Greater emphasis on the study of intact cellular networks in their physiological environment has led to rapid advances in intravital imaging of the central nervous system (CNS), while the peripheral system remains largely unexplored. To assess large networks of sensory neurons, we selectively label primary afferents with GCaMP6s in male and female C57bl/6 mice and visualize their functional responses to peripheral stimulation in vivo. We show that we are able to monitor the activity of hundreds of sensory neurons simultaneously, with sufficient sensitivity to detect, in most cases, single action potentials with a typical rise time of around 200 ms, and an exponential decay with a time constant of approximately 700 ms. With this technique we are able to characterize the responses of large populations of sensory neurons to innocuous and noxious mechanical and thermal stimuli under normal and inflammatory conditions. We demonstrate that the majority of primary afferents are polymodal with between 50–80% of thermally sensitive DRG neurons responding also to noxious mechanical stimulation. We also specifically assess the small population of peripheral cold neurons and demonstrate significant sensitization to cooling after a model of sterile and persistent inflammation, with significantly increased sensitivity already at decreases of 5°C when compared to uninflamed responses. This not only reveals interesting new insights into the (patho)physiology of the peripheral nervous system but also demonstrates the sensitivity of this imaging technique to physiological changes in primary afferents.

See Basbaum (doi:) for a scientific commentary on this article.Following nerve injury, sensory neurons become hyperexcitable and this is a key driver of neuropathic pain. Weir et al. develop a gene therapy approach that silences sensory neurons and reversibly normalizes pain thresholds in animal models of neuropathic pain. This new approach has significant translational potential.

Greater emphasis on the study of intact cellular networks in their physiological environment has led to rapid advances in intravital imaging in the central nervous system, while the peripheral system remains largely unexplored. To assess large networks of sensory neurons we selectively label primary afferents with GCaMP6s and visualise their functional responses in vivo to peripheral stimulation. We show that we are able to monitor simultaneously the activity of hundreds of sensory neurons with sensitivity sufficient to detect, in most cases, single action potentials with a typical rise time of around 200 milliseconds, and an exponential decay with a time constant of approximately 700 milliseconds. Using this sensitive technique we are able to show that large scale recordings demonstrate the recently disputed polymodality of nociceptive primary afferents with between 40-80% of thermally sensitive DRG neurons responding also to noxious mechanical stimulation. We also specifically assess the small population of peripheral cold fibres and demonstrate significant sensitisation to cooling after a model of sterile and persistent inflammation, with significantly increased sensitivity already at decreases of 5°C when compared to uninflamed responses. This not only reveals interesting new insights into the (patho)physiology of the peripheral nervous system but also demonstrates the sensitivity of this imaging technique to physiological changes in primary afferents.Significance StatementMost of our functional understanding of the peripheral nervous system has come from single unit recordings. However, the acquisition of such data is labour-intensive and usually ‘low yield’. Moreover, some questions are best addressed by studying populations of neurons. To this end we report on a system that monitors activity in hundreds of single sensory neurons simultaneously, with sufficient sensitivity to detect in most cases single action potentials. We use this technique to characterise nociceptor properties and demonstrate polymodality in the majority of neurons and their sensitization under inflammatory conditions. We therefore believe this approach will be very useful for the studies of the somatosensory system in general and pain in particular.

Transcriptional alterations are characteristic of persistent pain states, but the key regulators remain elusive. HDAC4 is a transcriptional corepressor that has been linked to synaptic plasticity and neuronal excitability, mechanisms that may be involved in peripheral and central sensitization. Using a conditional knockout (cKO) strategy in mice, we sought to determine whether the loss of HDAC4 would have implications for sensory neuron transcription and nociception. HDAC4 was found to be largely unnecessary for transcriptional regulation of naïve sensory neurons but was essential for appropriate transcriptional responses after injury, with Calca and Trpv1 expression consistently down-regulated in HDAC4 cKO compared to levels in the littermate controls (0.2–0.44-fold change, n = 4 in 2 separate experiments). This down-regulation corresponded to reduced sensitivity to 100 nM capsaicin in vitro (IC50 = 230 ± 20 nM, 76 ± 4.4% wild-type capsaicin responders vs. 56.9 ± 4.7% HDAC4 cKO responders) and to reduced thermal hypersensitivity in the complete Freund’s adjuvant (CFA) model of inflammatory pain (1.3–1.4-fold improvement over wild-type controls; n = 5–12, in 2 separate experiments). These data indicate that HDAC4 is a novel inflammatory pain mediator and may be a good therapeutic target, capable of orchestrating the regulation of multiple downstream effectors.—Crow, M., Khovanov, N., Kelleher, J. H., Sharma, S., Grant, A. D., Bogdanov, Y., Wood, J. N., McMahon, S. B., Denk, F. HDAC4 is required for inflammation-associated thermal hypersensitivity.