In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Trigeminal Ganglia

This research focuses on peripheral ganglia neural networks, aiming to understand the signals and intercellular interactions involved in pain, itch, and touch sensation. Neurons sense stimuli, but studying their activity in vivo under normal physiological conditions remains highly challenging. Our protocol allows the study of trigeminal ganglion neural activation at the population level directly in response to stimuli, which is physiologically important, but technically very challenging.

Data produced by this protocol serves as a powerful complement to behavior, cell culture, and immunohistochemistry data, allowing the investigation of the immediate effects of stimuli or drugs on an entire ganglion. To begin, place the anesthetized mouse on a heating pad to maintain the body temperature at 37 degrees Celsius, then position the head in a stereotactic mask with a tilt of approximately 15 degrees to the left. Apply ophthalmic ointment to the eyes to prevent dryness and irritation.

After shaving the right side of the mouse's head, make a rectangular incision of nine millimeters by five millimeters between the right ear and the right eye. Cut the tissue on the skull surface just ventral to the eye. Stop any bleeding using a hemostatic swab or a laboratory wipe.

Using a dental drill and a taper fissure bur, drill a hole approximately nine millimeters by five millimeters in the dorsal skull centered on the incision site. Tilt the head until the trigeminal ganglion is clearly visible. If there is bleeding on the trigeminal ganglion, aspirate the blood or remove it using a hemostatic swab or laboratory wipe.

Ensure the trigeminal ganglion is clearly visible without removing any cortical tissue. Move the animal and heating pad to the microscope stage. After confirming the mouse receives continuous isoflurane anesthesia, position the stereotaxic frame so that the objective of the microscope is nine millimeters directly above the cranial opening.

Insert the rectal thermometer and connect the power cord to the thermometer and heating pad. Use a 5X objective to locate the surface of the trigeminal ganglion with the microscope. Adjust the objective and the nose cone to flatten the ganglion surface and maximize the surface area in the focal plane.

Now, load the given microscope high-speed scanning protocol. Scan the trigeminal ganglion in short bursts of six to eight cycles. Apply orthogonal projections of the scans to create a movie, and check the image clarity and consistency between frames.

Load the microscope high-resolution scanning protocol and create a high-resolution image of the trigeminal ganglion. Again, load the high-speed scanning protocol used to create the orthogonal projection movie and record spontaneous activity for 80 cycles. Create the movie and verify that the images are clear and consistent enough for analysis.

To apply stimulation, set the microscope to scan for 15 to 20 cycles. For Von Frey stimulation targeting the V2 region of the trigeminal ganglion, hold the filament and apply it repeatedly to the ipsilateral area below the eye and above the mouth from immediately after Scan 5 until immediately after Scan 10. For V3 region stimulation, apply the filament repeatedly to the ipsilateral area just below the ear during the same scanning window.

For cold and heat stimuli, cool or heat a beaker of water to just below or above the desired temperature. Begin scanning, and immediately after Scan 5, use a plastic transfer pipette to apply the thermal stimulus to the ipsilateral area below the eye and above the mouth for the V2 region, or just below the ear for the V3 region. At the end of the experiment, inject 50 millimolar potassium chloride subcutaneously into the V2 or V3 regions, starting on Cycle 6 to activate, and identify all responsive neurons.

Open the orthogonal projection file by dragging and dropping it into the analysis software. Select the image type by clicking on Image, then Type, and choosing RGB color. To open the ROI Manager, click on Analyze, then Tools, and select ROI Manager.

Select active neurons by drawing an ROI using the ellipse tool in the toolbar. Add each selected region to the ROI file by clicking the add button in the ROI Manager window. Now, go to Analyze, select Set Measurements, and check the option for mean gray value.

In the ROI Manager window, click More, and then choose Multi-Measure to measure the intensity. When a new window labeled Results opens, save the CSV file by selecting File, then Save As.Open the CSV file in a spreadsheet software, and save it as a spreadsheet file. Calculate the calcium transient intensity using the given equation.

For the analysis, randomly sample approximately the same number of neurons from each ganglion to ensure that one ganglion does not dominate the analysis. Measure neuron diameter by using the line tool in the toolbar to draw lines along the longest and shortest diameters of each neuron. Then, calculate the average of those measurements.

Confocal imaging of the trigeminal ganglion enabled simultaneous visualization of over 3, 000 neurons, capturing both spontaneous and stimulus-evoked calcium transient across V2 and V3 regions. In the absence of stimulation, a subset of these neurons exhibited spontaneous calcium transients with distinct activity profiles across six individually tracked cells. Application of noxious heat to the maxillary region resulted in visible activation of multiple neurons, as indicated by bright fluorescence with synchronized increases in calcium transient intensity.

Noxious heat applied to the mandibular region similarly activated distinct neurons with variable patterns of calcium responses across individual cells. A significantly higher number of neurons responded to two-gram Von Frey stimulation compared to 0.4 grams in the V2 region. The calcium transient intensity in individual neurons increased more strongly with two-gram stimulation than with 0.4 gram, with a higher peak and broader response spread.

Mean calcium response intensity during the first stimulus frame was also significantly greater following two-gram stimulation compared to 0.4 gram.