Intracellular Calcium Responses Encode Action Potential Firing in Spinal Cord Lamina I Neurons

Abstract

Maladaptive plasticity of neurons in lamina I of the spinal cord is a lynchpin for the development of chronic pain, and is critically dependent on intracellular calcium signaling. However, the relationship between neuronal activity and intracellular calcium in these neurons is unknown. Here we combined two-photon calcium imaging with whole-cell electrophysiology to determine how action potential firing drives calcium responses within subcellular compartments of male rat spinal cord lamina I neurons. We found that single action potentials generated at the soma increase calcium concentration in the somatic cytosol and nucleus, and these calcium responses invade dendrites and dendritic spines by active backpropagation. Calcium responses in each compartment were dependent on voltage-gated calcium channels, and somatic and nuclear calcium responses were amplified by release of calcium from ryanodine-sensitive intracellular stores. Grouping single action potentialevoked calcium responses by neuron type demonstrated their presence in all defined types, as well as a high degree of similarity in calcium responses between neuron types. With bursts of action potentials, we found that calcium responses have the capacity to encode action potential frequency and number in all compartments, with action potential number being preferentially encoded. Together, these findings indicate that intracellular calcium serves as a readout of neuronal activity within lamina I neurons, providing a unifying mechanism through which activity may regulate plasticity, including that seen in chronic pain.

Molecular Determinants of Spinal Hyperexcitability in Rat and Human Models of Pathological Pain

“…Spinal cord lamina I neurons were recorded from in current-clamp configuration, with simultaneous two-photon calcium imaging as previously described (Harding et al, 2020 acquisition program (Carl Zeiss Microscopy, Germany). Laser power was kept between 0.3-0.7%, and gain was restrained to 650-850 for all calcium imaging experiments.…”

“…Fluorescence data were acquired using line scan acquisition (1024x1, 2x averaging) at a rate of 133 Hz, and saved as LSM files from Zen 2009 (Carl Zeiss Microscopy, Germany). Calcium imaging data were analyzed as previously described, utilizing a custom-made, semi-automated MATLAB toolbox (Mathworks, USA) for analysis, entitled CIAT (Calcium Imaging Analysis Toolbox) (Harding et al, 2020). Use of the semi-automated analysis toolbox, CIAT, which automatically calculates peak response from averaged calcium imaging trials, was designed to remove experimental bias.…”

“…Statistical analysis was only performed when group size was at least n = 5, which reflects the number of independent, non-technical replicate values. Sample sizes for experiments in Figures 1-3 were determined based on previous studies (Hildebrand et al, 2014;Harding et al, 2020) One-way repeated-measures ANOVAs were performed using SPSS (IBM SPSS Statistics 25.0, see supplemental tables 1 and 3). Before running each ANOVA, we examined Mauchly's test of sphericity, which indicated that the assumption of sphericity was violated in each case (see supplemental tables 1 and 3).…”

“…No significant effect of Z944 compared to control treatment was observed for HVA calcium currents elicited at 0 mV (n = 10 neurons for vehicle, 8 neurons for Z944) (Figure 1E), indicating specificity to T-type channels. Altogether, the presence of rapidly inactivating LVA calcium currents, and their selective inhibition by Z944 dependent upon voltage-gated calcium channels (Harding et al, 2020). Given our identification here of T-type currents in the majority of lamina I neurons, we next used a combination of current-clamp recordings and simultaneous two-photon calcium imaging to test whether Z944 can attenuate activity-induced calcium responses in these critical nociceptive spinal neurons.…”

“…Lamina I neurons were filled with the calcium-sensitive fluorophore Oregon Green Bapta-1 and the structural fluorophore Alexa Fluor-594 via the patch pipette and single action potential-induced calcium responses were measured in the soma and primary dendrites, as previously described (Figure 2A) (Harding et al, 2020). We found that, on average, perfusion of 2 µM Z944 significantly decreased action potential-induced calcium responses in both the soma and dendrites, as compared to the DMSO vehicle control (n = 8 neurons for vehicle, 7 neurons for Z944; Figure 2B-D).…”