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

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca²⁺ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca²⁺ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca²⁺ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca²⁺ entry into the cytoplasm or the release of intracellular Ca²⁺ stores. Pirt-GCaMP3-based Ca²⁺ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca²⁺ transients as well as the amplitude of Ca²⁺ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, Aβ, Aδ, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca²⁺ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

Primary sensory neurons directly innervate the skin and carry somatosensory information back to the central nervous system^1,2. Dorsal root ganglia (DRGs) are cell body clusters of 10,000-15,000 primary sensory neurons^3,4. DRG neurons present diverse size, myelination levels, and gene and receptor expression patterns. Smaller diameter neurons include pain-sensing neurons and larger diameter neurons typically respond to non-painful mechanical stimuli^5,6. Disorders in the primary sensory neurons s....

All procedures described here were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio.

NOTE: Once started, animal surgery (step 1) and imaging (step 2) must be completed in a continuous manner. Data analysis (step 3) may be performed later.

1. Surgery and securing the animal for right side L5 DRG imaging

NOTE: Bo.......

Figure 4: Representative images of L5 dorsal root ganglia of Pirt-GCaMP3 mice. (A,D) Single frame high resolution scans of L5 dorsal root ganglia of Pirt-GCaMP3 mice are shown. (B,E). Average intensity projections of 15 frames of Pirt-GCaMP3 L5 DRG ganglia from panel A and panel D, respectively.......

Persistent pain is present in a wide range of disorders, debilitating and/or reducing the quality of life for about 8% of people²⁹. Primary sensory neurons detect noxious stimuli on the skin, and their plasticity contributes to persistent pain⁸. While neurons can be studied in cell culture and explants, doing so removes them from their normal physiological context. Surgical exposure of the DRG, followed by Pirt-GCaMP3 Ca²⁺ imaging, permits the study of primary se.......

This work was supported by National Institutes of Health Grants R01DE026677 and R01DE031477 (to Y.S.K.), UTHSCSA startup fund (Y.S.K.), a Rising STAR Award from University of Texas system (Y.S.K.), and Craniofacial Oral-biology Student Training in Academic Research (COSTAR) National Institute of Health Grant 5T32DE014318 (J.S.).