A note on estimating absolute cytosolic Ca 2+ concentration in sensory neurons using a single wavelength Ca 2+ indicator

Background

Measuring changes in [Ca²⁺]_cyt is a commonly used method for determining the sensitivity of sensory neurons to different stimuli and investigating pro-nociceptive signalling pathways, with Fluo4 being a popular choice of single-wavelength Ca²⁺ indicator. As has been discussed in detail previously, ¹ one can estimate [Ca²⁺]_cyt from the optical properties of a fluorescent Ca²⁺ indicator using the following equation ²

[ C a 2 + ] c y t = K D F − F min F max − F

(1)

[ C a 2 + ] c y t = K D F F max − 1 R 1 − F F max

(2)

It is apparent that if R is large, as is the case for Fluo4 (R ≈ 85-100 ¹ ), then 1/R will become negligible (as 1/R << F/F_max), and equation (2) can be rewritten as

[ C a 2 + ] c y t = K D F F max 1 − F F max

(3)

The estimate of [Ca²⁺]_cyt provided by equation (3) does not require the experimenter to find F_min. F_max can be found easily at the end of each experiment by applying 0.1% Triton-X in a bathing solution containing 10 mM Ca²⁺. As R and K_D are properties intrinsic to the indicator, it is not necessary for them to be estimated for each experiment, ¹ though it is important to consider that these parameters are sensitive to the intracellular environment (e.g., pH, ionic composition). ⁵ Therefore, adding a straightforward step to the end of each Ca²⁺ imaging experiment can provide an estimate of [Ca²⁺]_cyt using Fluo4.

Estimating changes in [Ca²⁺]_cyt

Ionomycin is often used to calibrate Ca²⁺ signals measured in sensory neurons and while this calibration provides some useful information, it cannot be used to estimate absolute [Ca²⁺]_cyt. This is because ionomycin cannot raise [Ca²⁺]_cyt sufficiently to obtain a true F_max for the indicator. This is apparent if 50 mM KCl and 5 µM ionomycin are sequentially applied to sensory neurons in a bath solution containing 2 mM Ca²⁺ (Figure 1, trace i). Ionomycin application cannot have resulted in saturation of Fluo4 because the magnitude of Ca²⁺ transients evoked by KCl (black traces) exceeded those evoked by ionomycin. Similar results were obtained after raising bath [Ca²⁺] to 20 mM during the application of ionomycin, though the response to ionomycin was greater under these conditions (Figure 1, trace ii).OPEN IN VIEWER

While KCl is useful for identifying viable neurons, its use as a calibration for the magnitude of Ca²⁺ signals in sensory neurons is limited because the magnitude of the response to KCl is dependent on voltage-gated Ca²⁺ channel function and cytosolic Ca²⁺ buffering, which may not be comparable between different sensory neuron subpopulations ⁶ or different experimental conditions.

The absolute F_min or F_max for Fluo4 – as with other indicators – can be found by applying 0.1% Triton-X (to lyse cells) with either a high concentration of a Ca²⁺ chelator (1 mM EGTA) in Ca²⁺-free bathing solution (Figure 2(a), grey trace), or 10 mM Ca²⁺-containing bathing solution (Figure 2(a), black trace), respectively. The transient increase in F following the application of Triton-X/EGTA in Ca²⁺-free bathing solution (Figure 2(a), grey trace) partly represents liberation of Ca²⁺ from intracellular stores. It is important to note that in the case of equation (3), as F tends towards F_max, 1 – F/F_max will tend towards zero and, hence, estimates of [Ca²⁺]_cyt become unreasonably large. While F/F_max < 90%, F/F_max is approximately linear with [Ca²⁺]_cyt and provides a reliable measure of [Ca²⁺]_cyt (Figure 2(b)). Beyond 90%, F/F_max no longer provides a reliable metric of [Ca²⁺]_cyt and estimates become unreasonably large – as such, this has been dubbed the “zone of unreliability” (Figure 2(b), red shaded region ⁷ ). It is, therefore, important to ensure that F measured during an experiment remains within the approximately linear region of equation (3) and does not exceed 90% F/F_max.OPEN IN VIEWER

To test the utility of equation (3) for measuring [Ca²⁺]_cyt in sensory neurons, 50 mM KCl was used to induce depolarisation-dependent Ca²⁺ transients (n = 33 neurons, 2 mM bath Ca²⁺, Figure 2(c)). Estimates of [Ca²⁺]_cyt calculated using equations (1) and (3) were compared; the value for F_min used in equation (1) was found in the experiment shown in Figure 2(a). F_max was found for each neuron at the end of each experiment. The peak F evoked by KCl application remained below 90% of that evoked by Triton-X/10 mM Ca²⁺; peak F/F_max was 0.72 ± 0.02 (range: 0.45-0.87, Figure 2(d)). As such, reliable estimates of [Ca²⁺]_cyt were possible. Figure 2(e) and (f) show example traces of [Ca²⁺]_cyt obtained using equations (1) and (3), respectively. The data look qualitatively very similar. The congruence in the estimates of [Ca²⁺]_cyt found using equations (1) and (3) (Figure 2(g)) demonstrates that the dynamic range of Fluo4 is of sufficient magnitude to make its reciprocal negligibly small relative to F/F_max. The percentage error in the estimates of [Ca²⁺]_cyt provided by equations (1) and (3) was lowest during the peak of the KCl-evoked Ca²⁺ transient (0.97 ± 0.06%) and highest during baseline (4.36 ± 0.43%, Figure 2(h)). No difference in the estimated average baseline [Ca²⁺]_cyt was found between equations (1) and (3) (p = .26, Figure 2(i)). The estimates of peak [Ca²⁺]_cyt provided by equations (1) and (3) were also no different (p = .78, Figure 2(j)). Importantly, estimates calculated from equation (3) of both baseline (63.4 ± 2.3 nM) and peak (956.9 ± 88.1 nM) [Ca²⁺]_cyt agreed with previous estimates made using ratiometric Ca²⁺ imaging.^8–11 Changes in [Ca²⁺]_cyt evoked by 50 mM KCl are relatively large, but this method can also be used to calibrate smaller changes in [Ca²⁺]_cyt. The application of the algogenic mediator bradykinin (BK, 250 nM, 2 mM bath Ca²⁺) to sensory neurons raised [Ca²⁺]_cyt – calculated using equation (3) – from 60.3 ± 4.8 nM to 333.3 ± 39.6 nM (n = 13 neurons, Figure 2(k) and (l)).

Basal [Ca²⁺]_cyt

Multiple factors, including ageing ¹¹ and inflammation, ¹² can affect basal [Ca²⁺]_cyt in rodent sensory neurons. We have examined the effect of soma size on resting [Ca²⁺]_cyt in sensory neurons (Figure 3(a); soma area distribution shown in inset). Neurons were parsed into subgroups by soma area (A): small (A <400 µm², n = 86), medium (400 < A <1000 µm², n = 129) and large (A >1000 µm², n = 78). Although there was no difference in resting [Ca²⁺]_cyt between small- and medium-area neurons (73.0 ± 3.8 nM vs 74.3 ± 3.8 nM, p > .99, Figure 3(b)), basal [Ca²⁺]_cyt in large-area neurons (52.8 ± 2.6 nM) was lower than both small- (p = .0002) and medium-area (p < .0001) neurons (Figure 3(b)). Average basal [Ca²⁺]_cyt across all neurons in this sample was 68.2 ± 2.2 nM (n = 293 neurons from three independent DRG preparations), in agreement with previous estimates in rodent sensory neurons.^8–14 A modestly reduced resting [Ca²⁺]_cyt in large-diameter sensory neurons has been observed in rat, ⁶ but corroborating observations in mouse sensory neurons are lacking. It is not immediately clear why large-area sensory neurons would exhibit a lower resting [Ca²⁺]_cyt, though it could be due to more efficient [Ca²⁺] buffering. To test this possibility, another sample of neurons (n = 112 neurons from three independent DRG preparations) was stimulated with 50 mM KCl and the decay of evoked Ca²⁺ transients (expressed as the time constant for the decay of the transient) was analysed across different neuronal sizes. There was a modest negative correlation between soma area and time constant (r = −0.26, p = .0061, Figure 3(c)). KCl-evoked Ca²⁺ transients in small-area neurons exhibited a time constant of 13.0 ± 1.2 s (n = 39), compared to 8.0 ± 1.0 s (n = 23) in large-area neurons (p = .0068, Figure 3(d)). KCl-evoked Ca²⁺ transients in medium-area neurons exhibited an intermediate time constant (10.4 ± 0.8 s, n = 50) which was indistinguishable from that in small- (p = .25) and large-area (p = .26) neurons (Figure 3(d)). The more rapid decay of KCl-evoked Ca²⁺ transients in large-area neurons (compared with small-area neurons) is consistent with a greater capacity for Ca²⁺ buffering. ⁶ OPEN IN VIEWER

In summary, the method put forward by Maravall and colleagues ¹ provides a straightforward, easy-to-implement and – importantly – reliable protocol for estimating [Ca²⁺]_cyt in sensory neurons using a high dynamic range single-wavelength indicator without the need to estimate F_min. Estimates of [Ca²⁺]_cyt in sensory neurons made using this method with Fluo4 are well in-line with estimates made using other methods, including ratiometric Ca²⁺ imaging. F_min need not be estimated because the dynamic range of Fluo4 is sufficiently large, as evidenced by the close alignment of estimates of [Ca²⁺]_cyt between equations (1) and (3). Use of this method broadens the utility of Fluo4 and will enable more rigorous quantification of Ca²⁺ signalling in sensory neurons using Fluo4.

Preparation of sensory neurons

All animal work was carried out in accordance with the Animals (Scientific Procedures) Act 1986. Mice (C57Bl/6, Charles River) were housed in groups of up to six littermates under a 12 h light/dark cycle with bedding material, enrichment (e.g., igloos, tunnels, etc) and ad libitum access to food and water. All mice used were male aged 8-14 weeks.

Sensory neurons were prepared as described previously.^15–17 Briefly, dorsal root ganglia (DRG, T12-L5) were removed from the spinal column and enzymatically digested in collagenase (1 mg/mL) and trypsin (1 mg/mL). DRG were then mechanically dispersed by trituration through a pipette tip. Dispersed neurons were seeded onto glass-bottomed culture dishes coated with poly-d-lysine and laminin (MatTek, MA, USA). Neurons were incubated with supplemented L-15 growth medium (10% foetal bovine serum, 2.6% NaHCO₃, 1.5% d-glucose and 2% penicillin/streptomycin) at 37°C in 5% CO₂ and used for Ca²⁺ imaging no more than 24 h after plating.

Ca²⁺ imaging

Growth medium was aspirated from culture dishes and neurons were incubated with 10 µM Fluo4-AM for 30-45 min at room temperature (shielded from light). Neurons were then washed and bathed in extracellular bath solution containing (in mM): 140 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 4 d-glucose, 10 HEPES (pH 7.35-7.45 with NaOH; 290-310 mOsm). Ca²⁺-free bath solution was identical except for the omission of CaCl₂ and that it contained 1 mM EGTA and 2 mM MgCl₂. Dishes were mounted on an inverted Nikon Eclipse TE-2000S microscope and cells were visualised under brightfield illumination with a 10x air objective. Neurons were superfused with bath solution via a flexible inflow tube (placed adjacent to cells of interest) (AutoMate Scientific, CA, USA) fed by a gravity-fed perfusion system (Warner Instruments, CT, USA).

All imaging was carried out at room temperature. Images were acquired at 2.5 fps with 100 ms exposure using a Retiga Electro CCD camera (QImaging, BC, Canada). Fluo4 was excited by a 470 nm light source (Cairn Research, UK) and emission at 520 nm was recorded using µManager. ¹⁸ For experiments using 50 mM KCl as a stimulus, KCl was superfused for 30 s following a 10 s baseline. For experiments using 250 nM bradykinin as a stimulus, bradykinin was superfused for 30 s following a 20 s baseline. At the end of all experiments, 0.1% Triton-X in 10 mM Ca²⁺ bath solution was superfused to lyse cells and yield an estimate for F_max for each neuron. F_min was estimated in one experiment by applying 0.1% Triton-X to neurons bathed in Ca²⁺-free bath solution containing 1 mM EGTA.

Data analysis

Regions of interest were manually traced around neurons and the average pixel intensity per region per frame was calculated using ImageJ. After the subtraction of background fluorescence, measured fluorescence values for each neuron were calibrated to [Ca²⁺] using equations (1) and (3) using a K_D for Fluo4 of 325 nM.

Datasets were scrutinised to ensure that they met the assumptions of parametric analyses (normality tested using the Shapiro-Wilk test; equality of variances tested using F-tests) and, where appropriate, non-parametric, rank-based alternatives were used. Details of statistical tests used are in the figure legends. To find the time constant for the decay of KCl-evoked Ca²⁺ transients, data were fit with a one-phase exponential decay. The time constant gives the time for the transient to decay by a factor of 1/e, that is, ∼37% of peak. This is equivalent to T₅₀/ln(2), where T₅₀ is the half-life of the decay of the transient, that is, the time for the transient to decay by a factor of 1/2. Analysis was carried out in GraphPad Prism (GraphPad Inc.). Grouped data are displayed as mean ± standard error.