Two-photon optical interrogation of individual dendritic spines with caged dopamine

Abstract

We introduce a novel caged dopamine compound (RuBi-Dopa) based on ruthenium photochemistry. RuBi-Dopa has a high uncaging efficiency and can be released with visible (blue-green) and IR light in a two-photon regime. We combine two-photon photorelease of RuBi-Dopa with two-photon calcium imaging for an optical imaging and manipulation of dendritic spines in living brain slices, demonstrating that spines can express functional dopamine receptors. This novel compound allows mapping of functional dopamine receptors in living brain tissue with exquisite spatial resolution.

Figure 1

Structure (inset) and aliphatic section of the ¹H NMR spectra of RuBi-Dopa in D₂O before (top trace) and after (bottom trace) irradiation into the NMR tube with a 525 nm green LED. The doublets below 1.2 ppm belong to coordinated trimethylphosphine (PMe₃) in RuBi-Dopa (top trace) and in the aquo-complex formed after photoreaction (bottom trace). Signals of coordinated dopamine (B and C) are also apparent. The triplets A₁ and A₂, corresponding to the coordinated −NH₂, disappear after irradiation due to rapid exchange with D₂O. The signals of the free dopamine methylenes (B′ and C′) are indicative of the successful photorelease of the ligand.

Figure 2

UV–vis spectra of a 75 μM aqueous solution of RuBi-Dopa during photolysis at T = 25 °C and pH = 7. Samples were irradiated at 405 nm, and spectra collected from 350 to 700 nm. Inset: quantum yield calculation using eq 1.

Figure 3

Photolysis of RuBi-Dopa in two-photon regime. A solution containing 22 mM RuBi-Dopa was irradiated with an amplified Ti-Sa laser at 800 nm and the photoreaction followed through its emission band. Inset: time dependence of the emission during photolysis. Details are given in the text.

Figure 4

RuBi-Dopa uncaging onto dendritic spines. (A) RuBi-Dopa is bath applied to a cortical slice, and a layer five pyramidal neuron is loaded with 200 μM Fluo-4 Ca²⁺ indicator and 200 μM Alexa-488. Scale bar = 1 μm. (B) Experimental configuration: a Ti-Sapphire laser (820 nm) scans the spine head (at approximately 5–8 mW laser power on sample) first to monitor basal Ca²⁺ concentrations at the location marked with a circle in (A) and then moves to a nearby location (square), where the laser power is increased (to approximately 25 mW on the sample) to uncage dopamine from RuBi-Dopa for 4 ms. After uncaging the laser goes back to initial power and location on the spine head. (C) Spine fluorescent signals (bottom traces; individual traces in gray (n = 10), average trace in bold black, and ± StEr traces in black) showing a sudden increase of Ca²⁺ concentration after the two-photon uncaging of dopamine. The middle trace shows the uncaging pulse. The fluorescent signal detection, by a photomultiplier tube (PMT), was blocked for a few milliseconds before, during, and after the uncaging pulse with the use of an ultrafast shutter (top trace) to prevent potential PMT saturation and errors in signal detection.