around the fixed segments were cleanly removed while avoiding damage to the blood vessels. The head-end spinal clamp was fixed to the articular process of C6, and the tail-end spinal clamp was fixed to the articular process of T3.
The implementation of calcium imaging has led to numerous advancements in understanding the neuronal mechanisms of DRG related to visceral diseases13,14. Recently, Sun et al. demonstrated that Piezo1 or its downstream effector IL-6 in T-DRG neurons mediated a neurogenic inflammatory cascade after MI using cellular and molecular approaches. However, the functional relationship between DRG neurons and cardiac functions remains unknown due to technical difficulties. Synchronized recordings of cardiac function and activated T-DRG neurons were carried out, providing a more intuitive understanding of the correlation between neuronal activity and alterations in cardiac function. During these experiments, changes in heart rate in mice were observed using PENS at PC6, with concurrent calcium imaging being performed. The results showed that PENS-PC6 can increase calcium activity in T1-DRG and elevate heart rate. This approach not only opens the way for in vivo studies of sensory ganglia-related cardiac disorders but also provides an important tool to investigate the involvement of primary sensory neurons in the beneficial effects of PENS on cardiac function.
This research paradigm can also be combined with other research methods, such as optogenetics15,16and pharmacological interventions17. For example, Takuya Okada et al.14,15 generated holographic patterns of optogenetic stimulation using a spatial light modulator (SLM) and subsequently integrated this holographic stimulation with two-photon imaging. They conducted experiments to stimulate individual neurons and combined in vivo calcium imaging to observe the responses of surrounding neurons, aiming to investigate the functional connectivity between cells. Since neuronal coupling represents an important form of neuronal plasticity in the DRG, the combination of holographic stimulation and calcium imaging may elucidate interactions between neuron subtypes within sensory ganglia. DRG neuron-satellite glial cell (SGC) interactions may affect nociceptive transmission18. P2-purinergic receptors (P2Rs) are key elements in the two-way interactions between DRG neurons and SGC19. Chen et al.17 performed in vivo calcium imaging by utilizing α,β-MeATP (a selective P2R agonist) in the DRG and observed that α,β-MeATP elicited robust activation of small neurons, presumably nociceptors. They combined calcium imaging with pharmacological techniques in the DRG to investigate the role of P2R signaling within the DRG, which plays a significant role in the excitability and pain transmission of nociceptive neurons. Hence, this research methodology, which incorporates optogenetics and pharmacological approaches, will serve as a valuable tool for studying visceral sensory neurons transmitting cardiorespiratory inputs as well as somatic-visceral communications.
A technical limitation of this study is the inability to conduct chronic DRG calcium imaging in mice during their awake behavioral states. Anesthesia has the potential to diminish proprioception and visceral sensation signals, leaving the activity of T-DRG neurons in natural sensation contexts to be further investigated. As Chen et al. have developed an intervertebral fusion method for imaging lumbar DRG at cellular and subcellular resolution over weeks in awake mice, future studies should consider the approach of longitudinally monitoring the activity of neurons in T-DRG20.
The authors have nothing to disclose.
This study was funded by the National Natural Science Foundation of China (No. 82174518, 82074561, 82105029).
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 |
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