Electric Field Stimulation for the Functional Assessment of Isolated Dorsal Root Ganglion Neuron Excitability.

Genetically encoded calcium indicators have proven useful for characterizing dorsal root ganglion neuron excitability in vivo. Challenges persist in achieving high spatial-temporal resolutions in vivo, however, due to deep tissue imaging and motion artifacts that may be limiting technical factors in obtaining measurements. Here we report an ex vivo imaging method, using a peripheral neuron-specific Advillin-GCaMP mouse line and electric field stimulation of dorsal root ganglion tissues, to assess the sensitivity of neurons en bloc. The described method rapidly characterizes Ca2+ activity in hundreds of dorsal root ganglion neurons (221 ± 64 per dorsal root ganglion) with minimal perturbation to the in situ soma environment. We further validate the method for use as a drug screening platform with the voltage-gated sodium channel inhibitor, tetrodotoxin. Drug treatment led to decreased evoked Ca2+ activity; half-maximal response voltage (EV50) increased from 13.4 V in untreated tissues to 21.2, 23.3, 51.5 (p < 0.05), and 60.6 V (p < 0.05) at 0.01, 0.1, 1, and 10 µM doses, respectively. This technique may help improve an understanding of neural signaling while retaining tissue structural organization and serves as a tool for the rapid ex vivo recording and assessment of neural activity.

Behavioral Compensations and Neuronal Remodeling in a Rodent Model of Chronic Intervertebral Disc Degeneration.

Low back pain is associated with degeneration of the intervertebral disc, but specific mechanisms of pain generation in this pathology remain unknown. Sensory afferent nerve fiber growth into the intervertebral disc after injury-induced inflammation may contribute to discogenic pain. We describe a clinically relevant behavioral phenotype in a rodent model of chronic intervertebral disc degeneration which provides a means to map sensory neuron changes to a single affected lumbar intervertebral disc. Unilateral disc puncture of one lumbar intervertebral disc revealed a bilateral behavioral phenotype characterized by gait changes and decreased activity. Moreover, neurons extracted from the dorsal root ganglia in animals with intervertebral disc injury demonstrated altered TRPV1 activation in vitro independent of exogenous NGF administration. Finally, neuronal nuclear hypertrophy and elevated expression of p75NTR provide evidence of active adaptation of innervating sensory neurons in chronic intervertebral disc degeneration. Therefore, this model and findings provide the template for future studies to establish specific mechanisms of nociceptive pain in chronic intervertebral disc degeneration.