Imaging striatal dopamine release using a nongenetically encoded near infrared fluorescent catecholamine nanosensor

Abstract

Neuromodulation plays a critical role in brain function in both health and disease, and new tools that capture neuromodulation with high spatial and temporal resolution are needed. Here, we introduce a synthetic catecholamine nanosensor with fluorescent emission in the near infrared range (1000-1300 nm), near infrared catecholamine nanosensor (nIRCat). We demonstrate that nIRCats can be used to measure electrically and optogenetically evoked dopamine release in brain tissue, revealing hotspots with a median size of 2 µm. We also demonstrated that nIRCats are compatible with dopamine pharmacology and show D2 autoreceptor modulation of evoked dopamine release, which varied as a function of initial release magnitude at different hotspots. Together, our data demonstrate that nIRCats and other nanosensors of this class can serve as versatile synthetic optical tools to monitor neuromodulatory neurotransmitter release with high spatial resolution.

Fig. 1. Synthesis and testing of nIRCats.

(A) Schematic of optical catecholamine reporters, nIRCats. Pristine SWNTs are functionalized with (GT)₆ oligonucleotides to generate turn-on optical reporters for DA and NE. (B) Fluorescence spectra of nIRCats before (black) and after (red) the addition of 10 μM of DA in an in vitro preparation in phosphate-buffered saline (PBS; without tissue). Multiple emission peaks correspond to unique SWNT chiralities contained within the multichirality mixture. a.u., arbitrary units. (C) Nanosensor optical response to 100 μM DA, NE, GLU, GABA, ACH, serotonin (5-HT), histamine (HIST), octopamine (OCT), and tyramine (TYR) (data from in vitro testing). Black bars represent averages from n = 3 independent measurements, and error bars are calculated as SDs of the n = 3 measurements.

Fig. 2. Imaging and spatiotemporal analysis of DA release evoked by electrical stimulation in striatal tissue.

(A) Repeat images of the same field of view and ΔF/F of nIRCat signal after electrical stimulation of 0.3 mA in standard ACSF (top) and in ACSF and 10 μM nomifensine (bottom, +Nomifensine). Three example still frames are presented: “pre” is before electrical stimulation is applied, “stim” represents frame corresponding to peak ∆F/F following stimulation, and “post” is a frame after nIRCat fluorescence has returned to baseline. Scale bars, 10 μm. (B) Nanosensor fluorescence modulation scaled with single-pulse electrical stimulation amplitudes. Field-of-view mean traces and SD bands are presented for three stimulation amplitudes of 0.1, 0.3, and 0.5 mA. (C) Time traces of ∆F/F for 0.3-mA single-pulse electrical stimulation in standard ACSF (red) and in ACSF and 10 μM nomifensine (purple, +NOMF). Mean traces with SD bands are presented. (D) nIRCat ∆F/F responses are abolished in 0 mM Ca²⁺ ACSF and vary with extracellular (Ca²⁺). (E) A single frame from a time series gathered in the dorsomedial striatum showing the entire field of view, overlaid with regions of interest (ROIs) identified using per-pixel ∆F/F stack projections of nIRCat fluorescence modulation (see Materials and Methods). Color bar represents nIRCat labeling fluorescence intensity. Scale bar, 20 μm. (F) Frequency histogram of ROI sizes depicted in (E), exhibiting a log-normal distribution with a median ROI size of 2 μm. (G) A higher magnification view of an ROI with an effective radius of 5 μm. Maximum ∆F/F projection of the ROI shows the presence of smaller fluorescence hotspots within the ROI. Scale bar, 5 μm. (H) Overlay of representative normalized FSCV (gray) and nIRCat (blue) traces showing that nIRCat ROI signals exhibit heterogeneity in decay kinetics. Inset: An example of nIRCat experimental data (blue dots) fitted to first-order decay kinetics (red line) to compute decay time constants (τ). (I) Normalized frequency histogram of τs computed from FSCV and nIRCat individual ROI time traces. Data from n = 4 fields of view representing n = 2 biological replicates were pooled. Medians of each distribution: τ = 1.1 s (nIRCats) and τ = 0.4 s (FSCV).

Fig. 3. Imaging DA release in the presence of DA receptor agonists and antagonists.

(A) In vitro solution phase maximal ∆F/F (amplitude change at ~1128 nm) of nIRCat in the presence of 100 µM DA; the D2R antagonists sulpiride and haloperidol; D2R agonist quinpirole, and D1R antagonist SCH 23390; and drugs and DA. The addition of 1 μM drug quantities did not induce nIRCat fluorescence modulation in the absence of DA (****P < 0.0001 compared to DA ∆F/F). Subsequent addition of DA to drug-incubated nIRCat solutions produced ∆F/F responses indistinguishable from DA-only responses. Error bars represent SDs from n = 3 measurements. n.s., not significant. (B) Top: A schematic of the effect of D2R agonist and antagonist drugs on DA release. Bottom: Quinpirole suppressed nIRCat fluorescence modulation (****P < 0.0001), whereas sulpiride facilitated nIRCat fluorescence (***P = 0.001) in n = 3 biological replicates. Individual data point represents (∆F/F)_max ratio of the average trace collected in same field of view (post-/pre-drug application). (C and D) In brain slice, quinpirole (1 μM) suppressed nIRCat fluorescence modulation in response to a single-pulse electrical stimulation (0.5 mA, 3 ms; red trace) compared to pre-drug ACSF (black trace) but recovered following drug washout (purple and orange traces). (E and F) Sulpiride (1 μM) enhanced nIRCat fluorescence modulation in response to single-pulse electrical stimulation, yielding brighter nIRCat ∆F/F hotspots compared to drug-free ACSF. Scale bars, 10 μm. All error bands in (C) and (E) represent SD from the mean trace.

Fig. 4. Effects of quinpirole and sulpiride on nIRCat response at the level of ROIs (4 μm or smaller).

(A) ∆F/F of ROIs in ACSF and in ACSF with 0.25 μM and 1 μM of quinpirole. Each ∆F/F data point corresponding to an ROI is an average from n = 3 stimulation repeats. (B) Distribution of nIRCat response attenuation upon the addition of 0.25 μM (blue) or 1 μM (red) quinpirole for ROIs in (A). (C) Scatter plot of response to drug versus pre-drug ∆F/F amplitude for data in (A). (D) ∆F/F of ROIs in ACSF and following the addition of 1 μM of sulpiride. Each ∆F/F data point corresponding to an ROI is an average from n = 3 stimulation repeats. (E) Distribution of post-to-pre drug ∆F/F ratio for data in (D). (F) Scatter plot of response to drug versus pre-drug ∆F/F amplitude for data in (D). For (A) and (D), means and error bars (SD) are presented next to each distribution.

Fig. 5. nIRCat detection of striatal DA release evoked by optogenetic stimulation.

(A) Schematic of ChR2 expression in cortical glutamatergic terminals synapsing in the dorsal striatum. AMPA, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate; NMDA, N-methyl-d-aspartate; DAR, DA receptor. (B) No nIRCat fluorescence modulation was observed after stimulation of glutamatergic terminals. Inset: GLU release was confirmed by excitatory postsynaptic current (EPSC) on MSN. (C) Schematic of ChR2 expression in nigrostriatal dopaminergic terminals of the dorsal striatum. (D) Stimulation of dopaminergic terminals resulted in nIRCat fluorescence modulation. Stimulation protocol in (B) and (D) was five pulses (5P) at 25 Hz and a power flux of 1 mW/mm², and each pulse had a duration of 5 ms. (E) nIRCat ∆F/F in response to increasing number of pulses delivered at 25 Hz (5-ms pulse duration). (F) nIRCat ∆F/F in response to increasing pulse frequency (1, 10, and 25 Hz) of five pulses. Each pulse had a duration of 5 ms. (G) nIRCat ∆F/F in response to single pulses of 2-, 5-, and 10-ms duration. (H) Bath application of 1 μM of quinpirole suppresses DA release and results in depressed nIRCat ∆F/F. Drug washout rescues DA release and nIRCat ∆F/F. All error bands represent SD from the mean trace.