2.5. Establishing conditions for orthogonal two-color uncaging

Absence of optical crosstalk between two caged compounds when using specific wavelengths of light is the defining key parameter for orthogonal two-color uncaging. Crosstalk, however, can have a different meaning in chemistry or biology. From the chemist’s perspective, one could argue that any absorption inevitably leads to excitation and therefore uncaging so that crosstalk must occur if any spectral overlap is seen (Kotzur et al., 2009). Indeed, even with our newest probe DEAC454, the absorption is not truly zero at the λ_min in the UV range, while on the other hand, short wavelength probes like dcPNPP show zero absorption at the longer wavelength (Fig. 4a). Some photolysis is therefore measurable with highly sensitive techniques such as HPLC or NMR when irradiating DEAC454 with UV light. However, these probes are made for two-color uncaging in a biological context, so what is the readout for crosstalk here?

Two-color, one-photon uncaging of glutamate and GABA. (a) Optical densities of solutions of DEAC454-GABA (blue line) and dcPNPP-Glu (violet line) at working concentrations (here: 30 and 330 μM, respectively). The blue and violet dashed lines are the spectral outputs of the LEDs (here: 365 and 470 nm) used for uncaging. The black and red lines are the microscope filters.

(b) Left, representative current response from a hippocampal CA1 neuron to uncaging of dcPNPP-Glu with UV and blue light. Right, energy dosage/response curve for dcPNPP-Glu uncaging with UV and blue light. Increasing the uncaging duration with the UV LED increased the amplitude of the current response. No current could be elicited even with a high energy dosage of blue light (10 mW, 1000 ms).

(c) Left, representative current response from a hippocampal CA1 neuron to uncaging of DEAC454-GABA with UV and blue light. Right, energy dosage/response curve for DEAC454-GABA uncaging with UV and blue light. Increasing the uncaging duration with the blue LED increased the amplitude of the current response while only small currents could be elicited when using high energy dosages of UV light.

(d) The dcPNPP chromophore does not absorb blue light, so no biological actuation is possible with longer wavelengths used for optimal DEAC454 excitation. Thus, there is an “infinite power domain” for photolysis of DEAC454-GABA with such wavelengths (blue shading).

(e) Determination of the power domain for two-color, one-photon uncaging of DEAC454-GABA with zero biological crosstalk. As a result of the small absorbance of DEAC454 in the UV range, no biological crosstalk was detectable with UV light below 5 mW and 6 ms (pink shading). However, using such energies, dcPNPP-Glu was readily uncaged and elicited robust currents. At increased energy dosage, the power window was still >30x for the two caged compounds using UV light (grey shading).

The decisive factor for biological crosstalk is whether a certain energy dosage (uncaging power * duration) of UV light induces a functional response in the cell. This does not only depend on the quantitative measure of how much compound is photolyzed but whether the receptor to be studied is sufficiently activated by this amount to evoke a response. Even with the same caged probes and light sources, the crosstalk may be different when studying for example ionotropic vs. metabotropic receptors due their different sensitivity and since the response of the latter may be amplified by downstream signaling cascades (see Passlick et al., 2017). For each set of caged compounds, combination of wavelengths, and pair of receptors to be studied, the optical crosstalk should therefore be determined.

To measure optical crosstalk in biological systems, the general idea is to record energy dosage/response curves for both compounds separately with both wavelengths of light used (Fig. 4). The readout and detection limit are the critical factors here to be able to determine the conditions where two-color uncaging is possible without functional crosstalk. Therefore, conditions for determining the crosstalk might be different from the actual final experiment since the signals to be studied might not be readily detectable under those conditions. For example, when using physiological ion concentrations extracellularly and intracellularly, uncaging of GABA likely does not induce a detectable current since the reversal potential for chloride ions is close to the resting membrane potential of the cell. Therefore, despite opening of the GABA-A receptor and increasing the chloride conductivity of the cell by GABA uncaging, no net current might be measurable. To be able to determine the crosstalk of GABA uncaging with two wavelengths of light, experimental conditions need to be established where the detection limit is increased such that even small amounts of photolyzed GABA become detectable. To this end, in our two-color, one-photon uncaging approach, we must manipulate the extracellular and intracellular ion concentrations to increase the driving force for chloride ions. Further, we must optimize our recording conditions so that even small currents (here, >5 pA) may be measured. By doing so, we demonstrated that there was no functional biological crosstalk of GABA uncaging when using UV light energy dosages that elicited large AMPA-receptor mediated currents when dcPNPP-Glu was present (Fig. 4 and Passlick et al., 2017). Therefore, although the crosstalk may not be absolute zero in the chemical preparation (chemical crosstalk), it is functionally zero in the biological application (biological crosstalk) which demonstrates that caged GABA remains orthogonal in biological systems in which dcPNPP-Glu is being used.

Next, we describe the detailed procedure for determining the optical crosstalk for two-color, one-photon uncaging of dcPNPP-Glu and DEAC454-GABA to activate AMPA and GABA-A receptors, respectively. However, the same basic procedure also applies to other modes of two-color uncaging.

125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, 26 NaHCO₃, 10 glucose, 3 Na-pyruvate, 1.3 Na-ascorbate equilibrated with 95% O₂/ 5% CO₂.

Note: We and others include ascorbate and pyruvate in our ACSF, which are beneficial for cellular health during imaging and uncaging experiments.

For these recordings we include the following blockers and antagonists to look at pure AMPA and GABA-A receptor responses: tetrodotoxin (TTX, 1 μM) to prevent firing of action potentials, DL-AP5 (100 μM) to block NMDA receptors, CGP-55845 (3 μM) to block GABA-B receptors, and JNJ-16259685 (1 μM), MPEP (3 μM) and {"type":"entrez-nucleotide","attrs":{"text":"LY341495","term_id":"1257705759","term_text":"LY341495"}}LY341495 (30 nM) to block metabotropic glutamate receptors.

135 K-gluconate, 4 MgCl₂, 10 HEPES, 5 ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 4 Na₂-ATP, 0.4 Na₂-GTP, 10 Na₂ phosphocreatine, pH adjusted to 7.35.

Note: In combination with the above ACSF solution (section 2.5.1), the calculated AMPA receptor reversal potential is -1 mV. When neurons are clamped at -60 mV, the strong ionic driving force enables detection of small amounts of free glutamate from dcPNPP-Glu uncaging.

135 Cs-methanesulfonate, 4 MgCl₂, 10 HEPES, 5 EGTA, 4 Na₂-ATP, 0.4 Na₂-GTP, 10 Na₂ phosphocreatine, pH adjusted to 7.35.

Note: In combination with the above ACSF solution (section 2.5.1), the calculated GABA-A receptor reversal potential is -71 mV. When neurons are clamped at +10 mV, the strong ionic driving force enables detection of small amounts of free GABA by DEAC454-GABA uncaging.

This protocol describes the steps to determine the power window where two-color, one-photon whole-field (LED) uncaging of dcPNPP-Glu and DEAC454-GABA can be performed with zero functional crosstalk. We commonly perform these experiments by recording patch-clamp whole-cell currents from hippocampal neurons of acute mouse brain slices. However, the approach can easily be adapted to other preparations, caged compounds, or light sources.

Details on the hardware used here are given in section 2.4 (see also Passlick et al., 2017).

Calibrate the output power of the UV (365 nm) and blue LED (470 nm) and determine settings for different power levels, e.g. for 5 and 10 mW. If experiments will be performed on different microscopes, it may be useful to describe the power in mW/cm² for comparison.

Prepare mouse brain slices by standard protocols (Davie et al., 2006; Ting et al., 2014) (or other preparations such as cultured cells if preferred).

Dissolve 330 μM dcPNPP-Glu in carbogenated ACSF (including blockers; see 2.5.1), and recirculate the solution through the recording chamber.

Patch a cell with K-gluconate internal solution (see 2.5.2) in the whole-cell configuration, and hold it in the voltage-clamp mode at -60 mV.

Switch the light path at the microscope to uncaging.

Record an energy dosage/response curve for both LEDs: Either set the power of the LED to a fixed value and increase the uncaging duration, or set a fixed duration and increase the power of the LED while simultaneously recording the current response of the cell. We commonly record two eight-point power dosage/response curves with fixed power (5 and 10 mW) by increasing the uncaging duration (0/2/4/6/8/10/15/20 ms for both LEDs), respectively (Fig. 4b). Record these curves with both LEDs for the same cell and record at least three individual cells.

Perform the same type of experiment for DEAC454-GABA with the following modifications:

Dissolve 30 μM DEAC454-GABA in carbogenated ACSF (including blockers; see 2.5.1) and recirculate the solution through the recording chamber.

Patch a cell with Cs-methanesulfonate internal solution (see 2.5.2) in the whole-cell configuration and hold it in the voltage-clamp mode at +10 mV.

Record energy dosage/response curves for both LEDs as described under point 6 (Fig. 4c).

Measure the cellular current response to all stimuli from both LEDs for each cell separately and calculate the average current response for each stimulus.

Plot the measured cellular current response to dcPNPP-Glu and DEAC454-GABA uncaging against energy dosage (uncaging duration/power) with the blue LED (Fig. 4d) and the UV LED (Fig. 4e).

Determine the power window where no current is detectable from dcPNPP-Glu uncaging with blue light (Fig. 4d; infinite here since dcPNPP does not absorb any blue light) and DEAC454-GABA uncaging with UV light (Fig. 4e; finite since DEAC454 does absorb some UV light). This is the energy dosage range considered as zero functional crosstalk and should thus be used during the final experiment.