A fluorescent nanosensor paint detects dopamine release at axonal varicosities with high spatiotemporal resolution

Abstract

The neurotransmitter dopamine (DA) controls multiple behaviors and is perturbed in several major brain diseases. DA is released from large populations of specialized structures called axon varicosities. Determining the DA release mechanisms at such varicosities is essential for a detailed understanding of DA biology and pathobiology but has been limited by the low spatial resolution of DA detection methods. We used a near-infrared fluorescent DA nanosensor paint, adsorbed nanosensors detecting release of dopamine (AndromeDA), to detect DA secretion from cultured murine dopaminergic neurons with high spatial and temporal resolution. We found that AndromeDA detects discrete DA release events and extracellular DA diffusion and observed that DA release varies across varicosities. To systematically detect DA release hotspots, we developed a machine learning–based analysis tool. AndromeDA permitted the simultaneous visualization of DA release for up to 100 dopaminergic varicosities, showing that DA release hotspots are heterogeneous and occur at only ∼17% of all varicosities, indicating that many varicosities are functionally silent. Using AndromeDA, we determined that DA release requires Munc13-type vesicle priming proteins, validating the utility of AndromeDA as a tool to study the molecular and cellular mechanism of DA secretion.

Keywords: chemical signaling; dopamine; imaging; neurotransmission; sensors.

Fig. 1.

AndromeDA paint as a DA sensor. (A) Illustration of a cultured DAergic neuron painted with AndromeDA. Neuronal stimulation results in DA release, which interacts with AndromeDA, increasing nanosensor fluorescence and thus sensing the spatiotemporal pattern of DA release and diffusion. (B) Schematic of the nanosensors used in AndromeDA, each consisting of a (5, 6)-SWCNT-(GT)₁₀ complex. (C) Left: AndromeDA consists of a dense layer of individual nanosensors, visualized here using AFM. Right: Magnified image of a single nanosensor, taken from a lower-density preparation of nanosensors. (D) Left: Magnified view of an EGFP-positive axon with a single varicosity in view. Right: AndromeDA fluorescence in the same field of view at different points in time. Initially, NIR fluorescence is low due to the lack of extracellular DA (labeled Basal). Neuronal stimulation results in the appearance of a transient AndromeDA hotspot adjacent to the varicosity (labeled Hotspot). As DA diffuses, AndromeDA is activated over a wider area, resulting in a more generalized increase in NIR fluorescence (labeled Diffusion). Below: Side-view schematic showing a DAergic varicosity surrounded by AndromeDA on the glass coverslip (Left), and fluorescence trace (Right) showing the NIR fluorescence change associated with the hotspot image above it.

Fig. 2.

Imaging of DA release and diffusion. (A) Images of normalized AndromeDA fluorescence (Left to Right) prior to stimulation (No stim), during 20-Hz electrical stimulation, during 90 mM KCl stimulation, and during application of 100 µM DA reveal DA release in untreated neurons (Upper). Treatment with reserpine (1 µM, 90 min) decreases stimulus-dependent activation of AndromeDA (Lower). AndromeDA fluorescence was quantified at ROIs centered on EGFP-positive varicosities (examples shown as red circles). (B) Neurons were either untreated or pretreated with L-DOPA (100 µM, 45 min), and experiments were performed as in A. (C) Average AndromeDA fluorescence over time for neurons stimulated at 20 Hz with and without treatment with reserpine. (D) Peak fluorescence (mean ± standard error of the mean [SEM]) for each stimulus method. Reserpine decreases the evoked peak AndromeDA fluorescence compared with untreated neurons. n = 10 untreated experiments per condition. (E) Average AndromeDA fluorescence over time for neurons stimulated at 20 Hz with and without L-DOPA pretreatment. (F) Peak fluorescence (mean ± SEM) for each stimulus method. L-DOPA increases the evoked peak AndromeDA fluorescence compared with untreated neurons. n = 5 (untreated) and 11 (L-DOPA) experiments (scale bars, 10 µm). In line graphs, solid lines represent mean values, and shaded areas represent SEM. Statistical comparisons used two-tailed Welch’s t test. **P < 0.01, ***P < 0.001. Red arrows indicate the direction of changes in the AndromeDA signal in treated neurons compared to untreated controls. Note that the intensities of DA-treated images in A and B are above the displayed fluorescence scale. F = Fluorescence intensity.

Fig. 3.

Detection and analysis of AndromeDA hotspots at populations of discrete varicosities. (A) Field of view showing EGFP-positive DAergic axons. Five exemplary regions showing adjacent hotspots of AndromeDA fluorescence during 20-Hz electrical field stimulation are highlighted (white boxes, regions 1 to 5). (B) Left: Magnified images of exemplary EGFP-positive axonal varicosities. Middle: Normalized AndromeDA NIR response (ΔF/F₀) at the time point of the onset (t₁ to t₅) of the indicated hotspot (white arrowheads). The outline of the EGFP-positive axon is overlaid in white, illustrating the position of the hotspot in relation to the axon. A magnified view of the hotspot is shown in the right corner (scale bar, 1 µm). Region 6 shows a representative region without detectable DA release above the diffusive background DA. (C) The uppermost trace shows the mean fluorescence intensity over time within a selected region of the extracellular space that contained no varicosities or hotspots. Subsequent panels show fluorescence traces corresponding to the exemplary regions shown in B. For regions 1 to 5, the traces show the mean fluorescence intensity within the ROI of the representative hotspots. For region 6, which contained no hotspot, the trace shows the average fluorescence of the inset NIR in B. The 20-Hz electrical stimulation window is highlighted in pink. Arrowheads indicate hotspot fluorescence peaks. For the scale bars in C, the y axis represents hotspot fluorescence and the x axis represents time. Hotspot fluorescence traces were generated by subtracting the signal of the extracellular space (ΔF/F₀) from the mean signal within the hotspot ROIs (ΔF/F₀) to highlight AndromeDA activation due to local DA release above overall DA diffusion.

Fig. 4.

Hotspots are heterogeneous sites of activity-dependent DA secretion. Hotspots observed in untreated (black dots) and L-DOPA–treated (100 µM, 45 min, green dots) electrically stimulated neurons are shown. (A) Area of activated AndromeDA within single hotspots. (B) Peak fluorescence within single hotspots. (C) Scheme illustrating how distance and hotspot area are defined. (D) Distances between the center of individual hotspots and the nearest varicosity. (E) Varicosity-hotspot distance was larger when noncorresponding EGFP images were used (randomized). (F) Time points at which individual hotspots occur. Electrical stimulation from 10 to 20 s is highlighted (red). (G) Hotspots occur primarily during electrical stimulation. (H) Hotspot peak fluorescence is no different from that of hotspots observed in the absence of stimulation. Only hotspots from neurons treated with L-DOPA were analyzed due to the large number of hotspots available for analysis compared with untreated neurons. (I) Hotspots are the result of spontaneous neuronal firing. The representative trace shows AndromeDA fluorescence over time from an EGFP-positive varicosity within the ROI indicated (white arrowhead). During TTX application, no hotspots are evident, but washout of TTX results in the appearance of hotspots (black arrowheads). (J) Only a subpopulation of varicosities exhibits closely adjacent (<3 µm) hotspots. L-DOPA treatment causes a significant increase in the proportion of varicosities with adjacent hotspots. In E, dots represent the median of each experiment, and column height is the mean of all experiments (± SEM). In G, H, and J, dots represent the median of each experiment, and column height is the median of all experiments. In A, B, D, and F, hotspots are organized on the y axis into individual experiments (lines), and the hatched area shows the frequency distribution of all hotspots. In A–H, data represent 68 hotspots from n = 4 experiments (untreated) and 110 hotspots from n = 10 experiments (L-DOPA). Data were compared using either two-tailed Welch’s t test (E) or the Mann-Whitney test (G, H, and J). *P < 0.05, **P < 0.01. Hotspot traces were generated by subtracting the mean signal of the extracellular space (ΔF/F₀) from the mean signal within the hotspot ROI (ΔF/F₀) for each time point (scale bar, 10 µm).

Fig. 5.

AndromeDA activation decreases with distance from axons. (A) To examine how the local changes in AndromeDA fluorescence were influenced by distance from axons, AndromeDA fluorescence was quantified at different distances from axons. The first ROI was defined by thresholding the EGFP signal (white) of the DAergic axon and tracing it (ROI 1). The line defining the boundary of this ROI was then moved outwards by 2 µm from the axon to create ROI 2. This process was repeated to create concentric ROIs, as indicated in green and identified using numbers. (B) The change in the local normalized fluorescence within each ROI compared with a distant region containing no axons was quantified (Materials and Methods). This gave the local increase in AndromeDA fluorescence within each ROI. ROI 1, which is closest to the axon, shows a rapid, high-amplitude increase in fluorescence with electrical stimulation. As the local changes in AndromeDA fluorescence are analyzed farther from the axon, the amplitude of the response is decreased. This demonstrates that local AndromeDA activation is greatest immediately adjacent to axons and that the local DA level rises rapidly with stimulation. Traces in B represent average normalized fluorescence over time. ROI fluorescence values, which are colored according to the ROI they represent, were generated by subtracting the mean normalized (ΔF/F₀) signal of a selection of the extracellular space from the mean signal within the ROI (ΔF/F₀) for each time point. Data represent n = 7 independent coverslips.

Fig. 6.

Munc13-1 and Munc13-2 are required for evoked DA release. (A) Representative images of AndromeDA-painted ventral midbrain TH-EGFP neurons lacking expression of Munc13-1 and Munc13-2 (Munc13 DKO) compared with neurons from littermate control mice. Images show normalized AndromeDA fluorescence prior to stimulation, during 20-Hz electrical field stimulation, and during application of 100 µM DA. ROIs were defined around varicosities of EGFP-positive axons (red circles). (B) AndromeDA signal within ROIs over time for control and Munc13 DKO neurons with electrical stimulation. Munc13 DKO neurons exhibit no AndromeDA activation in response to electrical stimulation (red arrow indicates the loss of the AndromeDA signal in Munc13 DKO neurons). (C) Average maximal fluorescence peak during 20-Hz electrical field stimulation in control and Munc13 DKO neurons. For control neurons, n = 6, and for Munc13 DKO neurons, n = 4, where n represents independent experiments. In B, solid lines represent the mean and shaded areas represent SEM. In C, mean ± SEM is shown. Data were compared using two-tailed Welch’s t test. *P < 0.05. Note that the intensities of DA-treated images in A are above the displayed fluorescence scale.