Abstract
Imaging techniques may overcome the limitations of electrode techniques to measure locally not only membrane potential changes, but also ionic currents. Here, we review a recently developed approach to image native neuronal Ca2+ currents from brain slices. The technique is based on combined fluorescence recordings using low-affinity Ca2+ indicators possibly in combination with voltage sensitive dyes. We illustrate how the kinetics of a Ca2+ current can be estimated from the Ca2+ fluorescence change and locally correlated with the change of membrane potential, calibrated on an absolute scale, from the voltage fluorescence change. We show some representative measurements from the dendrites of CA1 hippocampal pyramidal neurons, from olfactory bulb mitral cells and from cerebellar Purkinje neurons. We discuss the striking difference in data analysis and interpretation between Ca2+ current measurements obtained using classical electrode techniques and the physiological currents obtained using this novel approach. Finally, we show how important is the kinetic information on the native Ca2+ current to explore the potential molecular targets of the Ca2+ flux from each individual Ca2+ channel.
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Keywords
- Calcium currents
- Calcium imaging
- Voltage sensitive dyes imaging
- CA1 hippocampal pyramidal neuron
- Olfactory bulb mitral cell
- Purkinje neuron
- Brain slices
- Action potential
- Synaptic potential
- Biophysical modeling
4.1 Introduction
Optical measurements have been historically designed to monitor the electrical activity of the nervous system, a task where the use of electrode techniques has clear limitations [1]. In the last two decades, the development of new organic voltage sensitive dyes (VSD), in parallel with the progress of devices to excite and detect fluorescence [2], allowed optical recordings of sub-cellular membrane potential (Vm) changes <1 mV with a signal-to-noise ratio (S/N) comparable to that of patch clamp recordings [3]. This achievement suggested that voltage imaging can be used to investigate voltage-dependent proteins, in particular voltage-gated ion channels, in their physiological environment. The principal function of an ion channel is to allow an ion flux through a membrane, i.e. to produce an ionic current. Thus, the study of the biophysics of ion channels is routinely performed by measurements of ionic currents in single-electrode or two-electrode voltage clamp [4]. A way to investigate the biophysics of isolated native ion channels is to perform excised patches from ex-vivo membranes [5]. Alternatively, ion channels can be expressed in foreign cells such as oocytes or mammalian cell lines [6] and studied by using patch clamp techniques [7]. Yet, the physiological role and function of voltage-gated ion channels must be investigated in their natural environment, i.e. in their native cellular compartment and during physiological changes of Vm. To this purpose, the voltage clamp electrode approach has serious limitations for several reasons. First, the ionic current is measured by maintaining the cell at a given artificial Vm and even if the cell is dynamically clamped the Vm change is never a physiological signal [8]. Second, the current measured with the electrode is the summation of the filtered currents from all different cellular regions, including remote regions where Vm is unclamped, and no information is available on the site of origin of the current [9]. Third, different ionic currents contribute to the physiological change of Vm producing a functional coupling among the different ion channels [10]. Thus, a single native ionic current must be pharmacologically isolated from the total current mediated by the other channels, but the block of these channels will make the Vm change non-physiological.
In the last few years, we designed a novel approach to measure physiological Ca2+ currents from neurons in brain slices [11]. The method is based on fast Ca2+ optical measurements using low-affinity indicators that can be combined with sequential [12] or simultaneous [13] Vm optical recordings. The latter measurements can be calibrated in mV [14] using cell-specific protocols. Individual cells are loaded with Ca2+ and Vm indicators using a patch clamp recording. In contrast to voltage-clamp current measurements, the current approach permits independent recordings of the Vm change and of the Ca2+ influx, i.e. the study of voltage gating during physiological Vm changes. Since the Ca2+ current is reconstructed by the measurement of Ca2+ locally binding to an indicator, this approach provides information on channels in different areas of the cell with a spatial resolution as good as the optical recording allows. Finally, the Ca2+ current is recorded without blocking all Na+ and K+ channels that are necessary to produce the physiological Vm change. The principle of obtaining an optical measurement of a fast Ca2+ current is based on the analysis of the dye-Ca2+ binding reaction in a cell, a scenario initially studied by Kao and Tsien [15]. According to their theoretical estimates and to our recent empirical measurements [16], the relaxation time of the dye-Ca2+ binding reaction is less than 200 μs for low-affinity indicators with equilibrium constant (KD) ≥ 10 μM such as Oregon Green BAPTA-5N (OG5N, KD = 35 μM, [17]) or Fura-FF (KD = 10 μM, [18]). Therefore, a fast Ca2+ current with duration of a few milliseconds can be reliably tracked by low-affinity indicators if fluorescence is acquired at sufficiently high speed. The goal of this methodological article is to provide an exhaustive tool for those scientists aiming at performing this type of measurement. The next section addresses in detail the problem of extracting the Ca2+ current kinetics from Ca2+ fluorescence measurements under different cellular buffering conditions. The following section is devoted to the technical aspects of how to set up combined Vm and Ca2+ optical measurements and to calibrate Vm signals on an absolute scale. We then illustrate some examples of combined Vm and Ca2+ current measurements and we finally discuss how to correctly interpret the results and how to use this information to significantly advance our knowledge on Ca2+ channels function. All data shown here were from experiments performed at the Laboratoire Interdisciplinaire de Physique and approved by the Isere prefecture (Authorisation n. 38 12 01). These experiments were performed at 32–34 °C using brain slices from 21 to 40 postnatal days old C57Bl6 mice of both genders.
4.2 Extracting Ca2+ Current Kinetics from Ca2+ Fluorescence Measurements
4.2.1 Biophysical Foundations of Ca2+ Currents Imaging
An optical measurement of a Ca2+ signal is ultimately a measurement of the Ca2+ indicator bound to Ca2+ ions, which is proportional to the Ca2+ fractional change of fluorescence (ΔF/F0) if the indicator is not saturated. If the kinetics of the Ca2+-binding reaction of the indicator is slower than the kinetics of the Ca2+ source, and imaging is performed at higher rate, the time-course of Ca2+ ΔF/F0 essentially tracks the kinetics of the chemical reaction. Alternatively, if the kinetics of the Ca2+-binding reaction is faster than the kinetics of the Ca2+ source, the Ca2+ ΔF/F0 signal tracks the kinetics of the Ca2+ source. It follows that the equilibration (or relaxation) time of the Ca2+-indicator binding reaction is a crucial variable to use the technique to investigate the biophysics and the physiology of the Ca2+ source. The relaxation of the Ca2+- binding reactions for early indicators was studied by Kao and Tsien [15] who established that the rate of association for all these molecules is limited by diffusion leading to an association constant of ∼6·108 M−1 s−1. Thus, both the equilibrium constant (KD) and the equilibrium time are determined by the dissociation constant, i.e. the lower is the affinity of the indicator the shorter is its equilibrium time. We have empirically demonstrated that indicators with KD ≥ 10 μM such as OG5N or FuraFF have relaxation time < 200 μs [16]. Since the kinetics of activation and deactivation of voltage-gated Ca2+ channels (VGCCs) during physiological changes of Vm (for instance action potentials), is governed by the kinetics of the Vm transient, it follows that the relaxation time for those indicators is shorter than the duration of the Ca2+ influx. Hence, since Ca2+ binds to the indicator linearly in time, the Ca2+ ΔF/F0 is proportional to the integral of the Ca2+ influx, i.e. to the integral of the Ca2+ current. In the cell, however, Ca2+ simultaneously binds to proteins that form the endogenous buffer and this binding is competing with the binding to the indicator. An endogenous buffer can be, in principle, at least as fast as the indicator in equilibrating. In this case, only a fraction of Ca2+ is bound to the indicator, but this fraction is proportional to the total Ca2+ entering the cell and therefore to the integral of the Ca2+ current. Alternatively, an endogenous buffer can equilibrate over a time scale that is longer than the duration of the Ca2+ current. In this case, Ca2+ first binds to the dye and later to the endogenous buffer, implying that part of Ca2+ moves from the indicator to the endogenous buffer during its relaxation time. Under this condition, the Ca2+ ΔF/F0 is not linear with the integral of the Ca2+ current over this time scale. To clarify this important concept we make use of two simple computer simulations shown in Fig. 4.1, produced by a model that takes into account the chemical reactions as well as an extrusion mechanism re-establishing the initial Ca2+ conditions over a time scale >100 ms. We analyse what hypothetically can happen if a Ca2+ current with Gaussian shape occurs in a cell filled with 2 mM OG5N. In the first simulation (Fig. 4.1a), the cell has only 1 mM of a fast endogenous buffer behaving with the same association constant of the indicator and KD = 10 μM. In the second simulation (Fig. 4.1b), the cell has additional 400 μM of a slower endogenous buffer with association rate ∼3 times slower than that of the indicator and KD = 0.2 μM. In the first case, the time derivative of the Ca2+ ΔF/F0 signal matches the kinetics of the Ca2+ current (Fig. 4.1a). In contrast, in the presence of the slower buffer, the time derivative of the Ca2+ ΔF/F0 signal has a negative component and does not match the kinetics of the Ca2+ current (Fig. 4.1b). In the next two paragraphs, we present the analysis strategies that can be applied to extract the kinetics of Ca2+ currents from Ca2+ imaging recordings.
4.2.2 The Case of Linearity Between Ca2+ Influx and Ca2+ Fluorescence Changes
The proteins expressed in a cell determine whether or not the time course of the Ca2+ ΔF/F0 signal is linear with the kinetics of the Ca2+ current. As previously demonstrated [11], in the case of linear behaviour, the Ca2+ ΔF/F0 signal must reach its peak and remain constant for a few milliseconds afterwards, i.e. for the entire duration of the current. As shown in the simulation of Fig. 4.1a, the kinetics of Ca2+ extrusion producing a slow decrease of the Ca2+ ΔF/F0 signal has negligible effect on the time derivative. Thus, the estimate of the Ca2+ current kinetics is reliably obtained by the calculation of the time derivative of the Ca2+ ΔF/F0 signal. This calculation, however, requires the signal noise to be smaller than the signal change between two consecutive samples. The classical way to achieve this necessary condition is to apply to the Ca2+ ΔF/F0 signal a “smoothing algorithm”, i.e. a temporal filter that reduces the noise of the signal with minimal distortion of its kinetics. At 20 kHz acquisition rate, we have found that the Savitky-Golay algorithm [19] is an optimal filtering tool permitting noise reduction of the signal without significant temporal distortion using time-windows of up to 20–30 samples [11]. The applicability of this strategy has however limitations, i.e. if the signal or the region of measurement are too small, or if the light is too dim, the smoothing of the signal might not be sufficient to reduce the noise down to the level permitting calculation of the time derivative. In this case, the alternative strategy to apply consists in fitting the raw or the filtered Ca2+ ΔF/F0 signal with a model function obtaining a noiseless curve that mimics the time course of the Ca2+ ΔF/F0 signal. A simple choice of function that resembles the time course of the Ca2+ ΔF/F0 transient is the sigmoid. In particular, we found that the product of three sigmoid functions always provides an excellent fit of the Ca2+ ΔF/F0 signal associated with a backpropagating action potential in CA1 hippocampal pyramidal neurons [16]. As shown in the example of Fig. 4.2a both strategies are faithful in correctly calculating the time derivative of the ΔF/F0 signal. In this example, a CA1 hippocampal pyramidal neuron was filled with 2 mM OG5N and the dendritic Ca2+ ΔF/F0 signal associated with a backpropagating action potential was recorded at 20 kHz and averaged over 16 trials. This high sampling frequency was necessary to avoid signal aliasing and therefore distortion of the kinetics of the current. The filtering strategy is the straightforward approach that enables the calculation of the time derivative, but it produces a curve with noise. The noise can be reduced (if possible) by increasing the number of trials to average or by enlarging the dendritic area from where fluorescence is averaged. The fitting strategy is less direct but it produces a noiseless curve and it is therefore the only possible approach when the noise of the Ca2+ ΔF/F0 signal is above a certain level, as quantitatively estimated in an original report [16]. In particular, this is the case when the current must be extracted from single trials or when the recording is obtained from small or relatively dim regions.
4.2.3 The Case of Nonlinearity Between Ca2+ Influx and Ca2+ Fluorescence Changes
The method of estimating the kinetics of a Ca2+ current by calculating the Ca2+ ΔF/F0 time derivative fails when Ca2+ unbinds from the indicator over a time scale that is longer than the current duration, but sufficiently short to distort the estimate of Ca2+ influx dynamics by fluorescence measurement. In other words, this method fails when the Ca2+ ΔF/F0 signal decays rapidly, after correction for bleaching, generating a negative component in its time derivative. Such a situation occurs, for example, where slow buffering is produced by Calbindin-D28k [20, 21] and Parvalbumin [22, 23]. As shown in the example of Fig. 4.2b, the Ca2+ ΔF/F0 signal associated with a climbing fibre excitatory postsynaptic potential (EPSP), recorded at 5 kHz from a dendritic region and averaged over four trials, decays rapidly after its maximum resulting in a negative component of its time derivative. The distortion from the linear behaviour produced by the slow buffers can be compensated by taking into account the kinetics of Ca2+ unbinding from the indicator. We have recently developed a successful method to achieve this goal [24]. The strategy is based on fitting the decay time of the Ca2+ ΔF/F0 signal with the result of a computer simulation of a model with a slow buffer. Initially the input current is the Gaussian function fitting the rising phase of the time derivative (that is still a good approximation of the initial part of the current). The kinetic parameters and the concentration of the slow buffer are set to obtain the best fit of the decay phase of the Ca2+ ΔF/F0 signal. Then, the kinetics of the Ca2+ current is obtained as summation of four Gaussian functions that maximise the match between the result of the computer simulation and the experimental Ca2+ ΔF/F0 signal. Although this new method provides only an indirect approximation of the kinetics of the Ca2+ current, this information is crucial at understanding the activation and deactivation of different types of VGCCs. In the dendrites of PNs, for instance, different Ca2+ current kinetics components are associated with the activation of P/Q-type VGCCs [25] and T-type Ca2+ channels [26] that can be in principle separated by pharmacological block of one component. Thus, the extrapolation of a curve that approaches the kinetics of the Ca2+ current can be used to quantitatively investigate the variability of channels activation at different dendritic sites, the modulation of channel activation due to physiological activity or to pharmacological action. Finally, it is important to say that such a strategy can be extended to estimate slower Ca2+ currents where the fitting procedure can be applied to the slower decay time due to Ca2+ extrusion [27].
4.3 Combining Membrane Potential and Ca2+ Imaging
4.3.1 Setting up Combined Voltage and Ca2+ Fluorescence Measurements
To combine Vm and Ca2+ optical measurements, the VSD and the Ca2+ indicator must have minimal overlap in the emission spectra. Water soluble voltage indicators with different excitation and emission spectra have been recently developed [28]. In particular, the red-excitable and IR emitting VSD ANBDQPTEA (or PY3283) is suitable for coupling with other optical techniques [29]. Nevertheless, the most used VSDs for single cell applications are still JPW3028 [30] and the commercially available JPW1114 [18]. These indicators have wide excitation spectrum in the blue/green region and they emit mainly in the red region. We have previously demonstrated that both indicators can be optimally combined with Fura indicators that are excited in the UV region and emit in the short green region [12]. In this case, VSDs were excited at 543 nm using a laser and Fura indicators were excited at 385 nm using a light emitting diode (LED) as shown in the scheme of Fig. 4.3a. Alternatively, simultaneous voltage and Ca2+ imaging can be achieved using Oregon Green, Calcium Green or Fluo Ca2+ indicators using blue light (470–490 nm) to excite both VSDs and Ca2+ indicators [31]. Simultaneous imaging of JPW1114 and OG5N was adopted to obtain the first combined measurement of Vm and Ca2+ currents using the configuration of Fig. 4.3b. This type of measurement, however, has several disadvantages. First, OG5N fluorescence has a small tail component in the red region [31] which can be negligible or not depending on the ratio of the two dyes at each site as well as on the ratio between the two signals. Thus, for example, it works in proximal dendrites of CA1 pyramidal neurons for signals associated with action potentials [11], where Vm fluorescence is stronger than Ca2+ fluorescence, but it does not in distal dendrites of cerebellar Purkinje neurons (PNs, data not shown), where Vm fluorescence is weaker than Ca2+ fluorescence. A second disadvantage is that the JPW1114 signal at 470 nm excitation is ∼4 times smaller than that at 532 nm excitation. If simultaneous recordings are not critical, one can replace them with sequential recordings obtained by alternating 470 nm and 532 nm excitation as used in a recent study [32]. Finally, a third disadvantage is that JPW1114 absorbs more in the blue range than in the green range, i.e. it exhibits toxic effects after fewer exposures. A crucial technical aspect to take into consideration while setting up combined voltage and Ca2+ fluorescence measurements is the ability to record the two signals simultaneously at high speed. To this purpose, the company RedShirtImaging (Decantur, GA) has developed a dual-head version of the SMQ NeuroCCD (Fig. 4.3c). This camera permits simultaneous image acquisitions from both heads at 5–20 kHz, i.e. at the required speed. A demagnifier developed by Cairn Research Ltd. (Faversham, UK) allows adjusting the size of the image before it is split in two images at the emission wavelengths of the two dyes. Thus, the alignment of the two heads of the camera allows obtaining, at each precise region of interest, the Vm and the Ca2+ signal.
4.3.2 Calibrating Membrane Potential Fluorescence Transients
The calibration of Vm optical signals on an absolute scale (in mV) is crucial to analyse the gating of Ca2+ channels at the same locations where Ca2+ recordings are performed. This is not, however, straightforward. Indeed, the fractional change of VSD fluorescence is proportional to Vm [33], but the linear coefficient between these two quantities depends on the ratio between the inactive dye and the active dye that varies from site to site. The inactive dye is bound to membranes that do not change potential and contributes only to the resting fluorescence, while the active dye is bound to the plasma membrane and contributes to the resting fluorescence, but also carries the signal. In particular, in experiments utilising intracellular application of the dye, inactive dye is the dye that binds to intracellular membranes and organelles. Since the sensitivity of recording varies from site to site, a calibration can be achieved only if a calibrating electrical signal that has known amplitude at all locations is available. Such a signal is different in different systems. In mitral cells of the olfactory bulb, the amplitude of an action potential is the same in the whole apical dendrite and it can be used to create a sensitivity profile of the measuring system [34]. Another type of calibrating electrical signal can be a slow electrical change spreading with minimal attenuation over relatively long distances. Such a signal can be used to reliably calibrate VSD signals in PNs [18]. An example of this type of calibration is reported in Fig. 4.4a. Starting from the resting Vm, that we assume nearly uniform over the entire cell, long current hyperpolarising or depolarising current pulses are injected to the soma via the patch pipette and the change in Vm is recorded. As shown by direct dendritic patch recording, the dendrite is hyperpolarised by the same amount of the soma [35]. Thus, the measurement of somatic hyperpolarisation can be used as voltage reference to calibrate the dendritic VSD fractional change of fluorescence (VSD ΔF/F0) optical signal, as shown in Fig. 4.4a. In contrast, a depolarisation step attenuates along the dendrite. A third type of calibrating signal is a uniform depolarisation over the entire dendritic tree using L-glutamate photolysis from 4-Methoxy-7-nitroindolinyl-caged-L-glutamate (MNI-glutamate) [14]. This calibration procedure is applicable to all membrane expressing a relatively large number of glutamate receptors, i.e. to dendrites with high densities of excitatory synapses. The calibration is based on the principle that if the ionotropic glutamate receptor becomes the dominant conductance in a particular neuronal compartment, its reversal potential will determine the membrane potential of the compartment. Thus, in the area where dominance of glutamate receptor conductance is obtained, the resulting Vm change will be the same and can be used to calibrate VSD signals. An example of this protocol to calibrate backpropagating action potentials in CA1 hippocampal pyramidal neurons is shown in Fig. 4.4b. The VSD ΔF/F0 signal associated with the backpropagating AP at different sites of the apical dendrites is variable and cannot be directly correlated with the absolute change of Vm. In the presence of 1 μM TTX, to block action potentials, L-glutamate is photoreleased to saturate glutamate receptors over the whole field of view. Since the recording is performed starting from the resting Vm, the size of the VSD ΔF/F0 corresponds to this potential in the whole illuminated area where Vm reaches the reversal potential of 0 mV. Thus, this information is used to extrapolate the Vm at each dendritic site.
4.4 Examples of Combined Voltage and Ca2+ Current Imaging
4.4.1 Ca2+ Currents Associated with Backpropagating Action Potentials in CA1 Hippocampal Pyramidal Neurons and in Olfactory Bulb Mitral Cells
In many neurons, action potentials generated in the axon hillock adjacent to the soma do not only propagate along the axon to reach neurotransmitter release terminals, but also backpropagate throughout dendrites to signal cell activation at the sites where the neuron receives the synaptic inputs. At least part of this information is given by the fast Ca2+ transients produced by activation of VGCCs caused by the dendritic depolarisation associated with the action potential. The analysis that can be performed using the present imaging method is therefore crucial at understanding signal processing in individual neurons, as well as the specific role and function of the diverse VGCCs activated in dendrites. The propagation of the action potential and the consequent activation of VGCCs may be very different in different neuronal systems. In CA1 hippocampal pyramidal neurons, action potentials attenuate along the dendrite and activate both high-voltage activated (HVA) and low-voltage activated (LVA) VGCCs [36, 37]. We have very recently demonstrated that HVA-VGCCs and LVA-VGCCs operate synergistically to stabilise Ca2+ signals during burst firing [32]. Somatic and dendritic action potentials, at nearly physiological temperature, have 1–4 ms duration as in the example shown in Fig. 4.5a. In agreement with this evidence, the kinetics of the Ca2+ current is similar to that of the action potential, with a peak delayed by a few hundred milliseconds from the peak of the action potential. In total contrast to the CA1 hippocampal pyramidal neuron, in olfactory bulb mitral cells the action potential does not attenuate along the dendrites [38]. In addition, as shown in the representative example of Fig. 4.5b, the somatic and dendritic action potential at near physiological temperature (32–34 °C) has duration <1 ms. Thus, in this system, the activation and deactivation of VGCCs is also faster leading to a Ca2+ current with shorter duration and shorter delay from the Vm waveform peak. This preliminary comparison between the two cases indicates that the role of VGCCs, activated by the action potential, is different in different systems. For example, Ca2+ currents are delayed by ∼100 μs in presynaptic terminals where the function of this signal is to trigger neurotransmitter release [39]. Here, the kinetics of the Ca2+ current was obtained by calculating the time derivative of the Ca2+ ΔF/F0 signal fit [16]. VGCCs contribute to the shape of the action potential directly and indirectly by activating K+ channels, but also provide a precise time-locked Ca2+ transient capable to select fast-activated Ca2+ binding proteins. The possibility to locally investigate, using combined Vm and Ca2+ current optical measurements, the physiological occurrence of Ca2+ signals mediated by VGCCs will contribute enormously, in the near future, to the understanding of complex signal processing in neurons.
4.4.2 Ca2+ Currents Associated with Climbing Fibre EPSPs in Cerebellar Purkinje Neurons
In contrast to pyramidal neurons of the cortex and hippocampus, and to olfactory bulb mitral cells, somatic/axonal action potentials in PNs do not actively propagate in the dendrites [40]. The dendrites of PNs, however, express P/Q-type HVA-VGCCs [25] and T-type LVA-VGCCs [26] that are activated by the dendritic depolarisation produced by climbing fibre EPSPs. As shown in the example of Fig. 4.6, the shape of the dendritic Vm calibrated in Fig. 4.4a is quite different in the soma and in the dendrite, mainly reflecting the absence of Na+ action potentials in the dendrite. In this system, the low-affinity Ca2+ indicator used to estimate the Ca2+ current was Fura-FF, since the larger Ca2+ signal produced by OG5N contaminated the optical Vm measurement. The prominent dendritic depolarisation produces a biphasic Ca2+ current, which is in this case obtained by applying our recent generalised method [24]. The fast and sharp component is nearly concomitant to the short period in which Vm > −40 mV and it is therefore likely mediated by HVA-VGCCs. The slower and more persistent component is instead mostly concomitant to the whole depolarisation transient and is therefore likely mediated by LVA-VGCCs, as demonstrated by selectively blocking T-type VGCCs (unpublished data not shown). The analysis of Ca2+ signalling associated with the climbing fibre EPSP is crucial for the understanding of synaptic plasticity in PNs [41]. Yet, while the role of the Ca2+ transient associated with the climbing fibre EPSP has been postulated to be auxiliary to the principal Ca2+ signal mediated by parallel fibre EPSPs, these first measurements of the Ca2+ current kinetics elucidate a quite precise timing of occurrence of the Ca2+ source that may eventually provide a less ambiguous explanation of the precise function of this spread dendritic depolarisation. In summary, the examples illustrated in this section show the potentials of this novel optical method in providing physiological information not available by using electrode techniques.
4.5 Data Interpretation and Future Prospective
The method described here has been developed to overcome the limitations of single-electrode or two-electrode voltage clamp techniques permitting the analysis of physiological Ca2+ currents of native Ca2+ channels. Indeed, in contrast to patch-clamp recordings, these Ca2+ optical currents can be measured in conditions of a physiological change of Vm and the measured currents are confined to the sites where they are recorded, as shown in apical dendrites of hippocampal pyramidal neurons [32]. The additional information on local Vm change, necessary to correlate the behaviour of the conductance with its biophysical properties, is obtained by combining VSD imaging. In cases of linear behaviour between Ca2+ influx and Ca2+ fluorescence changes the kinetics of the Ca2+ current can be extracted by calculating the time derivative of the Ca2+ ΔF/F0 signal using low-affinity Ca2+ indicators [11, 16]. In the case of nonlinear behaviour between Ca2+ influx and Ca2+ fluorescence, produced by Ca2+-binding proteins with slower kinetics with respect to the Ca2+ current, the kinetics of the Ca2+ current can be still correctly estimated by taking into account the faster unbinding of Ca2+ from the low-affinity indicator [24]. In this last section we address the question of how data, obtained using this technique, should be interpreted. In Ca2+ current recordings from channels expressed in heterologous systems using voltage clamp, Vm is controlled artificially and its change is therefore independent of the channel deactivation. Under physiological conditions, Ca2+ channels contribute to the Vm change directly, through the ion flux, and indirectly by regulating other conductances. It follows that the channel deactivation changes the Vm waveform. We have shown that in CA1 hippocampal pyramidal neurons this phenomenon produces a modulation of LVA-VGCCs by HVA-VGCCs [32]. More in general, a Ca2+ current mediated by diverse VGCCs is always the result of a synergy among all different ion channels contributing to the Vm waveform. It follows that in a Ca2+ current optical measurement, a single component of the current cannot be extracted simply by blocking the underlying channel, since this block may affect the residual current as well. This evidence has important implications in the study of transgenic animals carrying Ca2+ channel mutations. In this case, a certain phenotype is likely to result from the combined modification of function of many different channels, rather than from the specific Ca2+ influx component, making the study of these animals as models for disease challenging. In summary, the investigation of the role and function of individual Ca2+ channels must be performed in the global context of activation of all channels participating to the local Vm waveform.
Another important aspect of data interpretation is the relation of the kinetics of Ca2+ current with the putative molecular targets of Ca2+ ions entering the cell. While importance is normally given to possible molecular coupling between the Ca2+ channel and the Ca2+ binding protein, the kinetics of the Ca2+ current can be a potent selector of the molecular pathway which is activated. To illustrate this important concept we make use of computer simulations using the same theoretical framework for simple Ca2+-binding dynamics that we already used in the past [42]. We imagine the possible activation of two proteins: a “fast” protein with KON = 5.7·108 M−1 s−1 and KD = 10 μM, expressed at the concentration of 500 μM; and a “slow” protein with KON = 4·108 M−1 s−1 and KD = 0.4 μM, expressed at the concentration of 100 μM. In the first case, shown in Fig. 4.7a, the cell is receiving a fast Ca2+ current with ∼ 2 ms total duration which binds first to the fast protein and later to the slow protein. In the second case, shown in Fig. 4.7b, the cell is receiving a slower Ca2+ current that is smaller in amplitude but that carries approximately the same amount of Ca2+. In this case the slow protein binds to Ca2+ with a slower kinetics but the amount of the fast protein binding to Ca2+ is less than half with respect to the first case. These simulations indicate that the ability to activate for a molecular pathway triggered by the fast protein strongly depends on the kinetics of the Ca2+ current. Thus, the approach described here should drastically improve our understanding of the physiological function of Ca2+ channels by providing the possibility to explore the biophysics of native channels during physiological activity locally within the complex neuronal architecture. The examples of combined Vm and Ca2+ current optical measurements from CA1 hippocampal pyramidal neurons, olfactory bulb mitral cells and PNs reported here are representative of the types of exploration that can be performed using this novel approach.
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Acknowledgment
This work was supported by the Agence Nationale de la Recherche through three grants: (1) Grant WaveFrontImag, program number ANR-14-CE17–0006-01; (2) Labex Ion Channels Science and Therapeutics, program number ANR-11-LABX-0015; (3) National Infrastructure France Life Imaging “Noeud Grenoblois”; and by the Federation pour la recherché sur le Cerveau (FRC) through the grant Espoir en tête (in partnership with Rotary France).
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Ait Ouares, K., Jaafari, N., Kuczewski, N., Canepari, M. (2020). Imaging Native Calcium Currents in Brain Slices. In: Islam, M. (eds) Calcium Signaling. Advances in Experimental Medicine and Biology, vol 1131. Springer, Cham. https://doi.org/10.1007/978-3-030-12457-1_4
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