Article Figures & Data
Figures
- Figure 1.
Approach for calcium imaging of proprioceptors in the intact DRG. A, Schematic of the imaging preparation. Electrical stimulation of the sciatic nerve evoked action potentials in peripheral sensory neurons. Compound action potentials were recorded via a suction electrode on the proximal dorsal root. PV+ sensory neurons, consisting mainly of proprioceptors but also some mechanoreceptors, expressed GCaMP6s using a Cre-lox strategy. Two-photon calcium imaging data were acquired from single neurons in the intact lumbar 5 (L5) DRG. B, A representative image of a GCaMP6s-expressing DRG neuron responding to electrical stimulation. Yellow line indicates the region chosen for line scan analysis (at ∼700-Hz scanning rate). C, Heat map of the electrically evoked change in fluorescence across the line scan (oriented vertically) over time (horizontal axis). Optical recording begins 5 s before stimulus is delivered to establish pre-stimulus resting fluorescence. D, Compound action potentials recorded from the L5 dorsal root during the stimulus train. Action potentials are observed with every electrical pulse (0.1-ms pulses at 50 Hz for 0.5 s). Amplitude of the compound action potential remains stable throughout the stimulus. Area within dotted box is enlarged in the box on the upper right corner. E, Representative calcium transient evoked by electrical stimulation (box on gray bar). The average fluorescence across the length of the line scan at each time point composes the raw calcium transient. F, A smoothing algorithm removes high frequency signal fluctuations. Analysis scripts quantify four parameters of the transient: peak amplitude (peak), RT, decay constant (DT1), and recovery time (DT2). For descriptions of each parameter, see Results.
- Figure 2.
Linear regression analysis of calcium transient variables. A–H, Other than slope and peak amplitude (R 2 = 0.88), only a fraction of variability in one variable was predicted by other variables (R 2 range, 0.04–0.28). DT1, the time required to decay 63% from the peak fluorescence; DT2, the residual recovery time necessary to recover from DT1 to 90% of resting values. Resting F, fluorescence of the cell at rest, plotted as PMT a.u./100.
- Figure 3.
Histograms illustrating distributions of four parameters quantified from each cell imaged. A–D, Frequency (gray boxes) and cumulative (black line) histograms for each parameter. Sample size for each parameter is given in the upper right-hand corner. Distributions do not show evidence of multimodality.
- Figure 4.
Variation in calcium transients between cells, animals, and sexes. Each box plot represents data from 7–15 cells from one animal. Animals are grouped together by litter (labeled A-K). Shaded boxes indicate males and empty boxes indicate females. For each box plot: lower bar = first quartile, box bottom = 2nd quartile, horizontal line = median, x = mean, box top = 3rd quartile, top bar = 4th quartile, circles outside plot = data more or less than 1.5 times the interquartile range (box). A, Tall boxes and bars demonstrate that the peak amplitudes of calcium transients varied from cell to cell. Differing medians, means, and box heights show that peak amplitudes also varied from animal to animal. There are no differences between males and females or litters in the distribution or variability of peak amplitudes. B, In contrast to peak amplitude, resting fluorescence (Resting F; plotted as PMT a.u./100) is more consistent across cells, animals, and sexes. C–E, RT, DT1, and DT2 also demonstrate less variability between cells and animals than peak amplitude.
- Figure 5.
Effects of alterations in electrical activity and extracellular calcium concentrations on calcium transients. A, Example calcium transients from one cell in three different bath conditions: ACSF with normal (2 mM), high (4 mM), and low calcium (no calcium added). In addition to manipulating extracellular calcium concentrations by changing bath calcium, intracellular calcium concentrations were manipulated by stimulating different numbers of action potentials (APs). For reference, the standard stimulation of 25 APs used elsewhere in this study is shown in yellow. B–F, Plots of transient parameter changes with varying bath calcium concentrations and AP numbers. Each dot represents the average of six cells. Error bars represent the SEM. When less than three cells produced measurable transient parameters, error bars were not possible and thus omitted, i.e., at one and five APs. Resting fluorescence is plotted as PMT a.u./100.
- Figure 6.
Calcium transients evoked by nerve stimulation at various frequencies. A, Example transients from one cell following stimulation for 0.5 s at frequencies from 1 to 300 Hz. For reference, the standard stimulation of 25 action potentials used elsewhere in this study is shown in yellow. B, Resting fluorescence measured in arbitrary units does not change with increasing stimulation frequencies. C–F, Plots of transient parameter changes with varying stimulation frequencies. Error bars indicate SEM for responses evoked by stimulation frequencies of 10 Hz and higher.
- Figure 7.
GCaMP6s calcium transients do not significantly change with repeat imaging. A, Example calcium transients from one cell imaged at baseline (black) and 30 min after vehicle (ACSF with 0.1% DMSO, gray). B, Mean values (±SEM) from 13 cells imaged from three animals at baseline and after vehicle. Paired t tests found no significant differences after addition of vehicle were detected. C–F, Baseline and vehicle values for 13 cells (gray lines). Black dotted line denotes the average change from baseline for each parameter.
- Figure 8.
GCaMP6s calcium transients provide insights into the activity of SERCA. A, Example calcium transient from a cell at baseline recording (black) and after the addition of the SERCA-inhibitor, TG (gray). B, Mean values (±SEM) from 16 cells from two animals imaged at baseline and after addition of TG. Paired t tests revealed significant changes, which are indicated (****p < 0.0001). C–F, Plots for transient parameters at baseline and after the addition of TG for 16 cells. Black dotted line denotes the average change from baseline for each parameter.
- Figure 9.
Inhibiting PMCA activity by elevating bath pH to 8.8 increases the calcium transients of PV+ neurons. Calcium transients are restored when bath pH is returned to 7.3. A, Example calcium transients from a cell in normal solution (pH 7.3, black), in elevated bath pH conditions (pH 8.8, light gray), and after returning to normal bath pH (pH 7.3, darker gray). B–F, Profiles of transient parameter changes in 22 cells from normal bath pH values (7.3) to elevated pH (8.8) and after returning to normal pH (7.3). Black dotted line denotes the average change from baseline for each parameter. G, Mean values (±SEM) from 22 cells from two animals imaged at baseline and elevated pH. Paired t tests revealed significant changes, which are indicated (**p < 0.01, ***p < 0.001, ****p < 0.0001).
Tables
Main effect p Female (n = 9) Male (n = 9) Peak (%ΔF/F) 0.77 55 [27–145] (97) 60 [24–135] (82) RT (s) 0.95 1.22 ± 0.39 (97) 1.22 ± 0.30 (82) DT 1 (s) 0.54 2.74 ± 1.08 (95) 2.59 ± 0.68 (82) DT 2 (s) 0.62 9.15 ± 1.51 (92) 8.96 ± 1.37 (79) Median [IQR] or mean ± SD. Parentheses indicate number of animals (headings) or cells (table). One-way mixed effects ANOVAs compared the transient properties of males and females and found no significant differences (p > 0.01) between sexes for these four properties.
pH 7.3 pH 8.8 pH 7.3 Resting F (a.u.) 583 ± 86 (22) 661 ± 143 (22)*** 580 ± 80 (22) Peak (%ΔF/F) 28 [18–71] (22) 55 [25–84] (22)** 23 [14–49] (22) RT (s) 1.21 ± 0.27 (21) 1.63 ± 0.36 (21)**** 1.17 ± 0.28 (21) DT 1 (s) 2.77 ± 0.82 (19) 4.71**** ± 1.78 (19)**** 2.22 ± 0.47 (19) DT 2 (s) 8.78 ± 0.99 (16) 10.49** ± 2.30 (16)** 7.88 ± 1.56 (16) Median [IQR] or mean ± SD. Parentheses indicate number of cells. Different from baseline: **p < 0.01, ***p < 0.001, ****p < 0.0001. Changes in calcium transient parameters in response to elevation of bath pH to block PMCA activity. Paired t tests revealed that parameter values significantly increase when bath pH is changed to pH 8.8 and recover when bath pH is returned to pH 7.3.
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