Large ScaleIn VivoRecording of Sensory Neuron Activity with GCaMP6

Abstract

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.

Oral thermosensing by murine trigeminal neurons: modulation by capsaicin, menthol, and mustard oil

“…Fourteen per cent of oral thermosensitive ganglion neurons (154/1090 neurons) responded to both cool and elevated temperatures ( Fig. 1A) as also reported using similar functional imaging by Yarmolinsky et al (2016) in the trigeminal ganglion and Chisholm et al (2018) in dorsal root ganglia. Bimodal neurons generally showed temperature-dependent responses with increasing response amplitudes towards both temperature extremes.…”

“…Oral thermosensitive neurons were significantly smaller than the average size (23.4 ± 6.1 μm) of V3 trigeminal neurons ( Fig. 3B), consistent with dorsal root ganglia thermoreceptor neurons (Caterina et al 1997;Mizushima et al 2006;Facer et al 2007;Chisholm et al 2018). There were no significant differences in size among the thermoreceptor groups (P = 0.27-0.96, ANOVA).…”

Presynaptic GCaMP expression decreases vesicle release probability at the calyx of Held

“…Exogenous calcium buffering agents/chelators are routinely used to manipulate vesicle release probability by interfering with the coupling between endogenous calcium sensor and effector proteins (Eggermann et al, ), thereby affecting the synaptic transmission. The GCaMP family of proteins is a widely utilized tool in calcium imaging, and it has been optimized for sensitivity and speed (Akerboom et al, ; Badura et al, ; Chisholm et al, ; T. W. Chen et al, ; Xing & Wu, ). Unfortunately, GCaMP6m augments endogenous cytosolic Ca 2+ buffering and alters the transmission profile of infected synapses.…”

“…GECIs have a typical calcium binding domain, such as calmodulin or troponin C, and undergo conformational changes when bound to calcium, resulting in increased fluorescence of the fused protein probe (Barykina et al, ). The GCaMP family of proteins, in particular, is a widely utilized tool for calcium imaging (Akerboom et al, ; Badura, Sun, Giovannucci, Lynch, & Wang, ; Chen et al, ; T. W. Chen et al, ; Chisholm, Khovanov, Lopes, LaRussa, & McMahon, ; Xing & Wu, ). Targeted expression of these indicators has paved the way to more precisely explore the calcium activity in a variety of subcellular compartments like endoplasmic reticulum (ER; de Juan‐Sanz et al, ), mitochondria (Zhang & Ding, ), and in neuronal synapses (Xing & Wu, ).…”

Visualization of Trigeminal Ganglion Neuronal Activities in Mice

“…Genetically encoded calcium indicators (GECIs) are widely used to image neural activity in response to various stimuli. GCaMP, a prime representative of GECIs, has been widely used to study neuronal activity by allowing visualization of Ca 2+ transients in response to various stimuli (Chisholm, Khovanov, Lopes, La Russa, & McMahon, 2018;Miller et al 2018;Vrontou, Wong, Rau, Koerber, & Anderson, 2013). The GCaMP protein contains a calmodulin domain fused to an enhanced green-fluorescent-protein moiety.…”