Infrared light excites cells by changing their electrical capacitance

Abstract

Optical stimulation has enabled important advances in the study of brain function and other biological processes, and holds promise for medical applications ranging from hearing restoration to cardiac pace making. In particular, pulsed laser stimulation using infrared wavelengths >1.5 μm has therapeutic potential based on its ability to directly stimulate nerves and muscles without any genetic or chemical pre-treatment. However, the mechanism of infrared stimulation has been a mystery, hindering its path to the clinic. Here we show that infrared light excites cells through a novel, highly general electrostatic mechanism. Infrared pulses are absorbed by water, producing a rapid local increase in temperature. This heating reversibly alters the electrical capacitance of the plasma membrane, depolarizing the target cell. This mechanism is fully reversible and requires only the most basic properties of cell membranes. Our findings underscore the generality of pulsed infrared stimulation and its medical potential.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Infrared laser pulses evoke inward currents in wild-type oocytes via a water-heating mechanism.

(a) Currents recorded in voltage-clamped oocytes held at −80 mV evoked by infrared laser pulses of 0.1 ms (0.3 mJ) to 10 ms (7.3 mJ) duration and energy. Red lines above each trace indicate when the pulse was applied. (b) I–V response to the application of a 1 ms (2.8 mJ) pulse. (c) I–V response to the application of a 10 ms (7.3 mJ) pulse. (d) Q–V curves acquired in H2O-based SOS solution (H2O), D2O-based SOS solution (D2O), SOS solution in which NaCl has been replaced by KCl (KCl), SOS solution in which NaCl has been replaced by N-methyl-D-glucamine–methanesulphonate (NMG–MS), and SOS solution supplemented with 1 mM amiloride and 1 mM oubain (Am+Ou), 250 μM ruthenium red (RuR) or 1 mM GdCl3 (Gd3+). N=5 for each measurement. Where they are not visible, error bars (s.e.m.) are smaller than the corresponding symbols. Linear fits are shown for the H2O, D2O and Gd3+ data to aid in their comparison. (e) I–V response to a 1 ms (2.8 mJ) pulse after the oocyte has been exposed to pulses of energy >8 mJ. I–V responses were recorded at equally spaced voltages from −100 to +100 mV. Traces are spaced and coloured for clarity. The red bar and grey shading indicate the timing of laser stimulation. (f) Relative conductance (normalized sum of current magnitudes at holding potentials of −80 and +40 mV) of voltage-clamped oocytes before and after stimulation with pulses >8 mJ. N=5. (g) Local temperature responses acquired using calibrated pipet resistance to infrared pulses of 0.1 ms (0.3 mJ) (blue), 0.5 ms (1.4 mJ) (cyan), 1 ms per 2.8 mJ (orange), 2 ms (5.6 mJ) (black) and 10 ms (7.3 mJ) (red). The grey trace shows temperature response to a 10 ms (7.3 mJ) pulse in D2O solution. The insert shows a comparison of temperature response and current recording to a 10 ms (7.3 mJ) pulse. (h) Voltage responses recorded in current-clamped oocytes stimulated with the same set of pulses (similarly colour-denoted as in (g)). The resting potentials were −46±4.6 mV. The insert shows a zoomed-in view of the first 30 ms following the start of the laser pulse. All error bars are ±s.e.m.

Figure 2. Infrared evokes inward currents in untransfected HEK cells.

(a) I–V response in voltage-clamped HEK293T cells to the application of a 0.2 ms (0.7 mJ) pulse. (b) I–V response to a 1 ms (3.7 mJ) pulse. I–V responses were recorded at equally spaced voltages from −120 to +120 mV. Traces are spaced and coloured for clarity. The red bar and grey shading indicate the timing of laser stimulation. (c) Q–V curves acquired in HEK cells with H2O-based recording solutions (H2O), D2O-based bath solution (D2O), with 1 mM GdCl3 added to bath solution (Gd3+ out), 1 mM GdCl3 added to pipet solution (Gd3+ in) or 20 mM MgCl2 added to pipet solution with 0 mM MgCl2 in the bath (Mg2+ in). N=5 for each measurement. Error bars are ±s.e.m. Data are fitted with lines to aid in their comparison. (d) Voltage responses recorded in current-clamped HEK cells with 0.5 ms (1.9 mJ) (blue), 1 ms (3.7 mJ) (cyan), 1.5 ms (5.6 mJ) (orange) and 2 ms (7.4 mJ) (red) pulses. Insert provides a zoomed-in view of the first 30 ms following the start of a 1-ms pulse.

Figure 3. Infrared transiently alters the membrane electrical capacitance of artificial lipid bilayers and HEK cells.

(a) I–V current response in voltage-clamped artificial lipid bilayer comprising (1:1) PE:PC (PC:PE) in symmetric NaCl solution to 1 ms (2.8 mJ) infrared pulse. (b) I–V current response in voltage-clamped PE:PC bilayer in symmetric NaCl solution to 10 ms (7.3 mJ) infrared pulse. Traces are coloured for clarity. The red bar and grey shading indicate the timing of laser stimulation. Voltages ranged from −200 to +200 mV. (c) Q–V curves acquired in PE:PC lipid bilayers in response to 10 ms (7.3 mJ) infrared stimulation in H2O-based (H2O) and D2O-based (D2O) symmetric NaCl solution. N=5 for each measurement. (d) Changes in membrane electrical capacitance in a PE:PC bilayer induced by 1 ms (2.8 mJ) (purple), 2 ms (5.6 mJ) (cyan), and 10 ms (7.3 mJ) (red) infrared pulses, determined from current responses to a sinusoidal voltage input. (e) Maximum changes in equivalent capacitance at each pulse energy (N=5). (f) Changes in membrane equivalent capacitance in HEK cells induced by 0.2 ms (0.7 mJ) (purple), 0.5 ms (1.9 mJ) (cyan), 0.75 ms (2.8 mJ) (orange) and 1 ms (3.7 mJ) (red) infrared pulses determined from current responses to dual-sinusoidal voltage input. (g) Maximum changes in equivalent capacitance in HEK cells at each pulse energy (N=5). (h) Current responses of a PE:PC bilayer to voltage-clamp protocol (shown above current traces) starting with a holding potential at −80 mV, ramping to 0 or −160 mV, stepping back to −80 mV and ramping again to 0 or −160 mV after 1.1 s. In black traces, no infrared light is applied. In red traces, a 10 ms (7.3 mJ) infrared pulse is applied before the first ramp. Red traces are overlaid on black ones; four total traces are shown. (i) Zoomed view of the five boxed areas of (h), in the same relative spatial arrangement as they appear in (h). (j) Comparison of change in capacitive charge (integral of the difference between red and black traces in the first current ramp) and laser-induced charge displacement in individual traces collected using the paradigm of panel (h) using infrared pulse energies of 2.3–7.3 mJ. All error bars are ±s.e.m. In panels a, b, h and i the red lines and grey shading indicate the timing of infrared laser pulse.

Figure 4. Thermally induced changes in membrane electrical capacitance are consistent with capacitor theory.

(a) Simplified equivalent circuit diagram and current equation for a passive membrane. The membrane current (Im(t)) depends on the membrane voltage (Vm), the Thevenin conductance (gT) and potential (VT), bilayer surface charges (represented by Vs) and the temperature-dependent membrane capacitance (Cm(T(t))), highlighted in red. (b) Simulated current response (red) for a PE:PC bilayer in symmetric NaCl solution at a holding potential of +200 mV, based on the temporal profile of temperature response to a 10 ms (7.3 mJ) infrared pulse. An experimental current response for the same set of conditions is shown in black. (c) Simulated I–V response to a 10 ms (7.3 mJ) pulse at voltages ranging from −200 mV (blue) to +200 mV (red). (d) Q–V curves predicted by the model for PE:PC:PS bilayers with symmetric NaCl solution (Ctrl), and with 14 mM MgCl2 (Mg2+) or 1 mM GdCl3 (Gd3+) added to the 'outside' solution. (e) Simulated I–V response for the Mg2+ condition in (d). (f) Q–V curves measured experimentally in solutions matching the conditions modelled in (d). N=5 per measurement. (g) Representative I–V response for the Mg2+ condition in (f). (h) Q–V curves predicted by the model for PE:PC bilayers in symmetric NaCl solution (Ctrl) and with the 'outside' negative surface charge increased by 66% (ANS). (i) Simulated I–V response for the ANS condition in (h). (j) Q–V curves measured experimentally for PE:PC bilayers after (ANS) and before (Ctrl) the addition of 100 μM ANS. N=5 per measurement. (k) Representative I–V response corresponding to the ANS condition in (j). All I–V plots are for voltages ranging from −200 to +200 mV. In panels b, c, e, g, i and k the red bars and grey shading indicate the timing of the infrared laser pulse. All error bars in f and j are ±s.e.m.

Figure 5. Infrared depolarizes bilayers and elicits APs in artificial neurons.

(a) Voltage responses in current-clamped artificial bilayers containing PE:PC:PS (PE:PC:PS) in NaCl solution, with 15 mM MgCl2 added to the 'outside solution' (producing a reversal potential similar to that seen in oocytes and HEK cells). Responses were recorded for 1 ms (2.8 mJ) (blue), 2 ms per 5.6-mJ (cyan) and 10 ms (7.3 mJ) (red) pulses. (b) Maximum voltage responses to pulses with the indicated energies. N=3 per measurement. Error bars are ±s.e.m. (c) Voltage recordings from oocytes co-expressing voltage-gated sodium (Nav1.4 α,β) and potassium (Shaker) channels under loose voltage-clamp conditions, with (red) and without (black) a 1 ms (2.8 mJ) infrared laser pulse applied during a subthreshold voltage step. Red bars and grey shading indicate the timing of infrared laser pulse.