In Vivo Calcium Imaging of Neuronal Ensembles in the Intact Dorsal Root Ganglia of a Mouse

Take an anesthetized transgenic mouse with an exposed dorsal root ganglion, or DRG, a cluster of sensory neuron cell bodies that receive input from the hind limbs.             

The neurons in the DRG express a cytoplasmic calcium indicator.

Using a confocal microscope, visualize the DRG to image the collective activity of a large number of neurons.

Without external stimulation, the low intracellular calcium level keeps the indicators in their native conformation, resulting in a faint baseline fluorescence.         

Apply a mechanical or thermal stimulus to a hind paw, generating an action potential in the sensory neurons.

The signal propagates to the DRG, where voltage-gated calcium channels in the neurons open, allowing calcium influx.

The additional calcium ions bind to the indicators, inducing a conformational change that increases fluorescence intensity.

Observe a bright fluorescence in the DRG, with the intensity corresponding to the number of stimulated neurons exhibiting calcium influx. 

Place the mouse on a heated pad to maintain the body temperature at 37 degrees Celsius. Locate the lumbar enlargement by feeling the pelvic bone of the mouse. Then shave the back of the mouse above the area of lumbar enlargement. Using scissors, make a three-sided rectangular incision above the lumbar enlargement, and fold the skin away with forceps.

Use the 13-millimeter spring dissection scissors to make 3 to 4 millimeters incisions on the right side of the spine. Use scissors to cut back the skin and muscles to the sides to expose the spine. Then, using 8-millimeter scissors, cut away the muscle and connective tissue to clean the transverse process of the right side L5 DRG.

Try to minimize bleeding by using cotton or gel foam to absorb the blood. Cut open the right side L5 transverse process using Friedman-Pearson rongeurs or strong fine forceps, being careful to not touch the DRG. Next, move the mouse and heating pad onto the custom stage. Use stage tape to secure the animal and the heating pad in place.

Place the nose of the animal in the nose cone for continuous isoflurane anesthesia. Secure the right hind paw sticking out to a position off the stage for easy application of the stimuli. Secure the spine in place with the stage clamps over the skin on the vertebrae or the pelvic bone, just rostral and caudal to the L5 DRG. Adjust the clamps and the stage to make the surface of the DRG as level as possible.

Then, place the stage below the microscope so that the objective is directly 8 millimeters above the DRG when lowered. Insert the rectal thermometer. Connect the power lines to the heating pad in the rectal thermometer. Connect the nose cone to the isoflurane gas lines.

Use an upright confocal microscope with a 10x/0.4 DIC objective and the associated software for imaging. Use the green FITC filter settings of excitation at 495 nanometers, emission at 519 nanometers, and detection wavelength between 500 to 580 nanometers. Under the microscope, find the surface of the DRG. Adjust the clamps on the stage so the DRG surface is as level as possible and the maximum surface area is visualized in the focal plane.

Monitor the animal throughout the procedure to maintain isoflurane anesthesia without overdosing. To load the microscope rapid scanning protocol, use the typical settings of voxel size 2.496 by 2.496 by 16 microns, 512 by 512 pixels, 10-optical-slice z-stack, 1 Airy unit for 32 micrometers, 1% laser power of 488 nanometers and 5 milliwatts, pixel time- 1.52 microseconds, line time- 0.91 milliseconds, frame time- 465 milliseconds, LSM scan speed of 8, bidirectional scanning, PMD detector gain- 650 volts, and digital gain of 1.

Take a short eight-cycle scan of the DRG by clicking on Start Experiment under the Acquisition tab. Create a movie by making an orthogonal projection of scans at one scan per frame over time. Manually check for image clarity and imaging artifacts such as waves of brightness crossing the DRG. Adjust clamp position and optical section thickness, and repeat this step until a clear, high-quality movie is achieved.

Then load the microscope high-resolution scanning protocol using the typical settings of voxel size 1.248 by 1.248 by 14 microns, 1024 by 1024 pixels, six optical slice z-stack, 1.2 Airy unit or 39 micrometers, 5% laser power of 488 nanometers and 25 milliwatts, pixel time- 2.06 microseconds, line time- 4.95 milliseconds, frame time 5.06 seconds, LSM scan speed of 6, bidirectional scanning, PMT detector gain at 650 volts, and digital gain of 1.

Click on the Start Experiment button under the Acquisition tab to make a high-resolution image of the DRG. Load the microscope rapid scanning protocol, and record spontaneous activity in the DRG for 80 cycles. Generate an orthogonal projection movie, and verify that the image is of sufficient quality for analysis. For applying stimuli, set the microscope to perform 15 to 20 scans. Wait for scans 1 to 5 to complete to produce the baseline. Apply the stimulus during scans 6 to 10. Wait at least five minutes following each stimulus before applying the next one to prevent desensitization.

For a mechanical press, hold the algometer pincher with the paw between the paddles without touching the paw. Pinch the paw starting immediately after the end of scan 5 and stopping immediately after scan 10. Monitor the press force with an algometer and ensure that it does not exceed 10 g over the desired force.

For thermal stimuli, heat a beaker of water to just above the desired temperature. When the water is at the correct temperature, apply the stimulus immediately after scan 5 by immersing the pond in the water. Pull the beaker away immediately after scan 10.