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 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.
Protocol
Representative Results
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<sup …
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 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 |
References
- Kim, H., et al. Reduced tactile acuity in chronic low back pain is linked with structural neuroplasticity in primary somatosensory cortex and is modulated by acupuncture therapy. Neuroimage. 217, 116899 (2020).
- Wei, J. A., et al. Electroacupuncture activates inhibitory neural circuits in the somatosensory cortex to relieve neuropathic pain. iScience. 24 (2), 102066 (2021).
- Cui, X., et al. Referred somatic hyperalgesia mediates cardiac regulation by the activation of sympathetic nerves in a rat model of myocardial ischemia. Neuroscience Bulletin. 38 (4), 386-402 (2022).
- Fang, Y., et al. Cutaneous hypersensitivity as an indicator of visceral inflammation via c-nociceptor axon bifurcation. Neuroscience Bulletin. 37 (1), 45-54 (2021).
- Gao, X., et al. Calcium imaging in population of dorsal root ganglion neurons unravels novel mechanisms of visceral pain sensitization and referred somatic hypersensitivity. Pain. 162 (4), 106801081 (2021).
- Huang, T. Y., Belzer V Fau – Hanani, M., Hanani, M. Gap junctions in dorsal root ganglia: possible contribution to visceral pain. European Journal of Pain. 14 (1), 49.e1-49.e11 (2010).
- Wang, Y. L., Su, Y. S., He, W., Jing, X. H. Electroacupuncture relieved visceral and referred hindpaw hypersensitivity in colitis rats by inhibiting tyrosine hydroxylase expression in the sixth lumbar dorsal root ganglia. Neuropeptides. 77, 101957 (2019).
- Weng, Z., et al. Electroacupuncture diminishes P2X2 and P2X3 purinergic receptor expression in dorsal root ganglia of rats with visceral hypersensitivity. Neural Regeneration Research. 8 (9), 802-808 (2013).
- Spencer, N. J., Hibberd, T. J., Lagerström, M., Otsuka, Y., Kelley, N. Visceral pain – Novel approaches for optogenetic control of spinal afferents. Brain Research. 1693 (Pt B), 159-164 (2018).
- Mao, H., et al. Use of in vivo single-fiber recording and intact dorsal root ganglion with attached sciatic nerve to examine the mechanism of conduction failure. Journal of Visualized Experiments. 150, e59234 (2019).
- Ma, C., Nie, H., Gu, Q., Sikand, P., Lamotte, R. H. In vivo responses of cutaneous C-mechanosensitive neurons in mouse to punctate chemical stimuli that elicit itch and nociceptive sensations in humans. Journal of Neurophysiology. 107 (1), 357-363 (2012).
- Anderson, M., Zheng, Q., Dong, X. Investigation of pain mechanisms by calcium imaging approaches. Neuroscience Bulletin. 34 (1), 194-199 (2018).
- Yao, L., Ye, Q., Liu, Y., Yao, S., Yuan, S., Xu, Q., Deng, B., Tang, X., Shi, J., Luo, J., Wu, J., Wu, Z., Liu, J., Tang, C., Wang, L., Xu, N. Electroacupuncture improves swallowing function in a post-stroke dysphagia mouse model by activating the motor cortex inputs to the nucleus tractus solitarii through the parabrachial nuclei. Nat Commun. 14 (1), (2023).
- Tian, D., Liu, B., Huang, S., Shi, W., Tian, Y., Yang, Y., Zhang, D., Zhang, Z., Bu, F. Postoperative Ileus Murine Model. J Vis Exp. (209), (2024).
- Zhu, X., Li, C., Afridi, S. K., Zu, S., Xu, J. W., Quanquin, N., Yang, H., Cheng, G., Xu, Z. E90 subunit vaccine protects mice from Zika virus infection and microcephaly. Acta Neuropathol Commun. 6 (1), (2018).
- Zheng, Q., et al. Synchronized cluster firing, a distinct form of sensory neuron activation, drives spontaneous pain. Neuron. 110 (2), 209-220 (2022).
- Kim, Y. S., et al. Coupled activation of primary sensory neurons contributes to chronic pain. Neuron. 91 (5), 1085-1096 (2016).
- Li, Y., et al. P2Y12 receptor as a new target for electroacupuncture relieving comorbidity of visceral pain and depression of inflammatory bowel disease. Chinese Medicine. 16 (1), 139 (2021).
- Liu, Q., et al. Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus. Cell. 139 (7), 1353-1365 (2009).
- Chen, G. S., Su, X. L., Liu, Q., Wei, W. Research progress of moxibustion in treatment of irritable bowel syndrome. World Journal of Acupuncture-Moxibustion. 31 (02), 136-140 (2021).
- Scanziani, M., MHäusser, M. Electrophysiology in the age of light. Nature. 461 (7266), 930-939 (2009).
- Chen, T., et al. Top-down descending facilitation of spinal sensory excitatory transmission from the anterior cingulate cortex. Nature Communications. 9 (1), 1886 (2018).
- Cartarozzi, L. P., et al. In vivo two-photon imaging of motoneurons and adjacent glia in the ventral spinal cord. Journal of Neuroscience Methods. 299, 8-15 (2018).
- Shannonhouse, J., Gomez, R., Son, H., Zhang, Y., Kim, Y. S. In vivo calcium imaging of neuronal ensembles in networks of primary sensory neurons in intact dorsal root ganglia. Journal of Visualized Experiments. 192, e64826 (2023).
- Davalos, D., Akassoglou, K. In vivo imaging of the mouse spinal cord using two-photon microscopy. Journal of Visualized Experiments. 59, e2760 (2012).
- Chen, C., et al. Long-term imaging of dorsal root ganglia in awake behaving mice. Nature Communications. 10 (1), 3087 (2019).
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