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.
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.
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 growth1,2. 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 potentially carried out on primary sensory neurons using electrophysiological techniques or other neural recording methods3,4. Such research aids in comprehending the relationship between somatic and visceral input from specific target tissues or target organs, offering valuable insights into conditions related to acupuncture, visceral pain, autonomic nervous system regulation, and related medical conditions.
Being the first-order neurons in the somatosensory system, neurons in DRG are referred to as primary sensory neurons which have important roles in transducing information about the external environment as well as the internal state into electrical signals and transmitting signals to the central nervous system (CNS). Numerous studies have suggested that visceral nociception was dominantly relayed by sensory neurons whose cell bodies are in the DRG5,6. Although numerous researches have elucidated the cellular and molecular mechanism of DRG neurons in acupuncture-induced analgesic effect on visceral pain7,8, very little literature exists on its functional characteristics due to technical difficulties9. Several methods for recording neural activity in the DRG, such as peripheral fiber recording, single-cell electrophysiology recording, and in vivo calcium imaging, can be used to record the patterns and properties of the action potentials passed along axons10. Loosely patched glass electrode recording of the DRG has been one of the most widely used techniques to investigate the correlation between neuronal activities and different stimuli in vivo11. However, traditional methods such as electrophysiological recording cannot efficiently examine sufficient cell numbers and distinct specific cellular subtypes to identify visceral-responsive neurons in vivo.
In addition to encoding peripheral sensation, DRG neurons play a significant role in the transmission of acupuncture signals to the central nervous system. Traditional electrophysiological recording has already been widely applied to explore the regulation of acupuncture on abnormal activities of DRG neurons induced by pathological pain11. Appropriate segments of DRG need to be observed in relation to sensory innervation. Lumbar (L) 6 DRG was generally observed to investigate colon modulation4.
Recent advances in the development of optical and genetic methods make it possible to investigate the activity of large populations of genetically labeled neurons simultaneously12. However, there is still a lack of detailed calcium imaging methods for monitoring neuronal activity in DRG under visceral and somatic stimulation. Hence, this protocol explains the procedures for in vivo observation of responsiveness of L6 DRG neurons to intracolonic and acupuncture stimulation. The method described here can also be used to detect characteristics of somatic and visceral sensory neurons.
The broad application and promotion of calcium imaging deliver a very effective and practical tool for acupuncture research. Considering the advantages of calcium imaging mentioned above, this method ought to have been widespread and applied in acupuncture research. However, the utilization of calcium imaging in acupuncture research is still relatively uncommon. The key reason for this limitation may be the difficulty of operational and recording procedures. The primary purpose of this article is to give an overview of some critical points in the conduct of calcium imaging recordings of L6 DRG neurons in mice. Most importantly, we hope to promote the advancement and development of acupuncture research by using this cutting-edge tool in vivo.
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). The following procedure is optimized for either gender of mice weighing 20-35 g. The recommended age for mice undergoing the operation was between 8 and 14 weeks. Either genetically encoded calcium indicators (GECIs), such as Pirt-GCaMP6s mice, or normal C57 mice intrathecally injected with viral GECIs, were adopted to show the main populations of DRG neurons. Genetically engineered mice intrathecally injected with viral GECIs or crossed with Rosa26-GCaMP mice would show the specific population of DRG neurons. Laboratory gowns, gloves, and masks were worn throughout the protocol. After the experiment, the mice were euthanized with CO2 inhalation followed by cervical dislocation.
1. Pre-operative setup
2. Anesthetization
3. Tracheotomy
4. Exposure of lumbar vertebrae
5. Exposure of dorsal root ganglion
6. Immobilization and ventilation
7. Hardware and software setup for imaging
8. Acupuncture/visceral stimuli and imaging
9. Data analysis and processing
Following the above protocol, the lumbar-6 DRG of a transgenic Pirt-GCaMP6s mouse was exposed, and visceral CRD or somatic acupuncture stimuli were applied to the colorectum or receptive field. This experiment aimed to observe the number and types of neurons elicited by different visceral CRD and somatic stimuli.
As shown in Figure 2A, most of the neurons in the lumbar-6 DRG do not exhibit GFP fluorescence under baseline conditions. This baseline fluorescence may be influenced by two factors: the expression level of GCaMP and the possible damage done to the DRG during the surgery. CRD stimuli resulted in a rapid, transient increase in GCaMP fluorescence, and the numbers and intensities of GFP increased, as can be seen in Figure 2B. Similar changes occurred in Figure 2C when BL25 (Dachangshu) electroacupuncture (EA) was applied18. BL25 is located 3 mm beside the median dorsal line of the lower 4th lumbar spine, which is commonly used when there are lower intestinal disorders20. The selected and numbered cells are circled in Figure 2D using the imaging software.
As described in the above protocol, changes in fluorescence intensity above a threshold level of ≥30% F0 were considered as positive responses. Pseudo colors were added to show the merged image of neurons that responded to both CRD (red) and EA (green) in Figure 2E. There was one merged neuron that responded to both CRD and EA, as pointed out by the arrow in Figure 2E. The heat map and line chart in Figure 2F and Figure 2H represent the responses of all the neurons circled in the imaging software to CRD. Figure 2G displays a histogram chart of the different diameters of the responsive neurons to CRD. The heat map and line chart in Figure 2I and Figure 2K represent the responses of all the neurons circled in the imaging software to EA at BL25. Figure 2J displays a histogram chart of the different diameters of the responsive neurons to EA. In addition to analyzing the number and fluorescent intensities of the responsive neurons, it is possible to analyze different responses of neurons in the DRG of various sizes when different stimuli are applied, as shown in Figure 2G,J.
Once a single neuron is identified, it is also possible to analyze the responses of the same or adjacent cells to different stimuli. This allows for the examination of temporal and spatial interactions between different visceral and somatic stimuli at the DRG level.
Figure 1: Surgical procedure for lumbar-6 DRG. (A) The tracheotomy of the experimental animal. (B) Superior view displaying three exposed vertebrae, with the left side being rostral and the right side caudal. (C) The customized spinal clamp. Note the adjustable pad, allowing for precise alignment of the exposed DRG and the imaging objective, maintained at a perpendicular angle. The spinal clamps are controlled by universal ball units, facilitating fine-tuning of the recorded DRG and the objective. (D) Application of the spinal clamp and exposure of the DRG before imaging. (E) Enlarged images provide a closer look at the exposed left lumbar-6 DRG. (F) Depiction of the anesthesia ventilator and the custom stage used in this procedure. (G) The imaging process of the DRG under the objective lens. (H) A schematic representation of somatic and visceral stimulation during recording. Please click here to view a larger version of this figure.
Figure 2: Representative images and analysis of in vivo calcium imaging of lumbar-6 DRG neurons. (A–C) Representative images depict lumbar-6 DRG neurons during baseline, following CRD, and after BL25 EA stimuli. (D) Images illustrate labeled and numbered cells within a single lumbar-6 DRG after cell tracing using the imaging software. (E) Merged images display lumbar-6 DRG neuron ensembles responding to both CRD (in red) and EA (in green) stimuli. Scale bars = 100 µm. (F), (H), (I), (K) Heatmap and line chart display fluorescence intensity changes in the traced cells in response to CRD and EA stimuli. (G), (J) Depiction of the number of responsive cells of various sizes to CRD and EA stimuli. The error bars denote mean ± SEM. Please click here to view a larger version of this figure.
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 probes21. In the spinal cord, multiple laminectomy techniques have been developed for in vivo imaging of spinal neuron activity22,23. Although the DRG is in the vicinity of the spinal cord, in vivo imaging of DRG cells remains more challenging due to its location surrounded by connective tissue and muscles. A previous study has shown an approach to survey neural responses of L5 DRG under somatic stimuli24. Since L5 DRG neurons receive mostly somatic afferents instead of visceral afferents, questions remain about surgical procedures for surveying the activity of DRG neurons receiving somatic and visceral input.
The method described here allows for stable calcium imaging in genetically defined neurons of the DRG while applying acupuncture stimulation and visceral stimulation in intact mice. As is mentioned in most in vivo techniques, the achievable imaging stability strongly depends on the spinal stabilization device and proper anesthesia25. In this study, a custom-built spinal adaptor equipped with angle adjusters was used to conveniently angle the spine to obtain clear images of the DRG. A pair of Adson forceps was modified to serve as spinal vertebrae clampers attached to articulating arms, allowing enough space to lower an air lens over the exposed DRG. Alternative forceps can be applied as needed for different spinal cord segments to offer better stability. It is advisable to suspend the spinal cord and its imaging field slightly from the clamps, which also reduces respiratory displacement. Another important strategy to minimize fluctuations during imaging is the use of tracheal cannula anesthesia and muscle relaxants combined with mechanical ventilation.
This study can also be adapted for two-photon imaging, which may provide better cellular resolution in deeper tissues. Recently, Chen et al. developed an imaging technique that allows researchers to examine the activity of neurons in the DRG of awake mice over extended periods26. However, it is not yet readily available to most laboratories due to the difficulty in surgical preparation. Furthermore, although the method described here is not suitable for imaging DRG over weeks in awake mice, it has advantages in drug application and dorsal root stimulation since the animal's skin can be sewn to a ring to hold a pool for perfusion of ACSF.
There are also some limitations to this protocol. Restricting the recording to lumbar dorsal root segments limits the applicability of this technique to various ganglion segments that innervate different organs throughout the body, such as for the study of thoracic organs by recording thoracic DRGs. Calcium imaging videos were analyzed using the imaging software. There are challenges in calcium imaging data analysis, such as cell detection and Ca2+ signal extraction. Custom-written scripts in MATLAB seem to be an effective tool, but they require the researcher to have a programming background.
The authors have nothing to disclose.
This study was funded by the National Key R&D Program of China (No. 2022YFC3500702), the National Natural Science Foundation of China (No. 82230123, 82174281).
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 |
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