Name: In Vivo Thoracic Dorsal Root Ganglia (DRG) Calcium Imaging and ECG Recording for Studying Peripheral Nerve Stimulation (Video) | JoVE Journal
Uploaded: 2024-08-16T15:00:00.000+00:00
Duration: 6 min 34 s
Description: Learn in vivo calcium imaging of thoracic dorsal root ganglia with synchronized ECG recording in anesthetized mice. This method reveals how acupuncture at PC6 activates DRG neurons and modulates cardiac rhythms.

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5 minutes

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

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 &#38; 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 &#176;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 (5% isoflurane). The details of the reagents and the equipment used are listed in the Table of Materials.
1. Preoperative preparation
<ol>
	<li>Verify the proper functioning of the anesthesia ventilation device, imaging system, and electrocardiogram monitor and activate them sequentially.</li>
	<li>Administer an intraperitoneal injection of 1.25% tribromoethanol (0.2 mL per 10 g of body weight) to anesthetize the mouse). Assess the depth of anesthesia by firmly pinching the mouse&#8217;s hind foot to ensure there is no response.
	<ol>
		<li>If signs of pain occur during the surgical procedure, administer an additional intraperitoneal injection of 0.2 mL of tribromoethanol solution, or use an anesthesia machine to deliver 0.5%-1% isoflurane to prevent muscle spasms and ensure adequate anesthesia depth.</li>
	</ol>
	</li>
	<li>Remove the hair from the anterior neck and posterior back of the mice.</li>
	<li>Position the mouse in a supine position on a warming blanket and apply ophthalmic ointment to the mouse&#8217;s eyes to prevent drying.</li>
</ol>
2. Tracheotomy
<ol>
	<li>Disinfect the neck skin with iodine. Make a 1 cm vertical incision in the center of the skin anterior to the trachea.</li>
	<li>Expand the skin incision and displace the glands in front of the trachea. Expose the digastric muscle, bluntly separate it, and expose the trachea. Gently separate the trachea from surrounding tissues.</li>
	<li>Use spring scissors to create a transverse incision in the trachea. Insert an endotracheal tube into the trachea, directing it towards the lungs (Figure 1A). Secure the trachea with 3-0 surgical suture to prevent air leakage and accidental removal of the catheter.</li>
	<li>Replace the paratracheal muscles and glands over the trachea. Close the neck skin using 6-0 sutures.</li>
</ol>
3. Exposure of thoracic vertebrae
<ol>
	<li>Put&#160;the mouse in a prone position on a heated pad. Make a 2&#160;cm longitudinal incision in the center of the nape of the neck, extending from the C6 to T3 vertebrae.</li>
	<li>Carefully separate the fat and hibernating glands at the anterior aspect of the mouse&#39;s thoracic vertebrae, taking care to avoid the blood vessels beneath the glands. Use spring scissors to cut through the skin and muscle layers, including the trapezius muscle. Insert a retractor between the muscles to assist with further exposure.</li>
	<li>Remove the muscles attaching to the head clamp and the straight portion of the long neck muscles, exposing the spinous processes of T2.</li>
	<li>Displace the semispinalis and spinalis muscles to expose the vertebral arch from C6 to T3 (see Figure 1B). 
	NOTE: During the process of exposing the thoracic vertebrae, ensure special attention is given to preserving the spinous process of T2, as it serves as the primary point of force application for subsequent exposure of T1-DRG.</li>
</ol>
4. Exposure of T1-DRG
<ol>
	<li>Sever the connection between the vertebral arch plate and the articular process of T1. Use fine forceps to meticulously remove the left and right articular processes and mammillary processes of T1.</li>
	<li>After clearing away the overlying connective tissue, carefully expose either the left or right T1 DRG, ensuring the epineurium&#39;s integrity on the chosen side.</li>
	<li>Place a small cotton ball soaked in warm saline over the exposed DRG area to maintain moisture (see Figure 1C). 
	NOTE: Surrounding tissues of the DRG can be covered with a hemostatic sponge to prevent blood leakage during surgery. Change moistened cotton balls as needed to maintain a clear view of the surgical area when soaked with hemorrhagic effusions.</li>
</ol>
5. Thoracic vertebrae fixation and ECG detection
<ol>
	<li>Place the mouse on the stage of the custom spinal clamp with a heating pad (see Figure 1D).</li>
	<li>Secure the mouse using two clips attached to the articular processes of C6 and T3 to minimize movement due to the nerve stimulation (see Figure 1E).</li>
	<li>Connect the ECG monitor with the negative pole on the right upper limb, the ground wire on the right lower limb, and the positive pole on the left lower limb (see Figure 1F).</li>
	<li>Continuously replace the cotton balls soaked with saline until the surgical area is sufficiently clean. 
	NOTE: Ensure thorough clearance of muscles and tissues surrounding the articular processes of C6 and T3, especially in the region where the custom spinal clamp is applied for spinal fixation, to prevent displacement during subsequent experiments.</li>
</ol>
6. Hardware and software setup for imaging
<ol>
	<li>Position the spinal clamp with the secured mouse under the confocal microscope (see Figure 1G).</li>
	<li>Connect the respiratory anesthesia (0.5%-1% isoflurane) machine and heating pad and adjust relevant parameters based on the mouse&#39;s physical condition to ensure animal welfare.</li>
	<li>Place the 10x/0.32 long working distance air objective of the confocal microscope over the exposed T1-DRG for imaging (see Figure 1H).</li>
	<li>Capture the entire T1-DRG by adjusting the stage z-axis up and down, with a step size of 25 &#181;m and a resolution of 512 x 512 or 1024 x 1024 pixels.</li>
	<li>Perform XYZT scanning of the DRG with 8 stacks comprising 1-2 baseline states, 3-6 brush or PENS stimuli, and 7-8 post-stimuli states. 
	NOTE: To ensure a comprehensive and clear view of DRG morphology, position the DRG perpendicular to the lens. Adjust the tilt angle of each mouse using the customized stage according to practical requirements.</li>
</ol>
7. Somatic stimuli and imaging
<ol>
	<li>Apply brush stimulation to the upper limb of the mouse to assess the responsiveness of the imaged DRG neurons (see Figure 1H).</li>
	<li>Perform PENS-PC6 stimulation using a stimulator (see Figure 1F). 
	NOTE: PC6 is located 1-2 mm proximal to the palmar crease of the wrist, between the ulnar flexor carpi tendon and the superficial flexor digitorum tendon. The stimulation parameters are set to 15 Hz and 1 mA for 3-6 stacks.</li>
</ol>
8. Data analysis and processing
<ol>
	<li>In the confocal microscopy software, assess the calcium responses evoked by various stimuli by comparing the increase in green fluorescence intensity when neurons are stimulated by GCaMP upon calcium binding inside the cell (see Figure 2A-C).</li>
	<li>Use Fiji software to manually trace the visible cells and measure their size and relative fluorescence intensity (see Figure 2D).</li>
	<li>Express fluorescence intensity as the ratio of the maximum evoked fluorescence increase to the baseline level (Ft/F0), where the baseline (F0) represents the maximum fluorescence intensity measured during the baseline period.</li>
	<li>Define cell activation as an increase in fluorescence intensity (Ft/F0) &#8805;130% of F0 (see Figure 2E,F).</li>
	<li>Classify DRG neurons into small-sized neurons (&#60;20 &#181;m), medium-sized neurons (20-30 &#181;m), and large-sized neurons (&#62;30 &#181;m) based on their diameter11 (see Figure 2G) .</li>
	<li>Observe changes in heart rate caused by somatic stimulation using the ECG recording software (see Figure 2H). 
	NOTE: The heart rate of mice is easily influenced by external stimuli and respiratory anesthesia. When observing the heart rate response to a stimulus, wait for the heart rate to stabilize before administering the stimulus.</li>
</ol>

Calcium Imaging of Thoracic Segment T1 DRG for Cardiopulmonary Sensory Neurons

<ol><li>Sun, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Piezo1-mediated+neurogenic+inflammatory+cascade+exacerbates+ventricular+remodeling+after+myocardial+infarction%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Piezo1-mediated neurogenic inflammatory cascade exacerbates ventricular remodeling after myocardial infarction.</a> Circulation. 149 (19), 1516-1533 (2024).</li>
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The authors declare no conflicts of interest.

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&#39; 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 &#945;,&#946;-MeATP (a selective P2R agonist) in the DRG and observed that &#945;,&#946;-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.

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 &#39;Circulation&#39; 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.

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

in vivo thoracic dorsal root ganglia (drg) calcium imaging and ecg recording for studying peripheral nerve stimulation

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

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.

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.
<img alt="Endotracheal tube, spinal clamps; ECG detection, dorsal root ganglion dissection; experimental setup." class="xfigimg" src="/files/ftp_upload/67283/67283fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
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. <a href="https://www.jove.com/files/ftp_upload/67283/67283fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence imaging and data graphs analyzing neuron activation by PENS; microscopy and bar chart." class="xfigimg" src="/files/ftp_upload/67283/67283fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
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. <a href="https://www.jove.com/files/ftp_upload/67283/67283fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

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Research

JoVE Journal

Neuroscience

Source: Shannonhouse, J., et al.In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia. J. Vis. Exp. (2023).The video demonstrates an in vivo calcium imaging technique to monitor neuronal activity in the dorsal root ganglia (DRG) of a transgenic mouse. DRG neurons express a calcium indicator that increases fluorescence intensity upon binding to calcium. Mechanical or thermal stimuli applied to a hind paw generate action potentials that...

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

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