Simultaneous recordings of voltage and current waveforms from dendrites

In a typical ‘voltage‐clamp’ experiment, membrane channels are studied by artificial rapid steps of voltage and the resulting transmembrane current is thus unnatural (Fig. 1 A). That is to say, those current waveforms we observe in published articles do not exist in real biological conditions. The only way to gain insight into the dynamics of transmembrane current during a natural biological event, like an action potential for example, is to extract the physical properties of a channel by a series of voltage‐clamp experiments and then incorporate these properties into a computational model (Hodgkin et al. 1952). Only a model can simultaneously display voltage and current waveforms on the same time scale (Fig. 1 B). For example, during a model action potential (AP), the model sodium current shows an interesting ‘kink’ in the middle of the trace (Fig. 1 B, arrow) – a kink never seen in a real experimental measurement, because one cannot measure current (waveform) while measuring a natural membrane potential waveform. In the current issue of The Journal of Physiology, Jaafari and Canepari offer an interesting solution to this technical limitation. They record membrane potential waveform with voltage‐sensitive dye and calcium current waveform with a calcium‐sensitive dye (Jaafari & Canepari, 2016). To extract the waveform of the dendritic voltage‐gated calcium current, Jaafari and Canepari combine three strategies: (i) low affinity calcium indicator dye OGB‐5N (K _d = 20 μm), (ii) fast optical recording at 20 kHz frame rate, and (iii) mathematical transformation (derivative of ΔF/F). The resulting optical waveforms match the calcium current waveforms (Jaafari et al. 2014). This new exciting method for measuring transmembrane current waveforms optically has three very useful features.

1. Space‐clamp is not an issue. Recall that the current measured with the dendritic electrode is the sum of the filtered currents from all neuronal compartments, including remote compartments where membrane voltage is unclamped (Williams & Mitchell, 2008). The Jaafari and Canepari method effectively eliminates the space clamp problem because this method does not rely on voltage clamp. Also, the locally recorded calcium signal pertains to the local calcium current.

2. Thin dendrites are accessible. Some dendritic segments are so thin that they do not tolerate patch electrodes. Hence, calcium currents cannot be studied in thin dendritic branches using the dendritic patch method (Magee & Johnston, 1995). Optical recordings, on the other hand, do not have such limitations. Optical recordings can measure calcium current in all visible dendrites, in theory.

3. Multiple dendritic locations in one trial. The Jaafari and Canepari optical method measures calcium current at several dendritic segments simultaneously, during the same AP event. The calcium influx recorded optically from a region of interest reports the activity of calcium channels in that region only. So, there is an immediate spatial map of active currents (i.e. channels) emerging from the optical measurements (Jaafari & Canepari, 2016). It is necessary to note that, although useful in variety of experimental designs, such maps do not provide accurate information on channel density or their physical properties, because the AP waveform cannot explore the biophysics of the membrane conductances the way voltage steps do (Magee & Johnston, 1995). Also, ‘channel mapping’ will not work well in distal dendrites where AP amplitude is significantly attenuated. In the most distal dendritic regions, calcium channels may be present, but not responding to the somatic AP.

Figure 1. Voltage and current waveforms .

A, standard voltage‐clamp experiment. A sudden change in command voltage, from −70 to −20 mV, triggers fast inward sodium current. B, timing of the current injection pulse used to trigger AP (green trace) and current‐clamp ‘recording’ of an AP with underlying sodium current on the same time scale. Arrow marks a kink in the sodium current waveform. C, a ball‐and‐stick schematic diagram of a pyramidal neuron. Action potential propagates from the soma into the dendrite. Voltage‐sensitive dye signals (V _m) and calcium‐sensitive dye signals were recorded simultaneously from two regions of interest (ROIs 1 and 2). Calcium signals were transformed into calcium current, I _Ca. AP amplitude declines with distance from the soma (dashed black line), but voltage‐gated I _Ca is similar in two locations (dashed blue line).

In their article, Jaafari & Canepari (2016) report that in spite of the decrease in AP amplitude along the apical dendrite, the amplitude of the associated Ca²⁺ signal in the initial 200 μm dendritic segment does not change (Fig. 1 C). More specifically, using a dual voltage calcium imaging technique, the authors monitored AP voltage waveform and the activation of local calcium channels (calcium current waveform, I _Ca) at each dendritic segment on the way. Calibrated voltage‐sensitive dye signals indicated that AP amplitude decreases gradually with distance from the cell body (Fig. 1 C, black dashed line). The attenuated APs cannot open high voltage‐activated calcium channels in distal dendritic regions with the same rigour as in the proximal dendrite, where AP amplitude is high. In contrast, closer to the soma, a reduction in calcium influx though high voltage‐activated calcium channels is always perfectly compensated by the influx through low voltage‐activated calcium channels. The new method demonstrates that high voltage‐ and low voltage‐activated calcium channels operate synergistically to stabilise calcium signals associated with APs in the most proximal 200 μm dendritic segment (Jaafari & Canepari, 2016).

In the near future we may expect this type of optical measurement, optical measurement of I _Ca developed by Jaafari and Canepari, to be applied to thin dendritic branches of many neuronal types across the central nervous system. Similar measurements could perhaps be employed in the field of sodium currents. Development of a sodium‐sensitive dye with low K _d could potentially allow researchers to record the sodium current waveform (Fleidervish et al. 2010). In summary, we may soon be able to directly monitor sodium and calcium current waveforms during bursts of APs in different neurones and neuronal compartments, under control and test conditions.