Photoactivatable drugs for nicotinic optopharmacology

Abstract

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.

Figure 1. Development of PA-Nic and its utility for improved pharmacological studies of native nAChRs

(a) Chemical structure of nicotine (1) and the synthesis and photolysis of PA-Nic (9). (b) Chemical structures of ‘uncageable’ drugs 2–7. (c) Plot of mean HPLC chromatogram peak area vs. irradiation time (365 nm) provides the uncaging quantum yield (Φ_u); solid line is exponential fit; error bars indicate ±SD; n=2 independent samples. (d) Representative dark stability experiment of PA-Nic (9); n=3 independent samples. (e) Representative absolute absorption spectrum of PA-Nic (9, 10 μM); n=3 independent samples. (f) Light-evoked currents following PA-Nic epi-illumination photolysis. Voltage clamp traces from individual MHb neurons are shown for different methods of PA-Nic application (similar for >10 independent experiments). Photolysis: 1 s pulse, 0.12 mW/mm². Scale: 2.5 s, 100 pA (grey), 150 pA (black) (g–j) Controllable nicotine uncaging via PA-Nic epi-illumination photolysis. (g) Representative voltage clamp traces from a MHb neuron for light pulses (1 s) of varying intensity. Scale: 2.5 s, 75 pA (h) Resulting photochemical dose-response relation for peak currents (y=2.876x+2.1, R²=0.9921). Mean (n=9/6) peak of light-activated currents plotted vs. input flash intensity. (i) Representative voltage clamp traces during light pulses (0.12 mW/mm²) of varying duration applied to a MHb neuron. Scale: 2.5 s, 250 pA (j) Graphical analysis of summary pulse duration data in i. The Hill equation was fitted to the mean data (n_H (Hill slope)=2.0, duration at ½ max=0.3 s, R²=0.928) from n=5/3. All error bars indicate ±SEM.

Figure 2. PA-Nic enables subcellular mapping of nAChRs following chronic nicotine treatment

(a) PA-Nic uncaging and mecamylamine antagonism. Nicotine was uncaged in a ~1 μm peri-somatic spot with a 405 nm laser pulse (10 ms, 2.9 mW). Voltage-clamp currents are shown before (black trace, similar for >10 independent experiments) and 10 min after (red trace, single experiment) mecamylamine (10 μM) superfusion. Scale: 50 ms, 100 pA (b–c) Lateral spread of uncaged nicotine, estimated electrophysiologically. (b) A representative 2PLSM image of a MHb neuron is shown. Nicotine was uncaged (white circles; 10 ms, 1.5 mW) at the surface (1; 0 μm), and at 3.5 (2), 7.0 (3), and 10.5 (4) μm from the cell surface. Representative traces from a single neuron are shown. Scales on image: (lower left = relative intensity; lower right = 20 μm). Scale (current): 500 ms, 30 pA (c) The Hill equation was fitted to the mean (±SEM) data (n (Hill slope)=2.293, R²=0.9074) (n=6/5), resulting in an estimate of 4.5 μm for the lateral distance at ½ max amplitude. (d–e) Subcellular mapping of nAChRs in MHb neurons. (d) Representative 2PLSM image of a ChAT(+) MHb neuron, marked with uncaging positions (white circles; 50 ms, 2 mW) and the evoked response at that location. Scales: (lower left = relative intensity; lower right = 20 μm, upper right = 1 s, 60 pA) (e) Summary of position-dependent uncaging data for ChAT(+) MHb neurons (n=8/5) using PA-Nic (80 μM). Nicotine uncaging responses were recorded at the soma and at dendritic locations at the indicated linear distance from the soma surface. The mean (+SEM) and individual responses are shown. P values: Tukey post-hoc after 1-way ANOVA (F(4,69)=4.3, p=0.0036). (f–i) Interrogating chronic nicotine-mediated nAChR up-regulation with PA-Nic. (f) Representative traces are shown from a control and chronic nicotine-treated ChAT(+) MHb neuron stimulated via epi-illumination photolysis (0.12 mW/mm²) for the indicated durations. Scale: 200 pA, 2 s (g) The Hill equation was fitted to photochemical dose response mean (±SEM) data from control (n=7/2) or chronic nicotine-treated neurons (n=11/3) (control: n=1.7, duration at ½ max=0.3 s, R²=0.732; chronic nicotine: n=1.6, duration at ½ max=0.3 s, R²=0.89). (h–i) Chronic nicotine up-regulates nAChRs at all cellular locations tested. (h) Representative uncaging responses are shown from a control and chronic nicotine-treated neuron stimulated at the soma and at a dendrite ~30 μm from the soma using 405 nm laser photolysis (50 ms, 2 mW) of PA-Nic. Scale: 20 pA, 2 s (i) Scatter plots (mean ±SEM) of nicotine uncaging amplitudes at the indicated cellular location are shown for ChAT(+) control (n=6/3) and chronic nicotine-treated (n=14/4) neurons. P values: two-sided Mann-Whitney test.