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• W4300492090 abstract "Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional composite nanofilm that facilitates visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ≈ 3.2 µm (full width at half maximum [FWHM]) and are observed with a mean spatial frequency of one hotspot per ≈ 7.5 µm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots co-localized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals. Editor's evaluation This is a very exciting study that presents a novel approach to examining dopamine release with spatial precision that is so far unrivaled. This manuscript is also important and timely in the field of biosensor development and of potential interest to neuroscientists who study neurochemical release. It introduces a synthetic nanofilm with high spatiotemporal resolution and quantal sensitivity to dopamine measurement. By utilizing this technology to visualize sub-cellular dopamine efflux, the work provides new insights into the spatiotemporal dynamics and protein machinery of somatodendritic dopamine release. The authors identify hotspots for DA release and also provide evidence for DA release in the presence of TTX, suggesting the occurrence of quantal release. https://doi.org/10.7554/eLife.78773.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest To form the vast and complex network necessary for an organism to sense and react to the world, neurons must connect at highly specialized junctions. Individual cells communicate at these ‘synapses’ by releasing chemical signals (or neurotransmitters) such as dopamine, a molecule involved in learning and motivation. Despite the central role that synapses play in the brain, it remains challenging to measure exactly where neurotransmitters are released and how far they travel from their release site. Currently, most tools available to scientists only allow bulk measurements of neurotransmitter release. To tackle this limitation, Bulumulla et al. developed a new way to measure neurotransmitter release from neurons, harnessing a technique which uses fluorescent nanosensors that glow brighter when exposed to dopamine. These sensors form a very thin film upon which neurons can grow; when the cells release dopamine, the sensors ‘light up’ as they encounter the molecule. Dubbed DopaFilm, the technology reveals exactly where the neurotransmitter comes from and how it spreads between cells in real time. In particular, the approach showed that dopamine emerges from 'hot spots' at specific sites in cells; it also helped Bulumulla et al. study how dopamine is released from subcellular compartments that have previously not been well characterized. Improving the sensors so that the film could detect other neurotransmitters besides dopamine would broaden the use of this approach. In the future, combining this technology with other types of imaging should enable studies of individual synapses with intricate detail. Introduction Chemical neurotransmission generally falls under one of two broad categories: fast synaptic transmission or neuromodulation. Synapses that mediate rapid communication between most excitatory and inhibitory synapses in the brain primarily employ glutamate or γ-aminobutyric acid (GABA). Such chemical communication occurs at highly specialized synaptic structures that have nanoscale spatial organization and operate with millisecond temporal precision (Clements et al., 1992; Südhof, 2012; Choquet and Triller, 2013). In contrast, neuromodulators, including biogenic amines, neuropeptides, and hormones, operate at different spatiotemporal scales. Neuromodulatory synapses do not exhibit a close apposition to their partners but act on receptors that are extrasynaptically localized, and signal through G-protein coupled intracellular mechanisms (Greengard, 2001). This suggests that neuromodulators diffuse from their release sites in adequate quantities to influence target receptors (Agnati et al., 1995). Generally, the nature of the chemical synapse is less well understood for neuromodulators, which differ from their classical counterparts not only in their mechanisms of action on receptors but also in their secretory apparatus and spatiotemporal dynamics. For dopamine, one of the most important neuromodulators in the brain, the challenge is compounded by certain unique features pertinent to dopamine neurobiology. Dopaminergic neurons are known for their large size and exhibit highly ramified axonal arborizations and dense varicosities (Matsuda et al., 2009; Pacelli et al., 2015; Bolam and Pissadaki, 2012). Studies have sought to establish the molecular determinants of dopamine release from these dense axonal arbors, but these attempts do not rely on measurement of dopamine efflux with single release site resolution (Ducrot et al., 2021; Liu et al., 2018). Previous studies have demonstrated that dopamine neurons possess the machinery for co-release of other neurotransmitters, including glutamate and GABA, but to what extent, if any, co-release events spatially overlap remains insufficiently understood (Stuber et al., 2010; Sulzer et al., 1998; Tritsch et al., 2016; Tritsch et al., 2012; Fortin et al., 2019). Electrochemical and microdialysis assays in midbrain regions have shown that somatodendritic release of dopamine constitutes an important component of dopamine signaling, with notable implications in disease and behavior (Beckstead et al., 2004; Gantz et al., 2013; Björklund and Lindvall, 1975; Cheramy et al., 1981; Cragg et al., 1997; Ludwig et al., 2016). However, the spatial and temporal dynamics of dendritic release events and their regulatory mechanisms remain poorly characterized. In sum, there is a pressing need for tools with appropriate sensitivity, kinetics, and subcellular spatial resolution to explore mechanisms of neurochemical release, including that of dopamine. In this work, we developed an assay that facilitates visualization of the efflux of dopamine from active zones with synaptic resolution and quantal sensitivity. Here, we define efflux to mean the two-dimensional (2D) broadening of released dopamine quanta. In axonal arbors, we observed that dopamine release arises from a sparse set of varicosities, and we were able to assign the observed spatially defined effluxes to individually identified boutons. In dendrites and dendritic arbors of dopamine neurons, we similarly visualized spatially resolved effluxes of dopamine from putative dendritic active zones, which have been less well understood than release zones in axons. Our results show that dopamine neurons can sustain robust levels of release from their dendritic processes. Indeed, we demonstrate that dendritic release is fast and Ca2+ dependent, suggesting gating of dopamine release by a Ca2+-mediated release machinery, reminiscent of classical active zones. Dopamine efflux was observed at major dendrites, fine dendritic processes, and at junctions of the soma and dendrites but rarely directly from the cell body itself. Soma of dopamine neurons were observed to receive strong dopaminergic input due to release and diffusion from proximal dendrites. Retrospective super-resolution imaging at identified release sites shows that Bassoon, long established as a presynaptic scaffolding protein of the cytomatrix in presynaptic active zones, is also enriched at the vicinity of dopamine release sites in dendrites and dendritic arbors. The expression of vesicular SNARE protein synaptobrevin-2 correlated with dopamine release activity in dendritic segments, establishing its functional utility in dopamine neurons. Therefore, active zones in dendrites appear to utilize a constellation of presynaptic and SNARE-complex proteins that are responsible for coordinating release, similar to those observed in classical synapses. This study offers a technology that facilitates visualization of the spatial and temporal efflux of dopamine and deploys the technology to shed light on somatodendritic dopamine release, a facet of dopaminergic neurobiology that has been insufficiently characterized. Results Visualizing active zone dopamine efflux from axons and dendrites To a first-order approximation, once released from a synaptic active zone, the temporal evolution of the released chemical should approximate that of diffusion from a point source, characterized by an isotropic expansion from the point of release but constrained by transporter activity and local three-dimensional ultrastructure. Such a signal can only be fully measured if the sensing platform sufficiently samples the underlying signal in both the spatial and temporal domains. However, the inability of current technologies to measure chemical efflux sufficiently in the spatial domain limits our ability to study chemical synapses. We addressed this challenge by using near-infrared (NIR) fluorescent dopamine nanosensors to image single dopamine release sites from rat primary midbrain neuronal cultures. The nanosensors are assembled from oligonucleotide-functionalized, single-wall carbon nanotubes in solution phase (Beyene et al., 2018; Kruss et al., 2014) and have previously been used to image dopamine release in striatal acute slices and cultured cells but were lacking in synaptic information (Beyene et al., 2019; Kruss et al., 2017). In this study, we drop-cast glass coverslips with dopamine nanosensors to produce a 2D layer of a turn-on fluorescent, dopamine-sensitive surface that can effectively image dopamine diffusion from a point source (Figure 1A). Temporally, the sensors exhibit subsecond turn-on responses, which enabled real-time imaging of dopamine’s temporal evolution. We named the engineered surface as DopaFilm, a 2D engineered film that affords video-rate filming of dopamine spatiotemporal dynamics with subcellular spatial and millisecond temporal resolution. Figure 1 with 3 supplements see all Download asset Open asset Schematic of DopaFilm imaging protocol. (A) Schematic of DopaFilm. (B) Workflow for preparing dopamine neuron primary cultures from the rat midbrain regions highlighted in blue. Neurons are grown on dishes with an engineered, chemi-sensitive, and fluorescent surface (DopaFilm) between polylysine and glass surfaces. (C) Imaging DopaFilm fluorescence activity in cultured dopamine neurons. Immunocytochemistry and Airyscan super-resolution imaging are carried out following DopaFilm activity imaging. DopaFilm was validated by co-culturing rat primary midbrain dopamine neurons with cortical and hippocampal neurons on the nanofilm surface for up to 6 weeks (typical period for an experiment) (Figure 1B and C). Dopamine neurons in culture exhibited stereotyped morphology, with thick major dendrites arising from the soma that go on to ramify into dendritic arbors, and axonal processes that branch and arborize on a scale of millimeters from a single neuron (Figure 1—figure supplement 1). The electrophysiological properties of hippocampal neurons grown on DopaFilm were similar to neurons that were grown on sensor-free substrates, which suggested that the electrophysiological phenotype of neurons grown DopaFilm was not altered (Figure 1—figure supplement 2). DopaFilm fluoresced in the NIR to short-wave infrared (SWIR) regions of the spectrum (850–1350 nm) when imaged with a 785 nm excitation laser, permitting its multiplexed deployment with existing optical technologies with no spectral overlap (Figure 1—figure supplement 3). The surface exhibited an isotropic turn-on response when exposed to exogenous dopamine wash, suggesting uniform sensor coverage and response (Figure 1—figure supplement 3A). Dopamine wash experiments revealed DopaFilm has the sensitivity to detect 1 nM concentrations, remained stable over the duration of a typical experiment, and has an apparent dissociation constant (Kd) of 268 nM (Figure 1—figure supplement 3B-C). This compares to half maximal effective concentrations(EC50 )values of ~ 1 µM for D1-like and ~ 10 nM for D2-like dopamine receptors, suggesting that DopaFilm is sensitive to chemical secretions that have physiological relevance (Rice and Cragg, 2008). In order to utilize DopaFilm’s advantageous photophysical properties, we developed a custom microscope that is optimized for broad spectrum imaging in the visible, NIR and SWIR regions of the spectrum (400–1400 nm), with integrated widefield and laser scanning confocal capabilities. The optimizations in the NIR and SWIR regions facilitated imaging and recording of activity with exceedingly high signal-to-noise (SNR) ratios, attaining SNRs in the range of 5–50 for most experiments. In this study, most imaging experiments were carried out in widefield epifluorescence mode using a 40×/0.8 NA objective (N40X-NIR, Nikon). This gave us a field of view (FOV) of 180 × 230 µm. In axonal arbors, the FOV contained several hundred dopaminergic varicosities, whereas in cell body regions, we could simultaneously image activity around the soma, major dendrites, and dendritic arbors. Post hoc immunofluorescence super-resolution images were collected on Zeiss LSM 880 with Airyscan mode. We asked if dopamine neurons grown on DopaFilm can be evoked to release dopamine, and whether DopaFilm fluorescence transients can recapitulate the predicted spatiotemporal evolution of dopamine efflux. To drive this effort, we virally co-expressed TH-GFP (GFP expressed under the control of the rat tyrosine hydroxylase [TH] promoter) and Syn-ChrimsonR-tdTomato (the red shifted opsin, ChrimsonR, expressed under the control of synapsin promoter and fused to tdTomato for visualization). This co-expression paradigm facilitated identification and optical stimulation of putative dopamine neurons. About 79% of TH-GFP+ neurons were confirmed to be dopaminergic in retrospective immunofluorescence against TH, the rate-limiting enzyme in dopamine biosynthesis. On the other hand, all of the TH-immunoreactive neurons expressed the virally delivered TH-GFP transgene. During seeding, we optimized density of cells on DopaFilm such that mature neurons formed a monolayer of cells on the substrate. The seeding density allowed us to record activities arising from isolated dopamine neurons where no other neurons in the vicinity of the neuron of interest were TH+, ensuring that detected activity can be assigned to single identifiable processes with minimal crosstalk. We used 561 nm LED (5 pulses and 25 Hz) to depolarize dopaminergic neurons. A subset of dopamine neurons exhibited spontaneous spiking activity, and we applied no external stimuli in those cases. Furthermore, most dopamine neurons exhibited stochastic, temporally uncorrelated release events that appeared to be action potential independent, and those events were also included in our data. All imaging experiments were carried out 3–4 weeks postviral infection, and neurons were 4–5 weeks in vitro at the time of activity imaging. We first imaged in axonal arbors, where dopamine release is relatively better characterized through microdialysis and voltammetry measurements (Liu et al., 2018; Garris et al., 1994; Robinson et al., 2003; Robertson et al., 1991). Whenever targeted to TH+ neurons (further confirmed by retrospective immunofluorescence experiments), our stimulation protocol elicited robust fluorescence transients from DopaFilm (Figure 2A and B, Figure 2—animation 1), and the fluorescence hotspots co-localized with TH+ boutons in axonal arbors (Figure 2C). We observed diffusive broadening of the fluorescence hotspots in subsequent imaging frames (Figure 2A, +1 s), and fluorescence transients returned to baseline in the poststimulation epoch (Figure 2A post, Figure 2B). The observed spatiotemporal evolution of DopaFilm hotspots is consistent with that of release and diffusion from multiple point-like sources localized in a 2D plane, with estimated mass diffusivities of ≈ 1.1 ± 0.8 × 10–6 cm2 s–1 (mean ± SD), in reasonable agreement with estimated values of diffusion coefficient for dopamine (Cragg et al., 2001). Despite the high density of axonal varicosities in the FOV, fluorescence transients were observed to emanate from a subset of varicosities while another subset of varicosities produced no corresponding ∆F/F fluorescence hotspots (Figure 2C, red arrows). The percentage of release-competent boutons varied greatly in axonal arbors, ranging from 5% in some FOVs to 65% in others, with a mean of 32% of putative boutons participating in release. This sparse-release observation is in agreement with results from previous studies (Liu et al., 2018; Pereira et al., 2016). DopaFilm hotspot activities were also observed to co-localize with dendrites of dopamine neurons (Figure 2D and E) and were additionally noted to arise from dendritic processes that commingled with the soma (Figure 2F and G). The turn-on and clearance kinetics of the measured transients in axons were 0.46 ± 0.16 s (mean ± SD) for time to peak (τpeak) and 3.83 ± 0.8 s (mean ± SD) for first-order decay time constant (τoff) (Figure 2—figure supplement 1). The turn-on kinetics is slower than those reported for the genetically encoded dopamine sensors GRABDA (≈ 100 ms) and dLight (reported as τ1/2 of ≈10 ms followed by a plateau of ≈100 ms) (Patriarchi et al., 2018; Sun et al., 2018). On the other hand, decay kinetics appears to be slower than dLight (reported as τ1/2 ≈ 100 ms) and comparable to or faster than those reported for GRABDA (≈ 3–17 s for variants). For comparison, G-protein-gated inwardly rectifying K+ (GIRK) current-based dopamine dynamics measurements exhibited τpeak ≈ 250 ms, whereas carbon fiber recordings peaked in τpeak ≈ 300 ms (Marcott et al., 2014). This suggests that the kinetic properties of DopaFilm transients are comparable with the range of reported values from existing tools. The DopaFilm was responsive only to the activity of TH+ neurons. Stimulation of ChrimsonR+ in TH− neurons did not elicit fluorescence transients in DopaFilm, and neither evoked nor spontaneous activities were noted in neurons that lacked TH immunoreactivity despite being TH-GFP+ in the viral expression paradigm (Figure 2—figure supplement 2). Both evoked and spontaneous DopaFilm fluorescence transients were absent when imaging in extracellular ‘Ca2+-free’ media (Figure 2—figure supplement 3). Figure 2 with 4 supplements see all Download asset Open asset Visualizing active zone dopamine efflux from axons and dendrites. (A) ∆F/F images before stimulation (pre), at the time of stimulation (stim), 1 s after stimulation (+1 s) and after return to baseline (post). (B) Mean ± SD of ∆F/F traces from the imaging field of view in (A) averaged over n=5 repeat stimulation runs. (C) Tyrosine hydroxylase-GFP (TH-GFP) image of axonal arbor and near-infrared (NIR) ∆F/F image shown in (A) and overlay. The NIR ∆F/F frame corresponds to ‘stim’ and before diffusive broadening of the hotspots. (D, E) TH-GFP and ∆F/F activity from a dendrite of a dopamine neuron and overlay. Cell body not shown. Activity traces from dendrite are averaged over n=3 stimulations. (F, G) Spontaneous activity from dendrites around cell body of a dopamine neuron and maximum intensity projection of the ∆F/F stack and overlay. (F) Shows ∆F/F activity traces from regions of interest (ROI) numbered in (G). Red wedges in (B) and (D) = time of optical stimuli. DopaFilm hotspots localize to defined varicosities We next explored the consistency of DopaFilm ∆F/F hotspot dynamics over multiple stimulation epochs and asked if repeat stimulations in the same FOV generated DopaFilm hotspots that localize to the same set of boutons. We carried out imaging in an FOV where we applied the same optical stimuli to drive hotspot activity over separate imaging sessions. In axon terminals, we observed that repeat stimulations can be carried out with rest periods of ~ 2–3 min between stimuli, giving rise to a consistent set of DopaFilm hotspot activities, and suggesting that release-competent dopamine neuron boutons likely have a high probability of release (Figure 3A, Figure 3—figure supplement 1A-B). To evaluate the spatial specificity of the observed hotspots, we computed the intensity-weighted centroid of each DopaFilm hotspot and compared the centroids across multiple stimulation repeats. We found that hotspot centroids were remarkably consistent across repeat stimulations (Figure 3C). To determine if the hotspots localized to the same set of varicosities, we compared DopaFilm hotspot centroids with the centroid of a TH+ varicosity that spatially overlapped with the DopaFilm hotspot from the overlay image (Figure 3B). We defined dopamine varicosities as puncta where TH-GFP intensity is at least 3 × the mean intensity of TH-GFP expression along the process and computed the centroids of these TH+ boutons (Figure 3B center, green puncta, Figure 3D). Using an arbitrarily chosen origin as a reference point (0,0), we compared centroids of DopaFilm hotspots and TH+ boutons. Our results showed that ∆F/F hotspot centroids and bouton centroids matched with remarkable consistency across stimuli (no offset in some regions of interest [ROIs], <5 camera pixels for all ROIs, equivalent to <1.7 µm; Figure 3E). These experiments demonstrate that DopaFilm activity hotspots can be faithfully localized to the same set of boutons across stimulation epochs and are therefore likely driven by the efflux of dopamine from putative active zones of these boutons. The ability to localize synaptic dopamine efflux to specific boutons is unique to this study and to the best of our knowledge has not been demonstrated before. Figure 3 with 2 supplements see all Download asset Open asset DopaFilm hotspots localize to defined varicosities. (A) Repeat optical stimulation produced a consistent set of DopaFilm hotspots (n=5 of n=10 stimulations shown), thresholded for better visualization. (B) Hotspot colocalization with tyrosine hydroxylase-GFP (TH-GFP) varicosities (green) for stim #1. Green colors indicate GFP mean intensity >3 × the mean intensity along the process. (C) Centroid of hotpots (regions of interest [ROIs] 1–5). Each data point of an ROI corresponds to one evoked imaging run and there are five total per ROI. (D) Line profile for one of the varicosities is shown in (B). Line profile is calculated along the red curve shown in (B). (E) Parity plot for TH and DopaFilm hotspot centroids. Centroid of TH varicosities and averaged centroid of DopaFilm hotspots are plotted. Distance is calculated from the top left corner (=origin) of near-infrared (NIR) ∆F/F image shown in (B). (F, G) Spatiotemporal dynamics of imaging in artificial cerebrospinal fluid (ACSF) and ACSF + nomifensine (+10 µM NOM), respectively. (H) TH-GFP image of field of view in (F, G). Red arrows = no release. Scale bar = 10 µm. (I) ∆F/F traces of hotspots in ACSF (black) and ACSF + 10 µM NOM (red) and their mean traces in bold. (J) Box plots comparing the effect of NOM on peak ∆F/F (left) and full width at half maximum (FWHM; right) of traces shown in (I). Unpaired t-test, p<10–4 for both ∆F/F and FWHM data. See Methods for box plot definitions. It is notable that not all TH-GFP+ boutons produced a corresponding DopaFilm activity (Figure 2C, Figure 3B, red arrows indicate no activity) despite appearing to satisfy the morphological criteria for a varicosity. We considered the possibility that the failure to detect activity from some putatively silent boutons was the result of rapid dopamine clearance, before DopaFilm detection. To test if dopamine clearance was critical to ‘silent’ boutons, we applied saturating levels of nomifensine (NOM; 10 µM), a dopamine-specific reuptake inhibitor (Ki ≈ 100 nM). Application of NOM altered the clearance kinetics of DopaFilm transients, defined by a first-order decay time constant, τoff (mean ± SD of τoff for artificial cerebrospinal fluid [ACSF]: 4.38 ± 0.84 and for +NOM: 5.70 ± 1.47, p-value=3×10–4, Figure 3—figure supplement 1E) and dramatically affected the overall kinetic profile of ∆F/F traces, indicated by mean FWHM of 1.85 s before and 4.45 s after application of NOM (Figure 3F–J). Additionally, the measured peak amplitude of the ∆F/F trace was higher after application of NOM (mean peak ∆F/F = 15.7% before and 24.5% after application of NOM; Figure 3F–J). Intriguingly, NOM altered the turn-on profile of DopaFilm transients (Figure 3—figure supplement 1C-D). When imaging under normal ACSF, the clearance profile of dopamine followed an approximately monotonic decay that is punctuated by brief instances of upward deflection (blue arrows in Figure 3—figure supplement 1C). We posit that these are dopamine release events whose signal is arrested by dopamine transporters (DATs). Saturation of DATs by NOM permits these release events in the poststimulation epoch to be detected by DopaFilm. This suggests that DATs may therefore play a role in arresting spillover of dopamine during a train of release events. Importantly, application of NOM did not reveal any subthreshold activity at silent varicosities that could have gone undetected in pre-NOM imaging sessions (Figure 3F and G). Thus, we conclude that DopaFilm hotspot activity arises from dopamine release at varicosities, and that the absence of DopaFilm fluorescence transient is likely an indication of a lack of dopamine release at release-incompetent varicosities. When coupled with the fact that DopaFilm activity is not observed in TH− cells, and that activity is absent when imaging in Ca2+-free media, we conclude that DopaFilm ∆F/F activity is a consequence of release of endogenous dopamine from active release sites. The NOM manipulation of the temporal dynamics of dopamine release provides additional evidence that DopaFilm possesses the kinetics necessary to recapitulate the dynamic behavior of dopamine release, diffusion, and clearance. In our study, dopamine neurons are co-cultured with cortico-hippocampal neurons, and we explored if glutamatergic activity from neurons in co-culture could influence dopamine release. To investigate this, we carried out experiments in which α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor antagonist 2,3-dioxo-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX) and N-methyl-D-aspartate (NMDA)-type glutamate receptor antagonist D-2-amino-5-phosphonovalerate (D-AP5) were bath-applied to the co-culture system while imaging release from dopamine neurons. We first examined neurons from which DopaFilm activity can be detected from spontaneous spiking events in which we applied no external stimulus to generate activity. We imaged from these neurons under ACSF (our normal imaging buffer) and then bath-applied NBQX (10 µM). DopaFilm activities that were detected before application of NBQX were absent in the postdrug imaging sessions (Figure 3—figure supplement 2A-B). Application of NBQX was sufficient to abolish these activities. Additionally, we examined the extent to which glutamatergic currents contributed to dopamine neuron depolarization during evoked activity imaging. To investigate this, we carried out imaging before and after glutamate receptor blockade with a combined application of NBQX and D-AP5. Here, such treatment resulted in reduced dopamine release as measured by the peak amplitude of ∆F/F traces and the area under the curve of ∆F/F traces (Figure 3—figure supplement 2C-E). In sum, these results indicate that DopaFilm offers an opportunity for direct measurement of dopamine release under pharmacological perturbations and suggests that our in vitro culture system may permit simplified explorations of local chemical circuitries that control dopamine release in the absence of complex circuit effects that may be encountered in vivo. DopaFilm detects quantal release of dopamine We next sought to establish the limit of detection of DopaFilm. In in vitro experiments, we determined that DopaFilm exhibits high sensitivity to dopamine and can detect 1 nM concentrations in bath application experiments (Figure 1—figure supplement 3B). This suggested that DopaFilm may be sensitive enough to detect single events of quantized dopamine efflux from release sites. To determine the limit of detection from a practical sense, we carried out imaging experiments in a field of an axonal arbor of a dopamine neuron before and after bath application of tetrodotoxin (TTX), which inhibits action potential driven, synchronous neurotransmitter release while sparing stochastic and spontaneous release events. We first imaged activity in ACSF, o" @default.
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• W4300492090 date "2022-04-27" @default.
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• W4300492090 title "Decision letter: Visualizing synaptic dopamine efflux with a 2D composite nanofilm" @default.
• W4300492090 doi "https://doi.org/10.7554/elife.78773.sa1" @default.
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