Commonly used voltage sensitive dyes such as di-4-ANEPPS, di-8-ANEPPS and Di-2-ANEPEQ (JPW-1114) have wide excitation spectrum in the blue/green region and wide emission spectrum in the red/IR region (Fig. 4.2). If combined with Fluo, Calcium Green or Oregon Green Ca²⁺ indicators, both dyes can be excited by a blue wavelength (which, however, is not optimal for the voltage-sensitive dye) and green (Ca²⁺) and red (V_m) fluorescence can be recorded separately. Voltage sensitive dye excitation is optimal at 532 nm (Canepari et al. 2010). Thus, if combined with UV-excitable Fura indicators, the voltage-sensitive dye can be excited at its optimal visible wavelength and, as with the previous combination, the two emission signals can be recorded separately.

Although both types of combinations allow simultaneous measurement of V_m and Ca²⁺ fluorescence, many studies were initially performed through sequential recordings of the two optical signals. Combined V_m and Ca²⁺ imaging from individual neurons using blue-excitable/green-emitting Ca²⁺ indicators has been also used in several other studies. In the barrel cortex, the high-affinity indicator Oregon Green BAPTA-1 was combined with the voltage sensitive dye JPW-1114 in recordings from individual neurons and with the voltage indicator RH-1691 in in vitro and in vivo network recordings (Berger et al. 2007). In prefrontal cortical neurons, dendritic recordings were obtained combining JPW-1114 either with the high-affinity Ca²⁺ indicator Calcium Green-1 or with the low-affinity Ca²⁺ indicator Fluo-5F (Milojkovic et al. 2007).

The use of UV-excitable Fura indicators minimizes the spectral overlap with the voltage-sensitive dye. Additionally, the voltage-sensitive dye is not excited during Ca²⁺-imaging preventing a substantial photodynamic damage that can occur during relatively long recording periods (Canepari et al. 2008). UV-excitable Fura indicators with different equilibrium constants (K_d) are commercially available (see Table 4.1). This permits the choice of the most suitable indicator for the measurement of either the Ca²⁺ influx (high-affinity indicator) or the change in intracellular free Ca²⁺ concentration (low-affinity indicator).

In contrast to the indicators excited in the visible range, Fura indicators allow ratiometric measurements. When excited above their isosbestic wavelength (~360 nm), the changes of fluorescence associated with an increase in Ca²⁺ concentration are negative. Imaging above the isosbestic wavelength has several advantages. First, the resting fluorescence, which corresponds to nominally 0 Ca²⁺, is significantly higher than with dyes that increase their fluorescence with Ca²⁺. Second, the achievable imaging contrast is high because healthy cells are characterized by very low resting Ca²⁺, and the dye in adjacent structures, exposed to millimolar Ca²⁺ concentrations, is essentially not fluorescent. Third, since the dynamic range of the fluorescence is ~1 after subtraction of fluorescence at saturating Ca²⁺, these measurements allow a better quantitative estimation of the Ca²⁺ signals. An example of V_m and Ca²⁺ optical signals recorded sequentially from the two indicators in three different regions on the dendritic tree of a Purkinje neuron stained by JPW-1114 and Fura-FF is illustrated in Fig. 4.3. Using an excitation band of 387 ± 5 nm for Fura-FF (Fig. 4.3a), the Ca²⁺ increase associated with a climbing fibre excitatory postsynaptic potential (EPSP) corresponds to a negative ΔF/F₀ (Fig. 4.3b). Sequential V_m and Ca²⁺ recording using Fura Ca²⁺ indicators has been done in CA1 hippocampal pyramidal neurons (Canepari et al. 2007), in prefrontal cortex layer-5 pyramidal neurons (Milojkovic et al. 2007) and in cerebellar Purkinje neurons (Canepari and Vogt 2008). Sequential recording (as well as signal averaging) is meaningful only if repeated application of the same stimulation protocol results in the same response. This requirement must be confirmed experimentally by comparing individual recordings as shown in Fig. 4.3c. In this example, the V_m and the Ca²⁺ optical signals were recorded from a dendritic location on a cerebellar Purkinje neuron in response to four repetitions of climbing fibre activation separated by 1 min. The results showed that signals were practically identical in four individual trials (gray traces) permitting the correlation of V_m and Ca²⁺ optical signals recorded sequentially in response to the same stimulus.

An important aspect of combined V_m and Ca²⁺ imaging is the interpretation of Ca²⁺ optical signals, which depends on how much the Ca²⁺ indicator perturbs the physiological Ca²⁺ homeostasis. The buffering capacity of a Ca²⁺ indicator, K _dye , defined as the ratio between the dye-bound Ca²⁺ and the free Ca²⁺ in the presence of the indicator depends on the dissociation constant and on the concentration of the indicator (Table 4.1). The perturbation of the physiological Ca²⁺ introduced by the Ca²⁺ indicator can be evaluated by comparing the parameter K _dye with the endogenous buffering capacity of the cell (K _cell ). The interpretation of Ca²⁺ optical signals is simplified when K _dye is either much larger or much smaller than K _cell , i.e. when most of Ca²⁺ binds to the indicator or when the fraction of Ca²⁺ bound to the indicator is negligible compared to the total Ca²⁺ (Canepari et al. 2008). Without addressing in detail the issue of calibration of Ca²⁺ optical signals (Neher 2000) in this volume focused on V_m imaging, we will note that, in the first case, the ΔF/F₀ signal associated with Ca²⁺ increase is approximately linear with the total intracellular Ca²⁺ signal. In the second case, the time course of the ratio (F − F_min)/(F_max − F), where F_min and F_max are the fluorescence intensities at 0 and saturating Ca²⁺ respectively, is linear with the physiological intracellular free Ca²⁺ concentration change (Δ[Ca²⁺]_i). In many instances, Ca²⁺ signals are due to Ca²⁺ influx through a Ca²⁺ channel in the plasma membrane. The contribution to the change in membrane potential due to the Ca²⁺ influx depends on the Ca²⁺ permeability of the channel relative to its permeability to other ions, in particular to Na⁺ and K⁺. This is different for VGCCs, AMPA receptors, NMDA receptors and other Ca²⁺ permeable pores such as transient receptor potential (TRP) channels. Vice-versa, because opening or unblocking of Ca²⁺ channels often depends, in a non-linear manner, on the local membrane potential, the Ca²⁺ influx is often, but not always, larger when the voltage related depolarizing optical signal is larger (Canepari et al. 2007). Because this bi-directional relationship is complex, the analysis of V_m and Ca²⁺ signals from multiple sites on individual neurons is not straightforward and requires careful interpretation.

A notable application of combined V_m and Ca²⁺ imaging is the study of synaptic plasticity where the supra-linear Ca²⁺ signal plays an important role. The supra-linear Ca²⁺ signal occurs when two stimulation protocols are combined to evoke the coincident activity (pairing protocol) which results in a Ca²⁺ signal that is larger than the sum of the two Ca²⁺ signals associated with the application of individual stimulation protocols. A supra-linear Ca²⁺ signal is always caused by a supra-linear Δ[Ca²⁺]_i, but not necessarily by a supra-linear Ca²⁺ influx through the plasma membrane. Indeed, the supra-linear Ca²⁺ signal might be caused by Ca²⁺ release from internal stores or by the saturation of the endogenous Ca²⁺ buffer. However, in the latter case, if the buffering capacity of the dye dominates over the endogenous buffering capacity of the cell and the dye is not saturated, the presence of the indicator will cancel the supra-linear Δ[Ca²⁺]_i and no Ca²⁺ dependent ΔF/F₀ will be observed. Thus, in these conditions, detection of supra-linear Ca²⁺ dependent ΔF/F₀ signals which are not due to Ca²⁺ release from internal stores always corresponds to a supra-linear Ca²⁺ influx and it must always correlate with an increase in the depolarizing V_m signal. In contrast, if the buffering of the dye is negligible compared to the endogenous buffering of the cell, a supra-linear Δ[Ca²⁺]_i signal that is due to the saturation of the endogenous Ca²⁺ buffer can be measured reliably. The two different types of supra-linear Ca²⁺ signals not involving Ca²⁺ release from stores are illustrated by the two following examples.

In measurements from CA1 hippocampal pyramidal neurons (Canepari et al. 2007), cells were loaded with the voltage-sensitive dye JPW-3028 and 300 μM Bis-Fura-2. The purpose of these experiments was to analyze, over large regions of the dendritic tree, the V_m and Ca²⁺ optical signals associated with back-propagating action potentials, with excitatory postsynaptic potentials (EPSPs) and with pairing of these two membrane potential transients using an LTP induction protocol. Pyramidal neurons in the CA1 region are characterized by relatively low endogenous buffering capacity in the dendrites (~100) and even lower buffering capacity (~20) in the spines (Sabatini et al. 2002). Thus, the buffering capacity of 300 μM Bis-Fura-2 (K_d ~500 in the presence of Mg²⁺, see Table 4.1) is ~6 times larger than the buffering capacity of the dendrite and ~30 times larger than the buffering capacity of the spine. Ca²⁺ signals associated either with back-propagating action potentials or with EPSPs were mediated by Ca²⁺ influx through voltage-gated Ca²⁺ channels and/or through NMDA receptors. As shown in Fig. 4.4a, the pairing of the two stimulating protocols elicited a supra-linear Ca²⁺ signal. In the presence of the NMDA receptor blocker AP-5, since the measurements were done using a non-saturating concentration of a high-affinity indicator, the supra-linear Ca²⁺ signal must have been caused by supra-linear Ca²⁺ influx mediated by recruitment of additional VGCCs. In agreement with this expectation, at each site where a supra-linear Ca²⁺ signal was observed, the V_m related optical signal during the pairing protocol had a larger peak depolarization compared to the signals associated with unpaired stimulations (Fig. 4.4a).

In measurements from cerebellar Purkinje neurons (Canepari and Vogt 2008), cells were loaded with 1 mM of the Ca²⁺ indicator Fura-FF and with the voltage sensitive dye JPW-1114. Purkinje neurons have an exceptionally high dendritic K _cell estimated at ~2,000 (Fierro and Llano 1996). Therefore, the addition of a low affinity Ca²⁺ indicator such as Fura-FF (K_d ~10), even at millimolar concentrations, does not significantly alter the physiological homeostasis and it is possible to record changes in free intracellular Ca²⁺ concentration (Δ[Ca²⁺]_i signals Canepari et al. 2008). In this particular preparation, it was possible to calibrate V_m signals in terms of membrane potential. As shown in Fig. 4.4b, pairing the stimulation of the large climbing fibre synaptic potential with local parallel fibres stimulation generated a local dendritic supra-linear Δ[Ca²⁺]_i signal which was independent of Ca²⁺ release from stores (Brenowitz and Regehr 2005). Although the Δ[Ca²⁺]_i signal originated from Ca²⁺ influx through VGCCs, in Purkinje neurons, in contrast to CA1 pyramidal neurons, the V_m signal during the pairing protocol did not have larger peak depolarization compared to the V_m signals associated with unpaired stimulations (Fig. 4.4b). In this case the Ca²⁺ influx associated with the climbing fibre synaptic potential was the same in paired and unpaired conditions. The corresponding Δ[Ca²⁺]_i signal, however, was larger following paired stimulation because the Ca²⁺ influx associated with the parallel fibre stimulation locally and transiently saturated the endogenous Ca²⁺ buffer (Canepari and Vogt 2008). This supra-linear Ca²⁺ signal could be correctly interpreted only when recorded using a low-affinity indicator. When the cell was injected with 10 mM Bis-Fura-2, K _dye became ~10 times larger than K _cell and supra-linear Ca²⁺ signals were abolished (Canepari and Vogt 2008).

Sequential V_m and Ca²⁺ imaging produce valid results if identical responses are evoked in repetitive measurements. Many physiological processes, however, are stochastic, showing large trial-to-trial variability. In these cases, the study of the time variability contains important biological information and simultaneous V_m and Ca²⁺ optical measurement is required. Simultaneous V_m and Ca²⁺ imaging was initially achieved using the Ca²⁺ indicator Calcium Orange, which has excitation peak at ~550 nm and emission peak at ~580 nm (Sinha et al. 1995; Sinha and Saggau 1999), or with blue-excitable indicators (Bullen et al. 1997; Bullen and Saggau 1998). These possibilities may have, however, some inherent problems. First, the small overlap between the emission spectra of the two dyes may produce a “bleeding” of the V_m signal into the Ca²⁺ recording channel that contaminates the signal. In some situations, such emission cross talk can be mathematically corrected (Sinha et al. 1995). Second, while V_m recordings of synaptic or action potentials can be often achieved within short time, the complete time-course of the corresponding Ca²⁺ signal may require longer acquisition intervals (Canepari and Vogt 2008). In this case, the voltage sensitive dye is exposed to high intensity light for a longer time than what would be required for the V_m measurement alone, increasing both bleaching of the dye and phototoxicity. Simultaneous V_m and Ca²⁺ imaging was also performed using the combination of an absorption voltage sensitive dye with a fluorescent Ca²⁺ indicator. Using bath application of the absorption dye RH-482 and a low-affinity Ca²⁺ indicator Magnesium Green, Sabatini and Regehr could correlate the Ca²⁺ signal underlying synaptic release with the excitation of pre-synaptic terminals in cerebellar synapses (Sabatini and Regehr 1996; Sabatini and Regehr 1997). In these experiments, simultaneous Ca²⁺ and V_m measurements were done by using excitation light wavelength of ~500 nm and recording simultaneously fluorescence from the Ca²⁺ indicator Magnesium Green and the transmitted light from voltage-sensitive dye RH-482 at 710 nm from a single location with two photodiodes. The important limitation of this approach is that the absorption measurements from multiple sites on individual neurons are substantially less sensitive compared to fluorescence measurements (Antic and Zecevic 1995). In the above example, extensive temporal and spatial signal averaging over a large region of the cerebellar slice, which could be obtained with absorption signals, permitted signal detection from presynaptic terminals (Sabatini and Regehr 1997).