Infrared light elicits depolarizing currents in untreated oocytes

The large size of X. laevis oocytes (∼1 mm) enables simultaneous electrophysiological recording and optical stimulation of the cell with minimal potential for light-electrode artifacts (such as changes in seal or pipet resistance). Based on previous results indicating that infrared radiation increases cell excitability13,14, we first applied infrared laser pulses to oocytes expressing voltage-gated sodium (Na+) or potassium (K+) channels, searching for specific changes in their open probability upon irradiation. Contrary to expectations, we saw infrared effects that were independent of the type of expressed channels, and in fact were the same in wild-type oocytes as they were in oocytes expressing the ion channels. Consequently, we report here our results from wild-type oocytes.

Figure 1a shows that stimulation of wild-type oocytes with infrared laser pulses of 100 μs to 10 ms duration (pulse energies of 0.28 mJ to 7.3 mJ) elicited inward currents under voltage-clamp conditions. Current duration and amplitude corresponded to laser pulse width and energy. Infrared pulses lasting 10 ms, substantially longer than the voltage-clamp response time, allowed the natural shape of the current response to be resolved; a square-shaped current began with the onset of the laser pulse and ended immediately after the laser was turned off. Currents were inward at holding potentials from −100 mV to +100 mV (Fig. 1b,c) with a linear charge–voltage (QV) response reversing at an extrapolated 140±18 mV (Fig. 1d). Maximal current amplitudes of 86±5.4 nA were observed with 2 ms (5.6 mJ) pulses. With an optical fibre diameter of 400 μm and a penetration depth in water of <200 μm for 1889 nm light15, only ∼5% of the oocyte surface area is stimulated by infrared pulses. Stimulating an entire oocyte would thus be expected to elicit currents of up to 1.7 μA.

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 H 2 O-based SOS solution (H 2 O), D 2 O-based SOS solution (D 2 O), 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 GdCl 3 (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 H 2 O, D 2 O 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 D 2 O 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. Full size image

With pulse energies <8 mJ, stimulation elicited a consistent, transient current response over hundreds of trials. However, a few pulses at radiant energies exceeding 8 mJ were sufficient to irreversibly alter the oocyte's response to infrared. Subsequent to this energy barrier being breached, even lower-energy pulses produced a longer-lasting current reversing close to 0 mV (Fig. 1e). High-energy stimulation also tended to make oocytes more leaky (Fig. 1f). Presumably, this irreversible high-energy effect represents a form of damage to the oocyte membrane (indeed, local discolouration was sometimes seen on the oocyte surface after the experiment) and we did not investigate it further, confining pulse energies for the rest of our study to <8 mJ.

The large positive reversal potential for infrared-induced currents suggested an ionic conductance selective for Na+ or calcium (Ca2+). However, we found that current magnitudes and reversal potentials were not significantly affected by eliminating channel-permeable ions from extracellular solution (using N-methyl-D-glucamine as a cation and methanesulphonate as anion) or replacing Na+ with K+ in the physiological recording buffer (Fig. 1d). Responses were also not significantly affected by ion channel and transporter inhibitors ruthenium red, ouabain and amiloride. Gadolinium (Gd3+) was observed to shift the apparent reversal potential further in the positive direction rather than inhibiting the current.

It has previously been suggested that water is the primary chromophore responsible for absorbing infrared light and converting it into energy for cell excitation13. At 1889 nm, H 2 O has an absorption coefficient of 60.6 cm−1 (ref. 15). The absorption coefficient for heavy water (D 2 O) is approximately fivefold lower at this wavelength16. Replacing H 2 O with D 2 O in the extracellular recording solution produced a 65.3±4.1% decrease in response to infrared laser pulses, confirming the role of water in the excitation mechanism (Fig. 1d).

We measured the time course of local infrared-induced temperature changes in aqueous buffer using calibrated pipet resistance17. Laser pulses produced roughly linear increases in temperature of up to 22.2±0.6°C (for a 7.3 mJ pulse), followed by a decay to baseline with a time constant of ∼100 ms (Fig. 1g), matching the heat relaxation time for water. In D 2 O solution, temperature changes were reduced by about 70% (Fig. 1g). Comparing the time course of the temperature change to the oocyte current elicited by infrared reveals that oocyte currents correspond more closely to the rate of change in solution temperature than to the absolute temperature.

In current-clamp mode, infrared pulses depolarized oocytes from their resting potential by 0.6±0.03 mV (5.6 mJ pulse, Fig. 1h). The resting potential was recovered within 500ms, followed by an overshoot hyperpolarization of ∼40% of the depolarization magnitude. Adjusting for the 5% fractional stimulation of the oocyte, a depolarization of 11–12 mV would be expected for full-cell excitation.

Infrared light elicits depolarizing currents in mammalian cells

To determine whether the infrared-induced currents observed in oocytes were unique to that preparation, we performed infrared stimulation experiments in whole-cell-clamped HEK cells. Laser pulses of 200 μs (0.7 mJ) and 1 ms (3.7 mJ) elicited current responses similar to those seen in oocytes (Fig. 2a,b). Maximal current amplitudes of 73±20 pA were observed with 1 ms pulses. Currents were inward at all potentials examined, with an apparent reversal at 146±10.8 mV.

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 H 2 O-based recording solutions (H 2 O), D 2 O-based bath solution (D 2 O), with 1 mM GdCl 3 added to bath solution (Gd3+ out), 1 mM GdCl 3 added to pipet solution (Gd3+ in) or 20 mM MgCl 2 added to pipet solution with 0 mM MgCl 2 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. Full size image

Owing to the small size of HEK cells relative to the optical fibre diameter and light penetration depth, both the cell and the recording pipet were irradiated by light pulses. Infrared pulses applied directly to a pipet tip reduce pipet resistance, and may also reduce seal resistance. These effects were observed to confound QV curves measured with pulse energies >2.8 mJ (Supplementary Fig. S1). Thus 0.7 mJ pulses were used for QV analysis.

Replacing the H 2 O in extracellular solution with D 2 O reduced the observed current response in HEK cells by 75.8±12.6% (Fig. 2c). Similar to oocytes, extracellular application of GdCl 3 produced a positive shift in the reversal potential. Applying GdCl 3 in the intracellular solution produced the opposite effect. Increasing the concentration of magnesium chloride (MgCl 2 ) in the pipet solution also negatively shifted the reversal potential (Fig. 2c).

In current-clamp mode, infrared light depolarized cells by up to 2.7 mV (1ms, 3.7-mJ pulse, Fig. 2d). The extent of depolarization appeared to saturate with longer pulses in the 0.5–2 ms range, potentially due to concomitant changes in pipet, seal or membrane resistance. Our measurement may therefore underestimate the change in membrane potential one would obtain in unpatched cells. Undershooting repolarization similar to that seen in oocytes was observed after the initial response.

Infrared light changes the capacitance of artificial bilayers

Our observation of infrared-induced currents in untreated oocytes and HEK cells, and their failure to respond to channel and transporter blockers, led us to consider a general membrane-related mechanism. In particular, we noted that the time course of the current response to infrared pulses tracks the rate of change in temperature and not the temperature itself (Fig. 1g). This led us to hypothesize that temperature alters the electrical capacitance of the membrane, producing a current proportional to the derivative of capacitance, and thereby to the derivative of temperature, with respect to time.

We tested this possibility in artificial lipid bilayers, which are a good minimal model of living membranes because they have no proteins and minimal membrane conductance compared with cells. We tested the effects of infrared pulses on voltage-clamped bilayers prepared from 1:1 phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Infrared pulses elicited currents of up to 406±44 pA (1 ms, 2.8 mJ), which reversed, notably, near 0 mV (Fig. 3a–c). The shape of the observed current was similar to those seen in oocytes and HEK cells. Replacing H 2 O with D 2 O in the recording buffer reduced the response by 79.3±12.6% (Fig. 3c).

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 H 2 O-based (H 2 O) and D 2 O-based (D 2 O) 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. Full size image

If the infrared-induced temperature jumps causes an increase in membrane capacitance, such an increase would be expected to persist after the initial laser-induced current, with a decay time resembling that of the absolute temperature of the bilayer and surrounding solution. We used a sinusoidal voltage-clamp paradigm18 to directly measure changes in bilayer electrical capacitance. Our measurements revealed infrared-induced increases in capacitance of up to 6.6±0.2% (7.3 mJ pulse) that decayed on a timescale of 100–200 ms (Fig. 3d,e), consistent with the rate of thermal relaxation.

Infrared-induced changes in membrane capacitance were similarly measured in HEK cells in the whole-cell configuration. Infrared dose-dependent increases in membrane capacitance of up to 1.6±0.2% (3.7 mJ pulse) were observed (Fig. 3f,g). Capacitance increases decay on a timescale of ∼200 ms.

To further illustrate the relationship between laser-induced currents and changes in membrane capacitance, we designed a voltage-clamp protocol in which voltage ramps were applied to bilayers 8 ms and 1.1 s after an infrared pulse. Increases in ramp current magnitude relative to control were seen in both positive and negative directions immediately after the laser pulse, but are absent in ramps applied 1.1 s later (Fig. 3h,i). The magnitude of such increases was correlated with the magnitude of laser-induced charge displacement (Fig. 3j).

Capacitive effect is consistent with classical theory

The total electrical capacitance of a lipid membrane in electrolyte solution reflects a combination of the core capacitance of the phospholipid bilayer and the in-series capacitance of ionic double layers on each side of the membrane19. This total capacitance can be modelled using the Gouy–Chapman–Stern (GCS) theory of double layer capacitors20. This textbook theory, derived from the Poisson equation, models a charged surface in contact with electrolyte solution. Taking into account the relevant dielectric constants and ionic composition, GCS calculates the electrical capacitance of the system by balancing the electrical and thermal forces affecting the spatial distribution of ions near the charged surface. Although this classical theory has well-known limitations21, it is a useful starting point from which to understand the capacitance changes observed in our experiments.

We used the GCS model to simulate currents arising from temperature-dependent capacitance changes (illustrated by the equivalent circuit in Fig. 4a and Supplementary Fig. S2). Using the coupled equations (1) and (2) below (modified from Genet et al.22), we solved numerically for surface potentials Φ o and (Φ i −V m ) on each side of the bilayer as a function of membrane potential and temperature. This allowed us to calculate net membrane capacitive charge. >

It is noteworthy that temperature appears explicitly in two places in Eqs. (1) and (2) and implicitly in the temperature dependence of the aqueous dielectric permittivity ɛ T sol (ref. 23). Using this model, we simulated currents arising from infrared-induced temperature jumps (such as shown in Fig. 1g). For a 1:1 PE:PC bilayer in symmetric monovalent electrolyte, the model predicts a current response time course similar to those we measured experimentally (Fig. 4b,c). A reasonable set of parameter values produced simulated current magnitudes within 25% of the measurement.

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 (I m (t)) depends on the membrane voltage (V m ), the Thevenin conductance (g T ) and potential (V T ), bilayer surface charges (represented by V s ) and the temperature-dependent membrane capacitance (C m (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 MgCl 2 (Mg2+) or 1 mM GdCl 3 (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. Full size image

The model predicts a 0-mV reversal potential for infrared-induced currents when lipid surface charge and electrolyte composition are the same on both sides of the bilayer. However, this symmetry is predicted to be broken by placing multivalent cations on only one side of a symmetrically negatively charged bilayer, or by having an asymmetric surface charge. We modelled and experimentally tested scenarios (Fig. 4d–g) in which a symmetrically negatively charged lipid bilayer (1:1:1 PE:PC:PS (phosphatidylserine)) had MgCl 2 or GdCl 3 added to only the 'external' side of the membrane. Both model and experiment showed the reversal potential shifting positive. This model and artificial bilayer result is in agreement with our observations in oocytes and HEK cells, where adding multivalent cations Gd3+ or Mg2+ to one side of the membrane shifted the reversal potential so as to produce greater currents towards the other side (Figs 1d, 2c).

We also modelled and experimentally tested a scenario in which the surface charge of a 1:1 PE:PC bilayer in symmetric buffer is made more negative on one side by adding the amphipathic anion 1-anilino-8-naphthalenesulphonate (ANS) to the external solution (Fig. 4h–k). Again, there was qualitative agreement between model and experiment for a positive shift in the reversal potential. Experiments with ANS are confounded by its gradual voltage-dependent permeation across the bilayer. This may partly explain the different shape of the infrared-induced current response in ANS-treated bilayers relative to theory and other experimental conditions.

Infrared pulses depolarize bilayers by up to 9 mV

Artificial bilayers are a good system in which to test infrared-induced changes in membrane potential. Unlike oocytes, most or all of the membrane is irradiated by the light; unlike HEK cells, the light does not irradiate a pipet electrode thereby introducing changes in circuit resistance. We made passive measurements of membrane potential in artificial bilayers under conditions where their positive reversal potential mimics those of oocytes and HEK cells (that is, PE:PC:PS with asymmetric MgCl 2 ). Infrared-induced depolarizations of up to 8.7±1.0 mV were observed (Fig. 5a,b). This magnitude of depolarization is consistent with the extrapolation of oocyte voltage changes to a condition in which the entire oocyte surface is stimulated (11–12 mV). Notably, the undershoot repolarization observed in oocytes and HEK cells was absent in artificial bilayers, which is consistent with a higher ratio of membrane capacitance to membrane conductance in bilayers as compared with oocytes and HEK cells, as illustrated by equivalent circuit simulations (Supplementary Fig. S2).

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 MgCl 2 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. Full size image

Infrared light elicits APs

Infrared stimulation has been shown in vivo to elicit APs and corresponding downstream effects in nerves. As neuronal cell bodies, processes and associated glia express a variety of proteins that could be involved in transducing infrared effects, we wanted to test the ability of infrared stimulation to elicit APs in a stripped-down 'artificial neuron'. Oocytes coexpressing voltage-gated sodium (Nav1.4 α,β) and potassium (Shaker) channels can fire APs when depolarized past threshold from a pre-pulse of −90 mV using a loose voltage clamp.

When depolarized to potentials just below threshold, oocytes failed to fire APs. Under these conditions, a single infrared laser pulse of 1 ms (2.8 mJ) at or shortly before peak depolarization was sufficient to elicit an AP (Fig. 5c). For this effect to work, oocytes had to be within 0.5–1 mV of their firing threshold. As <5% of the oocyte surface is exposed to infrared light, preparations where the laser irradiates a larger fraction of membrane surface (such as neurons in vivo) may permit infrared pulses to elicit APs from cells at membrane voltages much further below the threshold, as illustrated for a Hodgkin–Huxley model neuron in Supplementary Fig. S3.