In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

Our research centers on the neurons and the circuits of somatosensory system involved in narrating analgesic effects of acupuncture on visceral pain while trying to adjust the neuron responses of neuro DRG to acupuncture stimuli. Traditional methods, such as electro recording, cannot efficiently examine the sufficient cell numbers and distinct specific cellular ties to identify the visceral or somatic responsive neurons in vivo. Studying the functional roles of DRG neuron in the analgesic effect of acupuncture has been difficult due to technological constraints.

We introduced a general approach to observe the responsiblity of a number six DRG neuron to intracolonic and acupuncture simulation in vivo. The protocol outlines the procedures for observing the activity of DRG neurons in vivo in response to somatic and viscera input. This is an improvement over the previous study, which only focused on surveying neuron responses in L5 DRG under the somatic stimuli.

In the future, with the right setup, DRG in vivo CAS imaging can be done in thoracic or cervical segments, providing a more powerful tool to study visceral nociception and acupuncture effects. This method is essential for acupuncture researchers to learn. To begin, place the tracheostomized mouse in the prone position on the heating pad.

Make a seven centimeter long midline incision in the lower back and extend it from the fifth lumbar to the first sacral vertebrae. Then, carefully move aside the longissimus lumborum muscles to expose the spinous processes of the three vertebrae. Move the inner parts of the bilateral longissimus lumborum, dorsal lumbar spine muscles, and dorsal lumbar semispine muscles to expose the successive three articular and mammilary processes with the recording segment in the middle of the vertebrae.

Now, securely immobilize the animal with tooth forceps on the bilateral articular processes of the neighboring vertebrae. Use rongeur forceps to remove the left or right articular and mammilary processes of the lumbar four or six vertebrae. Carefully expose the left or right lumbar six DRG after clearing the connective tissues above it.

Ensure to keep the epineurium intact on the chosen side of the lumbar six vertebrae. Cover the exposed DRG areas with small cotton balls soaked in saline to maintain the active condition. Now, connect the low-flow anesthesia delivery system to the tracheal cannula to intubate the mice.

Position the animal with the exposed DRG onto a heating pad situated on the stage of a customized spinal clamp. Using two clamps on the spinal columns of the neighboring vertebrae, secure the animal to minimize movement during testing. Then, replace the saline-wetted cotton balls until the surgical area is adequately clean.

Insert the customized spinal clamp into the custom-designed microscope stage. For imaging, position a long working distance air objective lens from a confocal microscope over the exposed DRG. Capture timelapsed Z-stacks of the intact lumbar four or six DRG with 25 micrometers per step and 512 by 512 or 1, 024 by 1, 024 pixel resolution.

Perform XYZT scanning of the DRG with four to eight stacks, encompassing one to three baseline states, two to three acupuncture or visceral colorectal distension stimuli, and one to two post-stimuli states. Then, apply brushing or pinching stimuli to the animal's lower back, hind limb, or hind paw to evaluate the most sensitive receptive field of the imaged DRG neurons. Administer acupuncture stimuli manually or using a commercially available acupoint and nerve stimulator.

Employ colorectal distension with a self-made air pressure gauge to induce visceral stimuli. Using a confocal microscope, measure the increase in green fluorescence of the neuronal GCaMP upon binding to intracellular calcium following somatic or visceral stimuli. Using imaging software, lighten the video using the color option.

Manually delineate visible cells and ascertain cell size and relative fluorescence intensity. Express fluorescence intensity as a ratio of the increase in maximum evoked fluorescence to the basal level. The lumbar six DRG neurons generally lack baseline GFP fluorescence, possibly influenced by GCaMP expression levels and surgical damage.

Colorectal distension stimuli induced a rapid, transient increase in GCaMP fluorescence. Similar responses were observed with BL25 electroacupuncture application. Heat maps and line charts showed responses of all circled neurons to colorectal distension and BL25 electroacupuncture.

The histogram shows the diameters of responsive neurons to colorectal distension and BL25 electroacupuncture.