In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

The general goal of our lab is to understand the peripheral mechanisms of pain. And my project specifically investigates pain associated with trigeminal nerve injury. The preparation that I'm showing today allows us to characterize changes in sensory coding and identify afferent subpopulations contributing to neuropathic pain.

So we have shown that there are differences between the trigeminal and somatic afferents in response to nerve injury. Nav 1.1 specifically is preferentially upregulated in the trigeminal nerve, suggesting that selective blockers for Nav 1.1 can be used to identify different afferent subpopulations that contribute to neuropathic pain. So transgenic mice are used most extensively at the moment in combination with a variety of imaging and omic-based strategies.

Translating data generated in mice to other species, if not humans. Mice are really small, they're difficult to train, and there's a growing list of differences between mice, rats, and humans. So using rats may help address several of these challenges.

Pain mechanisms and potential therapeutic targets differ in craniofacial structures, as in those above the neck and other somatic and visceral structures i.e. those below the neck. Results with this preparation suggest that there's a component of neuropathic pain, which is previously thought to reflect changes in the central nervous system, but may actually also reflect changes in the peripheral nervous system.

To begin, use a 25 microliter Hamilton syringe to intraperitoneally inject anesthetized rat pups with 15 microliters of agene. Next, shave the hair and whiskers of anesthetized six to eight week old pups, mount the rat on a stereotaxic frame with ear bars and place a heating pad underneath to maintain the body temperature of the animal. Next place a gauze dipped in ice cold saline on the rat's head to minimize bleeding.

With a size 15 scalpel, make an incision on the midline of the skin and muscle over the skull. Pull away at the skin and muscle to expose the skull with a one by four round drill bit, carefully perforate the skull cap to expose the forebrain. Then use rongeurs to cut through the skull.

With a size 15 scalpel, make an incision in the brain and olfactory bulb. Use a spatula to carefully disconnect the dura from the skull. Then gently lift the severed brain to display the trigeminal ganglion and the skull base.

Cauterize any bleeding with a cautery pen. To image the GCaMP6s, place a skull cavity underneath the objective of the microscope. Then bring the terminal ganglion into focus.

Next, apply mechanical stimuli to one square centimeter of the face. Use a 20 x objective to visualize the neurons of the trigeminal ganglion that are responsive to the stimulus. To collect the fluorescent stato over time with the acquisition software, press the stream acquisition button under the acquire menu, then set the number of frames to 300 and acquisition time to 90 seconds.

Set the camera parameters to acquire images at frame rate. Obtained stable baseline recordings over three minutes. A larger volume of viral titer resulted in increased neuronal infection of the rat pups.

Light mechanical simulation activated only the V2 region of the trigeminal ganglion. Resting fluorescence was relatively low in most neurons with few spontaneous increases. Natural stimuli, specifically punctuate stimulation, elicited the most robust neuronal responses.

Brush stimuli in chronic constriction injury models elicited a twofold increase in magnitude of peak response on the injured side relative to the contralateral side. When two stimuli were applied in series, there was a significant amplification in response to the second stimulus.