Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain

Abstract

We recently introduced fluorescent false neurotransmitters (FFNs) as optical tracers that enable the visualization of neurotransmitter release at individual presynaptic terminals. Here, we describe a pH-responsive FFN probe, FFN102, which as a polar dopamine transporter substrate selectively labels dopamine cell bodies and dendrites in ventral midbrain and dopaminergic synaptic terminals in dorsal striatum. FFN102 exhibits greater fluorescence emission in neutral than acidic environments, and thus affords a means to optically measure evoked release of synaptic vesicle content into the extracellular space. Simultaneously, FFN102 allows the measurement of individual synaptic terminal activity by following fluorescence loss upon stimulation. Thus, FFN102 enables not only the identification of dopamine cells and their processes in brain tissue, but also the optical measurement of functional parameters including dopamine transporter activity and dopamine release at the level of individual synapses. As such, the development of FFN102 demonstrates that, by bringing together organic chemistry and neuroscience, molecular entities can be generated that match the endogenous transmitters in selectivity and distribution, allowing for the study of both the microanatomy and functional plasticity of the normal and diseased nervous system.

Fig. 1.

Structure and photophysical properties of FFN102. (A) Structure of FFN102. (B) Absorption spectra of FFN102 acquired at a range of pH values (2–10). (C) The excitation spectra of FFN102, measured at an emission wavelength of 453 nm, displayed a maximum at 340 nm at pH 5.0 (red), whereas a maximum at 370 nm was observed at pH 7.4 (blue). (D) The emission spectra of FFN102 at both pH 5.0 (red, λ_ex = 340 nm) and pH 7.4 (blue, λ_ex = 370 nm) displayed a maximum at 453 nm.

Fig. 2.

FFN102 labels dopaminergic terminals in the dorsal striatum and midbrain neurons of acute mouse brain slices. (A) GFP signal (λ_ex = 910 nm) expressed under the control of the TH promoter. (B) Labeling of FFN102 (λ_ex = 760 nm). (C) Overlap of TH-GFP and FFN102 suggests a high level of colocalization (yellow); 91.1 ± 1.9% (mean ± SEM; n = 3) of puncta labeled with FFN102 were also labeled with TH-GFP. (D) Overlap of two images of FFN102 acquired before and after the TH-GFP image showed 94.8 ± 0.9% (mean ± SEM; n = 3) colocalization. (E) Effect of 6-OHDA lesion on the uptake of FFN102 in the dorsal striatum of acute mouse brain slices. A statistical difference (P < 0.05, t test; n = 3) in the number of labeled terminals was observed in the control hemisphere (Right, 221 ± 10; mean ± SEM) compared with the lesioned hemisphere (Left, 3 ± 2; mean ± SEM). (Scale bar, 10 μm.) (F) FFN102 fluorescent labeling of dopamine neurons in the substantia nigra compacta (SNc) and their dendrites (arrows) projecting into the substantia nigra reticulata (SNr). The asterisk indicates an apparent blood vessel. (Scale bar, 50 μm.) (G) FFN102 accumulation in slices from TH-GFP (λ_ex = 930 nm) mice at the level of the ventral tegmental area (VTA) reveals high degree of FFN102 and GFP colocalization. (Scale bar, 20 μm.) (H) Quantification of FFN102 and GFP colocalization in SNc and VTA illustrates the predominant labeling of dopaminergic neurons. (I) Schematic illustration modified from Franklin and Paxinos (16) showing the approximate regions at which the images were acquired.

Fig. 3.

DAT dependency of FFN102 uptake in the dorsal striatum of acute mouse brain slices. (A) Treatment with 1 μM nomifensine decreased FFN102 uptake (Right, 12 ± 3 puncta; mean ± SEM) compared with untreated slices (Left, 202 ± 9 puncta; mean ± SEM). (B) Treatment with 5 μM cocaine resulted in a similar decreased uptake of FFN102 (Right, 14 ± 2 puncta; mean ± SEM) compared with control slices (Left, 222 ± 15 puncta; mean ± SEM). (C) FFN102 uptake was significantly reduced in the absence of DAT, as seen by the higher number of fluorescent puncta observed in wild-type slices (WT) (Left, 217 ± 7; mean ± SEM), compared with slices from DAT-deficient mice (DAT-KO; Right, 9 ± 3; mean ± SEM). Values obtained for treated and untreated slices (A and B), as well as those obtained for WT and DAT-KO (C), were statistically different (P < 0.05, t test, n = 3). (Scale bar, 10 μm.) (D) FFN102 concentrations of 4–40 µM inhibited the reuptake of dopamine released by electrical stimulation, as measured by cyclic voltammetry in the dorsal striatum (P < 0.05, two-tailed Student t test). The height of the signal mostly represents dopamine release, whereas the decay is mainly dependent on DAT-mediated reuptake. (E) FFN102-positive cells were found in the midbrain (SNc shown) of WT (Left) but not DAT-deficient (Right) mice (n = 3). (Scale bar, 20 µm.)

Fig. 4.

AMPH induces release of FFN102 from dorsal striatum and midbrain. (A and B) Images of an acute striatal slice loaded with FFN102 taken 0 and 10 min after perfusion with ACSF containing no AMPH (A) or 1 µM AMPH (B). (C) Quantification of FFN102 puncta fluorescence loss in the absence and presence of 1 μM AMPH, normalized to the intensity at time 0 (expressed as mean ± SEM; n = 3). (D) Schematic illustration of midbrain AMPH-induced FFN102 release experiments. FFN102-loaded midbrain slices were perfused with ACSF for 15 min in the imaging chamber and images were acquired 0, 20, and 40 min thereafter. One-half of the slices were perfused with ACSF containing 10 µM AMPH during the 20- to 40-min period, whereas the other half was perfused with regular ACSF throughout the entire experiment. (E) Representative images of FFN102-filled SNc DA neurons acquired after 0, 20, and 40 min of ACSF (Upper) or ACSF plus 10 μM AMPH (Lower) perfusion. (F) Quantification of fluorescence intensity shows a decreased rate of fluorescence loss between the 20- and 40-min time points (ΔF2) compared with the 0- to 20-min period (ΔF1) in control slices, but increased rate in AMPH-treated slices. (G) Fluorescence over time after normalization. Note the more rapid decrease in fluorescence in the presence of AMPH. *P < 0.05 compared with control slices (t test; n = 28–29 cells from 6 slices per treatment). For F and G, results are displayed as mean ± SEM. (Scale bar: A and B, 10 µm; E, 20 µm.)

Fig. 5.

Calcium-dependent release of FFN102 in the dorsal striatum of acute mouse brain slices in response to electrical stimulation. (A–C) Normalized mean fluorescence intensity of fluorescent puncta (A), regions that do not include the puncta, i.e., “background” (B), and fluorescent puncta after background subtraction (C). Mean values are shown for unstimulated slices (red) and slices stimulated at 10 Hz in the absence (blue) or presence of 200 µM CdCl₂ (green). Fluorescence intensities for each curve were normalized to the corresponding intensity values at the time point before the onset of stimulation (t = 0). Each point represents a mean ± SEM (n = 5). (D) Relative fluorescence over time of representative individual destaining (blue) and nondestaining terminals (black). For calculation of half-time (t_1/2) values, each curve was fit with a one-phase exponential decay function. For the exponential curves shown, t_1/2 values were calculated to be 59.5, 76.5, 100.4, 146.3, and 310.3, with R² values of 0.89, 0.97, 0.92, 0.93, and 0.95, respectively. (E) Relative signal over time of destaining (blue) and nondestaining puncta (black), after background subtraction, in slices stimulated at 10 Hz. Each curve represents the mean ± SEM (n = 5). For A–E, stimulation was initiated at time 0 s. (F) Histogram of t_1/2 values of individual FFN102-labeled terminals that destain under 10-Hz stimulation. (Bin size, 20 s.)