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Photoactivatable drugs for nicotinic optopharmacology


Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

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Figure 1: Development of PA-Nic and its utility for improved pharmacological studies of native nAChRs.
Figure 2: PA-Nic enables subcellular mapping of nAChRs after chronic nicotine treatment.


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We thank members of the Drenan and Lavis laboratories for helpful advice and discussion, and T. Lerner (Northwestern University, Chicago, Illinois, USA) for contributing viral reagents. This work was supported by the Howard Hughes Medical Institute (to S.B., J.J.M., and L.D.L.), the US National Institutes of Health (NIH) (grants DA035942 and DA040626 to R.M.D., MH099114 to A.C., DA037161 to H.A.L., NS054850 to D.J. Surmeier, and GM103801 and GM48677 to J.M.M.), the PhRMA Foundation (fellowship to M.C.A.), the Arnold and Mabel Beckman Foundation (Beckman Young Investigator Award to Y.K.), the Bernice E. Bumpus Foundation (Early Career Innovation Award to Y.K.), the Rita Allen Foundation (to Y.K.), the Searle Scholars Program (to Y.K.), the Alfred P. Sloan Foundation (Sloan Research Fellowship to Y.K.), NINDS (grant NINDS F32 NS103243 to N.M.B.), the JPB Foundation, and Northwestern University.

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Authors and Affiliations



R.M.D., M.C.A., H.A.L., S.B., K.R.G., and L.D.L. conceived the project. M.C.A., N.M.B., D.L.W., X.-T.J., J.J.M., Y.W., C.P., G.Z., V.J.K., J.J.M., A.C., Y.K., R.M.D., S.B., and L.D.L. planned and/or executed experiments. D.L.W., Y.K., J.M.M., and K.R.G. contributed essential reagents and expertise. R.M.D., M.C.A., S.B., and L.D.L. wrote the paper with input from all other authors. R.M.D. and L.D.L. supervised all aspects of the work.

Corresponding authors

Correspondence to Ryan M Drenan or Luke D Lavis.

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Competing interests

K.R.G. is an employee of Thermo Fisher Scientific and has stock options. All other authors declare no competing interests.

Integrated supplementary information

Supplementary Figure 1 Synthesis and characterization of other caged pharmacological agents.

(ag) Structures, exact mass (E.M.) and LC-MS traces of photoactivatable (PA) pharmacological agents and parent drug, before photolysis (–hv), and after photolysis (+hv); arrows show major ionic species from each peak. (a) PA-Nic (9) to nicotine (1) with coumarin byproducts 10 and 11. (b) PA-Cev (12) to cevimeline (2). (c) PA-PNU (13) to PNU-282,987 (3). (d) PA-Mil (14) to milameline (4). (e) PA-Oxo (15) to oxotremorine (5). (f) PA-Fen (16) to fentanyl (6). (g) PA-Esc (17) to escitalopram (7). (h) Representative plot of normalized HPLC chromatogram peak area of PA compounds 9,1217 vs. irradiation time (405 nm) to determine relative uncaging quantum yield (Φu); n=2 independent samples. (i) Representative evaluation of chemical (‘dark’) stability of PA compounds 9,1217 (note scale) in the absence of light; n=3 independent samples. (j) Normalized absorption spectrum of PA compounds 9,1217. (k) Table listing absorbance maximum (λmax), extinction coefficient (ɛ), uncaging quantum yield (Φu), fluorescence emission maximum (λem), and fluorescence quantum yield (Φf) for compounds 917. (lm) Quaternization of the coumarin cage with a minimal tertiary amine elicits a 14 nm bathochromic shift in λmax. (l) Structures of model compounds 18 and 19. (m) Representative normalized absorption spectra of 18 and 19; n=3 independent samples.

Source data

Supplementary Figure 2 PA-Nic epi-illumination validation studies.

(a) Chemical structure of RuBi-Nic (20). (b) Location of MHb in mouse brain near bregma –1 mm to –2 mm. (c) MHb subregions. Recordings were made from MHb neurons in the ventral inferior (VI) subregion. Other ventral MHb subregions: central (VC) and lateral (VL). (d) Validation of ChAT-Cre::Ai14 mice for targeted recordings from MHb cholinergic neurons. Coronal sections from ChAT-Cre::Ai14 mice containing MHb were co-stained with anti-ChAT and anti-DsRed antibodies (single experiment). Scale: 175 μm. (e) Comparison of light-evoked responses using PA-Nic and RuBi-Nic in a MHb neuron. Epi-illumination flash PA-Nic (~405 nm, 1 s, 0.12 mW/mm2, 80 μM superfusion, similar for > 10 independent experiments) and RuBi-Nic (470 nm, 15 s, 0.12 mW/mm2, 1 mM local perfusion, single experiment) uncaging responses are plotted on the same time and current scale. Scale bars: 2 s, 60 pA. Inset: RuBi-Nic response re-plotted to show the small inward current deflection. Scale bars: 2 s, 5 pA (f) RuBi-Nic is unstable in brain slice experiments. Voltage clamp recordings before/during RuBi-Nic (100 μM) superfusion for n=2 independent ventral tegmental area cells are shown. No light flashes were delivered. Scale bars: 100 pA, 2 min. (g) No PA-Nic uncaging with blue or green light. PA-Nic was applied to (n=4/2) voltage-clamped MHb neurons, and blue (~470 nm) or green (~560 nm) light flashes (100 ms, 0.06 mW/mm2) were applied. Representative traces are shown for one cell. Scale: 500 ms, 5 pA. (hi) The main photochemical by-product of PA-Nic photolysis does not act as an agonist or antagonist at nAChRs. (h) The monoalkylcoumarin byproduct (10) was applied via pressure ejection to a voltage-clamped MHb neuron (100 μM, 12 psi, 125 ms, similar for 2 total independent experiments). Scale: 1 s, 5 pa. (i) ACh (100 μM) was applied to a voltage-clamped MHb neuron before (black trace) and after (red trace) superfusion of by-product (10; 100 μM). Representative trace shown. Scale: 250 ms, 20 pA. Inset: before-after plot summary data with two-sided paired t-test (n=4/2). (j) PA-Nic does not antagonize nAChRs in MHb neurons. ACh (100 μM) was applied to a voltage-clamped MHb neuron via pressure ejection before (black trace) and after (red trace) superfusion of PA-Nic (80 μM). Representative trace shown. Scale: 250 ms, 15 pA. Inset: before-after plot summary data with two-sided paired t-test (n=5/1). (k) Repeated nicotine uncaging using PA-Nic pressure ejection. PA-Nic (100 μM) was applied locally to a MHb neuron via pressure ejection (500 ms, 12 psi), followed immediately by a light flash (1 s pulse, 0.12 mW/mm2) with the microscope field stop aperture fully restricted, for several trials (similar for 2 total independent experiments). Scale bars: 125 ms, 60 pA. (l) PA-Nic voltage clamp responses are antagonized by nAChR antagonists. Representative voltage clamp traces are shown for light-evoked currents before (black trace) and 10 min after (red trace) superfusion of a nAChR antagonist cocktail (10 μM mecamylamine, 100 nM MLA, 10 μM DHβE, 20 μM SR16584). PA-Nic was applied locally to the cell via pressure ejection followed by a 1 s flash (0.12 mW/mm2). Scale: 2 s, 45 pA. Inset: before-after plot summary data with two-sided paired t-test (n=4/2). (m) Representative light-evoked currents from the same neuron are shown for different holding potentials. PA-Nic was applied locally to the cell via pressure ejection followed by a 1 s flash (0.12 mW/mm2). Scale: 2 s, 200 pA. (n) Current-voltage relation: currents during nicotine uncaging (1 s pulse, 0.12 mW/mm2) at various holding potentials. Data show mean±s.e.m. (n=3/2). A linear regression (y = 0.0123x – 0.03, R2 = 0.997) extrapolates to a reversal potential of ~ +2 mV. (op) Calibration of PA-Nic responses. Summary data (mean±s.e.m.) is shown for MHb voltage clamp responses to pressure ejection application of the indicated concentration of nicotine (n=12/2; o) or ACh (n=30/7; p).

Source data

Supplementary Figure 3 PA-Nic one- and two-photon laser flash photolysis validation studies.

(a) 2-Photon fluorescence action cross-section (δf) spectra for PA-Nic and GCaMP6f; points denote mean; n=2 independent samples. (b) Plot of normalized mean HPLC chromatogram peak area of PA-Nic vs. irradiation time (810 nm, 760 nm, or 720 nm) to determine 2-photon uncaging action cross-section (δu); found δu = 0.094 GM at 810 nm, 0.059 GM at 760 nm, and 0.025 at 720 nm; error bars indicate ±s.d.; n=3 independent samples. (c) 2-Photon fluorescence action cross-section (δf) and 2-photon uncaging action cross-section (δu) spectra for PA-Nic; points indicate mean; error bars indicate ±s.d.; n=2 independent samples. (d) Representative 2-photon laser scanning microscopy image of a patch-clamped, dye-filled MHb neuron soma, including three peri-somatic uncaging positions (spot diameter: ≤0.8 μm; similar for >10 independent experiments). Scale (image): 6 μm; Scale (current): 50 ms, 50 pA. Evoked currents (720 nm, 60 mW, 5 ms) are shown for each position (trials recorded at 30 s intervals). (e) Laser pulses delivered in the absence of PA-Nic fail to elicit currents. MHb neurons (n=5/4, 3 peri-somatic locations/cell) were voltage clamped at –70 mV and superfused with a PA-Nic free ACSF medium. ACSF (no PA-Nic) was locally applied to the neuron via pressure ejection and laser pulses (10 ms) were delivered to a peri-somatic location. The excitation wavelength was incremented as indicated and power was held constant (30 mW). Data show individual cell responses and mean±s.e.m. (fg) 2-photon photolysis currents as a function of pulse duration and laser power. Voltage-clamped MHb neurons were superfused with ACSF containing 100 μm PA-Nic and 1 μm atropine. (f) 2P photolysis responses (left) at a single peri-somatic location in response to PA-Nic photolysis using 3, 10, and 20 ms pulse durations at fixed laser power (80 mW; 760 nm). Traces show an average of 5-10 sweeps per condition. Mean±s.e.m. data (right): 15 uncaging locations, n=5/4. P value: Dunn’s post-hoc after Friedman’s test (χ2(3)=9.733, p=0.008) (g) 2P photolysis responses at a single peri-somatic location (left) in response to PA-Nic photolysis with a 10 ms pulse duration at 10, 20, 30, and 80 mW laser power. Traces show an average of 5–10 sweeps per condition. Mean±s.e.m data (right): 15 uncaging locations, n=5/3. Scale bars (fg), 10 pA, 50 ms. P value: overall 1-way ANOVA (F(3,53)=17.2). (h–i) Small inward currents during pulses with high laser power. In the absence of PA-Nic, patch-clamped MHb neurons were stimulated with 405 nm laser flashes (50 ms, peri-somatic uncaging position, n=4/2) using a range of laser powers: h shows average traces for n=4 neurons at the indicated laser power. Scale bars: 50 ms, 1.5 pA; i shows a plot of the peak inward current for these n=4 neurons at the indicated laser power. (jk) Relationship between laser power and inward current for PA-Nic photolysis. PA-Nic (80 μM) was applied via superfusion to voltage-clamped MHb neurons, and nAChR-mediated currents were evoked via 405 nm laser flashes (10 ms, peri-somatic position, n=4 neurons) using a range of laser powers. j shows voltage recordings from a representative cell during PA-Nic photolysis using the indicated laser power. Scale bars: 1 s, 16 pA. k shows mean±s.e.m. peak inward current for PA-Nic photolysis-evoked responses at the indicated laser power (n=4/3). A single-phase exponential function was fitted to the data (R2=0.537). (lm) Relationship between laser pulse duration and inward current for PA-Nic photolysis. PA-Nic (80 μM) was applied via superfusion to voltage-clamped MHb neurons, and nAChR-mediated currents were evoked via 405 nm laser flashes (1 mW, peri-somatic location, n=6 neurons) using a range of pulse durations. l shows voltage recordings from a representative cell during PA-Nic photolysis using the indicated pulse duration. Scale bars: 1 s, 40 pA. m shows mean±s.e.m.; normalized to maximum for each cell, n=6/3) peak inward current at each pulse duration. The Hill equation was fitted to the data (nH (Hill slope)=1.0, duration at ½ max=100 ms, R2=0.943).

Source data

Supplementary Figure 4 Upregulation of nAChRs in MHb ChAT(+) neurons.

(ab) Chronic nicotine treatment up-regulates nAChRs in MHb neurons of C57BL/6 mice. (a) Representative ACh (100 μM)-evoked currents from a control or chronic nicotine-treated MHb neuron from C57BL/6 mice treated with control or chronic nicotine via nicotine-laced drinking water. Scale: 500 ms, 200 pA. (b) Bar/dot plot showing mean+s.e.m. and individual responses to pressure-applied ACh (100 μM) in MHb neurons (# of neurons/mice: n=11/2 control; n=11/2 nicotine). P value: two-sided unpaired t-test. (cd) Chronic nicotine treatment up-regulates nAChRs in MHb ChAT(+) neurons. (c) ChAT-expressing MHb neurons in brain slices during patch clamp recordings. Representative Dodt contrast (left) and 2PLSM (right) image of tdTomato expression in ChAT(+) MHb neurons from ChAT-Cre::Ai14 mice (arrow) is shown (similar for >10 independent experiments). Scale: 20 μm. (d) nAChR functional up-regulation in ChAT(+) MHb neurons. ChAT-Cre::Ai14 mice were treated with control or chronic nicotine for 4-6 weeks via their drinking water, and ACh (100 μM)-evoked currents were recorded from visually-identified ChAT(+) neurons (# of neurons/mice: n=9/2 control; n=17/3 nicotine). Bar/dot plot shows mean (+s.e.m.) and individual responses. P value: two-sided unpaired t-test.

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Supplementary Figure 5 Action potentials and Ca2+ mobilization studied with PA-Nic.

(ab) PA-Nic can be used to drive action potential firing via nAChRs. (a) MHb VI neurons were held in current clamp (I=0) configuration during epi-illumination photolysis of PA-Nic. A restricted field stop aperture permitted nicotine uncaging directly over the recorded VI neuron (i), or at 100 (ii) to 200 (iii) μm from the recorded cell. A representative (similar for 4 independent experiments) trace is shown for a recording from a MHb VI neuron (right panel). Photolysis: 33 ms, 0.12 mW/mm2. Scale: 1 s, 15 mV (b) Before-after plots showing the peak action potential firing rate in individual MHb VI neurons (n=4/2) at baseline and following nicotine uncaging at position i, ii, or iii as indicated in a. 2-way RM ANOVA of treatment (2; baseline vs. flash) x location (3; (i), (ii), (iii)): significant main effects of treatment [F(1,9)=23.17, p=0.001], location [F(2,9)=7.155, p=0.0138], and a significant treatment x location interaction [F(2,9)=5.416, p=0.0286]. P values (Bonferroni multiple comparison): 0.0012 (i), 0.3962 (ii), >0.7541 (iii). (c) GCaMP6f 2-photon Ca2+ imaging setup. AAV-Flex-GCaMP6f was microinjected into MHb of ChAT-Cre mice via stereotaxic surgery. (de) GCaMP6f-expressing MHb neuron characteristics in acute slices. (d) Flash photolysis was only conducted in neurons exhibiting spontaneous Ca2+ cycling between high-Ca2+ (box/image 1) and low-Ca2+ (box/image 2) states. Scale: 15 s, 1.0 ΔF/F. Image scale: 8 μm. (e) Neurons with spontaneous Ca2+ cycling behavior are sensitive to nicotine. A representative trace (n=5/3) from a Ca2+ cycling MHb neuron showing spontaneous Ca2+ cycling and a sustained increase in Ca2+ following superfusion of nicotine (100 μM). Scale: 1 min, 1.0 ΔF/F. (fh) All-optical analysis of nAChR activity with 2PLSM Ca2+ imaging in MHb neurons. (f) Ca2+ signals in GCaMP6f-expressing ChAT+ MHb neurons in acute slices were imaged via 2PLSM and 405 nm laser flashes were delivered before and after local-perfusion of PA-Nic (1-2 psi, 2 mM). (g) Representative (n=6/5) traces from a Ca2+-cycling MHb neuron showing 405 nm laser flash-evoked increases in Ca2+ before (green trace) and after (magenta trace) superfusion of a nAChR antagonist cocktail (1 μM DHβE, 1 μM SR16584, 100 nM α-Ctx MII). Photolysis: 5 ms, 2 mW. Scale: 2.5 s, 1.0 ΔF/F. (h) Before-after plot of Ca2+ signals (ΔF/F) under the conditions indicated (n=6/5). P values: two-sided paired t-test.

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Supplementary Figure 6 IPN and homomeric α7 nAChRs analyzed with PA-Nic photolysis.

(a) Sagittal view of ChAT-Cre::Ai14 mouse brain showing MHb, fasciculus retroflexus (FR), and interpeduncular nucleus (IPN). 2PLSM maximal intensity projection of a representative dye-filled IPN neuron during electrophysiological recording (bottom left panel, scale: 16 μm). Schematic of imaging experiment in b, where an Alexa 488-filled IPN neuron is imaged via 2PLSM adjacent to ChAT+ cholinergic fibers from MHb (bottom right panel). (b) Alexa 488-filled IPN neuron surrounded by ChAT+ fibers in IPN of ChAT-Cre::Ai14 mice (scale: 12 μm). The soma (1) and a dendrite (2), surrounded by ChAT+ nAChR-expressing fibers are shown in xy, xz, and yz planes (at right). xy scale: 5 μm; xz and yz scale: 12 μm. Similar for 2 total independent experiments. (c) Targeted recordings of PA-Nic photolysis responses in IPN GABA neurons were enabled by GAD2-Cre::Ai14 mice, which express tdTomato in GAD2+ neurons. An IPN-containing coronal section from a GAD2-Cre::Ai14 mouse was stained with anti-DsRed antibodies (scale: 120 μm; similar for 2 total independent experiments). (d) PA-Nic photolysis elicits slow inward responses in IPN GABA neurons. A representative peri-somatic PA-Nic laser flash photolysis (405 nm, 50 ms, 2 mW) response is shown (black trace; scale: 4 pA, 4 s) compared to an averaged proximal dendrite response in MHb neurons (n=7; red trace; scale: 30 pA, 4 s) using identical stimulation parameters. The MHb response decay was fitted to a double exponential and extrapolated to match the duration of the IPN response (grey line). Inset: the same IPN and MHb responses are shown on the same scale (30 pA, 4 s). (e) Relationship between peak current and area under curve (AUC) is monotonic for IPN nicotine uncaging responses. A quadratic polynomial function (black line) was fitted (R2=0.59; grey lines=95% confidence intervals) to the peak current vs. AUC data plot (n=12/8). (fg) Subcellular PA-Nic uncaging in IPN GABA neurons. Summary of position-dependent uncaging peak response (f) and area under the curve (AUC; g) data for n=12/8 GAD2+ IPN neurons using PA-Nic (80 μM). Nicotine uncaging responses (50 ms, 2 mW) were recorded at the soma and at dendritic locations at the indicated linear distance from the soma, and are expressed normalized to the soma response. Bar plot shows mean (+s.e.m.) (h) PA-Nic photolysis responses are mediated by nAChRs. Representative IPN PA-Nic laser flash photolysis responses (405 nm, 50 ms, 2 mW) before and after application of nAChR antagonist cocktail (20 μM SR16584, 10 μM DHβE). Scale: 4 pA, 4 s. (i) Before-after scatter plot of PA-Nic photolysis response pharmacological blockade, two-sided paired t-test (n=4/3). (j) Stratum radiatum interneuron recordings. A Dodt contrast image of a typical (similar for 2 total independent experiments) stratum radiatum (SR) interneuron (arrowhead) is shown in proximity to the PA-Nic local perfusion pipette at left. The SR is ventral to the CA1 pyramidal cell layer (stratum pyramidale; SP). Scale: 24 μm. (k) Representative methyllycaconitine (MLA)-sensitive, ACh-evoked current in an SR interneuron. Scale: 12 pA, 1 s. (l) Before-after scatter plot of SR interneuron ACh (1 mM) responses and MLA blockade, two-sided paired t-test (n=5/2). (m) Representative MLA-sensitive PA-Nic photolysis response (250 ms, 0.12 mW/mm2) in an SR interneuron. Scale: 7 pA, 1 s. (n) Before-after scatter plot of SR interneuron PA-Nic photolysis responses and MLA blockade, two sided paired t-test (n=8/4).

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Banala, S., Arvin, M., Bannon, N. et al. Photoactivatable drugs for nicotinic optopharmacology. Nat Methods 15, 347–350 (2018).

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