The dopamine membrane transporter plays an active modulatory role in synaptic dopamine homeostasis

Abstract

Modulatory mechanisms of neurotransmitter release and clearance are highly controlled processes whose finely-tuned regulation is critical for functioning of the nervous system. Dysregulation of the monoamine neurotransmitter dopamine can lead to several neuropathies. Synaptic modulation of dopamine is known to involve pre-synaptic D2 auto-receptors and acid sensing ion channels. In addition, the dopamine membrane transporter (DAT) which is responsible for clearance of dopamine from the synaptic cleft, is suspected to play an active role in modulating release of dopamine. Using functional imaging on the C. elegans model system, we show that DAT-1 acts as a negative feedback modulator to neurotransmitter vesicle fusion. Results from our fluorescence recovery after photo-bleaching (FRAP) based experiments were followed up with and reaffirmed using swimming-induced paralysis (SWIP) behavioral assays. Utilizing our numerical FRAP data we have developed a mechanistic model to dissect the dynamics of synaptic vesicle fusion, and compare the feedback effects of DAT-1 with the dopamine auto‐receptor. Our experimental results and the mechanistic model are of potential broader significance as similar dynamics are likely to be used by other synaptic modulators including membrane transporters for other neurotransmitters across species.

Graphical Abstract

The pH-sensitive fluorescent sensor remains OFF inside neurotransmitter vesicles due to low pH. Upon fusion of the vesicle with the synaptic membrane, the sensor is exposed to the neutral extra-cellular pH which switches it to the ON configuration. FRAP imaging of vesicle fusion dynamics reveals an active role for the dopamine membrane transporter in synaptic dopamine homeostasis.

A key issue in neuroscience is understanding the precise rapport mechanisms between neurotransmitter release and neuronal activity. Dopamine is an amine neurotransmitter that influences motor control, cognition, motivation and reward. Dopamine is synthesized by dopaminergic neurons whose cell bodies are located in the midbrain of many mammals with cortical projections (reviewed in Shultz, 2007; Liss and Roeper, 2008). Dysregulation of dopamine signaling is associated with multiple neurological and mood disorders such as, Parkinson’s, Schizophrenia, and attention deficit hyperactivity disorder (ADHD). Understanding the various mechanisms that modulate synaptic dopamine levels via release and reuptake is critical for understanding neural function in health and disease. Molecular processes involved in dopamine synthesis, synaptic vesicular packaging, vesicular fusion and release, and in the reuptake mechanisms are evolutionarily conserved, from simple invertebrates to mammals. Comprehensive and in-depth studies of synaptic modulatory mechanisms and their interactions used by dopaminergic neurons can reveal a clearer picture of the finely-tuned synaptic modulation.

The complex circuitry of mammalian nervous system makes it particularly challenging to disentangle inter-digitated mechanisms of neurotransmitter homeostasis. The invertebrate nematode model of Caenorhabditis elegans with its well-defined 302-neuron nervous system has been utilized to elucidate dopamine modulatory mechanisms (Nass and Blakely, 2003; Chase and Koelle, 2007; Voglis and Tavernarakis, 2008; Formisano et al., 2020). As in humans, dopaminergic signaling in C. elegans is known to affect multiple behaviors including locomotion, non-associative and associative learning (Chase et al., 2004; Kindt et al., 2007; Voglis and Tavernarakis, 2008; Mersha et al., 2013; Ardiel et al., 2016). There are 8 dopaminergic neurons in the C. elegans hermaphrodite: 2 anterior deirid neurons (ADE) and 4 cephalic neurons (CEP) located towards the anterior, and 2 posterior deirid neurons (PDEs), plus an additional 6 neurons that are present only in the male tail (White et al., 1986; Chase and Koelle, 2007). Tyrosine hydroxylase (encoded by cat-2) catalyzes tyrosine to levodopa, which is then converted to dopamine by an aromatic amino acid decarboxylase (encoded by bas-1). Dopamine is packaged for release into acidified vesicles by a vesicular monoamine transporter (encoded by cat-1) (Loer and Kenyon, 1993; Duerr et al., 1999). Dopamine-laden vesicles fuse with the plasma membrane constitutively at a low basal rate to release dopamine, with increased rates evoked by depolarization (Chase and Koelle, 2007; Ramirez and Kavalali, 2011). The dopamine membrane transporter (DAT-1), a sodium-dependent symporter present on the pre-synaptic membrane, is responsible for clearance and re-uptake of dopamine from the synaptic cleft into the pre-synaptic neuron for storage and later release (Jayanthi et al., 1998). Deletion of dat-1 causes accumulation of extracellular dopamine into the synaptic cleft resulting in a swimming-induced paralysis (SWIP) behavioral phenotype (McDonald et al., 2007; Hardaway et al., 2012). Dynamic regulation of the dat-1 gene influences DAT-1 expression and is likely to modulate behavioral effects observed by its deletion (Felton and Johnson, 2014; Robinson et al., 2019).

Previously, the DAT transporter protein was believed to “simply clear” the extracellular dopamine through reuptake; it is now suspected to play a more active role in the activity of the pre-synaptic neuron (Giros and Caron, 1993; Gowrishanker et al., 2014; Refai and Blakely, 2018). Dopamine uptake by DAT is coupled with intracellular translocation of one Cl− and two Na+ ions, thereby driving the neuronal membrane towards depolarization. Evidence from multiple biological model systems have been used in efforts to understand DAT function. Ex vivo studies with mammalian HEK cells have demonstrated that membrane depolarization decreases trafficking of DAT to the membrane surface via CaM Kinase-II-dependent endocytosis (Richardson et al., 2016). Over-expression of DAT in mice results in decreased extra-cellular dopamine and increased locomotor hyperactivity in response to amphetamines (Salahpour et al., 2008). Increased activity of DAT and tyrosine hydroxylase has also been shown to correlate with D2 auto-receptor activation at saturating levels of ex vivo dopamine in Xenopus oocytes (Mayfield and Zahniser, 2001), and in vitro protein-protein interaction between DAT and the presynaptic D2 auto-receptor has been suggested to facilitate DAT recruitment to the cell surface (Lee et al., 2007). Interestingly, this recruitment tends to decrease in C. elegans mutants deleted in the D2 auto-receptor, upon ethanol exposure (Pandey et al, 2021). Optogenetic and voltammetric studies with rat hypothalamic dopamine neurons in culture suggest that auto-regulatory characteristics of these neurons are attributable to the re-uptake of synaptic dopamine by the dopamine transporter (Stagkourakis et al., 2019). In short, a wide range of studies underscore the importance of DAT on dopaminergic signaling and on the resulting dopamine-associated behaviors.

Fusion of the acidic dopamine-laden vesicles with the synaptic membrane causes co-release of H+ ions along with the neurotransmitter, causing a local drop in pH. This activates an acid sensing ion channel (ASIC-1 in C. elegans) that facilitates a positive feedback loop reinforcing further release of dopamine (Voglis and Tavernarakis, 2008). The role of ASIC channels in neuronal function appears to be conserved in both vertebrates and invertebrates (reviewed in Hill and Ben-Shahar, 2018). Presence of dopamine in the synaptic cleft can also activate auto-receptors (DOP-2 in C. elegans) that form a negative feedback loop to the vesicle fusion process via an inhibitory Gαi pathway (Pandey & Harbinder, 2012) in line with previously described D2-like pharmacological properties reported for DOP-2 (Suo et al., 2004). It has been suggested that the DAT transporter may influence synaptic vesicular fusion through functional interactions with other modulators such as the D2-type dopamine auto-receptors and acid-sensing ion channels (Ford, 2014; Bermingham et al., 2016; Formisano et al., 2020). However, direct evidence of the indicatory feedback role of the DAT transporter in dopamine modulation is lacking. The ability to image functional synaptic termini with a vesicle fusion-based optical indicator in live animals has been exploited to describe the feed-forward role of ASIC-1 and DOP-2 in modulating synaptic vesicle fusion dynamics at dopaminergic synapses (Miesenbock et al., 1998; Voglis and Tavernarakis, 2008; Formisano et al., 2020; Pandey et al., 2021).

Using a fluorescence recovery after photo-bleaching (FRAP) approach, in the present study we have utilized a pH-sensitive fluorescent reporter to quantitate synaptic vesicle fusion, as a surrogate for measuring the rate of constitutive neurotransmitter vesicle fusion at dopaminergic synapses in live animals. Analysis of our FRAP imaging data from individual synaptic termini in live animals suggests that the DAT-1 transporter forms a negative feedback loop pathway to modulate constitutive fusion of neurotransmitter vesicles. We also discuss our results from swimming induced paralysis, a behavioral phenotype that correlates paralysis with the presence of excessive extracellular dopamine. In addition, we put forth a novel mechanistic model that dissects the biphasic nature of synaptic vesicle fusion kinetics. Our findings on the modulatory role of the C. elegans DAT-1 transporter are of potential broader significance as similar control mechanisms are likely to be used by membrane transporters for other small molecule neurotransmitters.

Methods:

Strains and maintenance:

Wild-type (N2 Bristol), dat-1 (ok157)III and dop-2 (vs105)V deletion mutant strains were obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota, Twin Cities, MN). A transgenic strain expressing synaptobrevin-1 (snb-1) fused to a pH-sensitive reporter (SEpHluorin; described in Miesenbock et al., 1998 and Voglis and Tavernarakis, 2008) in the dopaminergic neurons was a gift from Dr. Nektarios Tavernarakis (Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Crete, Greece). Deletion strains were crossed into the transgenic strain expressing the SNB-1::SEpHluorin fusion protein to monitor synaptic vesicle fusion by fluorescence recovery after photo-bleaching, described below. Worm strains were grown and maintained at 20˚C on nematode growth media (NGM) seeded with Escherichia coli strain OP50 as previously described (Brenner, 1974).

Microscopy and FRAP data analyses:

SEpHluorin is a pH sensitive green fluorescent optical reporter that displays very limited fluorescence inside the acidic environment of synaptic vesicles; however, upon vesicle fusion with the synaptic membrane the fluorescent domain of SEpHluorin is exposed to the near neutral pH of the extracellular environment of the synaptic cleft, which unleashes its bright fluorescence (Miesenbock et al., 1998). Fusion rates of SNB-1::SEpHluorin sensor protein at individual synapses originating from worm dopaminergic neurons were examined on a confocal microscope (Zeiss LSM 710). FRAP imaging and image analyses was carried out essentially as previously described in the worm system (Samuel et al., 2003; Voglis and Tavernarakis, 2008; Formisano et al., 2020). In brief, young adults grown at 20^oC were mounted onto 5% agarose pads and treated with 2 microL of 20 mM Levamisol, a mild anesthetic. Individual dopaminergic synaptic puncta were first located with the 10X objective, after which the magnification was switched to estimate their baseline fluorescence. Initial pre-bleach intensity was recorded by capturing three control images (one frame every two seconds) from a rectangular region of interest (ROI) within the synapse under observation. Next, the ROI was bleached to ~50% of initial intensity using a 15mW argon laser (488nm wavelength), and emission was filtered through a 500–550nm BP filter. Immediately after photo-bleaching, 60 images were captured every 2 seconds to monitor fluorescence recovery. A pixel depth of 16-bit was used to obtain emission intensity data from the confocal microscope associated Zen digital imaging software (Carl Zeiss AG, Oberkochen, Germany). The fluorescence was quantified by measuring the fluorescence intensity in three regions: at the synapse of interest Fi; at an unbleached region Fc (another fluorescent synapse from the dopaminergic cell body region) and at a background region Fb (a non-fluorescent region). The data was transferred to the open-source Image-J/Fiji software for alignment of images and correction for any XY movement (Schindelin et al., 2012). Next, images were converted into numerical data using Zen software, and fluorescence intensity values for each synapse was normalized and quantitated with consideration to unbleached and to background non-fluorescent regions. Data from each region of interest was averaged and used to normalize intensity points such that: Normalized Value = (Fi - Fb_avg ) / (Fi * Bleach Correction). After correction for background and photobleaching the data was fitted to a zero to 100 scale in which the bleached intensity equals to zero and the maximum fluorescence intensity, i.e. the pre-bleach intensity, equals to 100 percent.

For statistical fittings with our mechanistic model (detailed in Box-1) FRAP data from wild-type and the two deletion strains were fitted to the equation $\mathit{F}\left(\mathit{t}\right)={\mathit{F}}_{\mathit{s}}\left(1-\mathit{f}\mathit{r}\mathit{a}\mathit{c}{\mathit{e}}^{-\mathit{d}\mathit{t}}-\left(1-\mathit{f}\mathit{r}\mathit{a}\mathit{c}\right){\mathit{e}}^{-\left(\mathit{f}+\mathit{\lambda }\right)\mathit{t}}\right).$ For 95% confidence intervals on synaptic vesicle fusion rates we performed bootstrapping by sampling without replacement from the singe-cell FRAP recoveries and fitting above equation to obtain values of f+λ.

Box-1: A mechanistic model for biphasic synaptic vesicle fusion dynamics.

Fusion of acidified neurotransmitter vesicles expressing the pH sensitive GFP reporter with the synaptic membrane causes exposure of the reporter to the neutral pH of the synaptic cleft, leading to activation of its fluorescence signal. Fluorescence recovery at the synapse is dependent upon (i) the vesicle fusion rate and (ii) diffusion rate of the fluorescence signal from the reporter. A qualitative representation of these two processes is shown on the top right of this box (higher resolution figure is uploaded with manuscript figures). These dynamic signals lead to the observed double-exponential recovery to a fast recovery time-scale (τ_F) that correlates with the diffusion rate of the fluorescent reporter outside the experimental region of interest (ROI), and a slow recovery time-scale (τ_S), that is a function of the docking rate of synaptic vesicles. In order to dissect the two phases observed with synaptic vesicle fusion dynamics, we have used a mathematical modelling approach to develop a mechanistic model that resolves the double-differential recovery kinetics.

SWIP behavior assays and data analyses:

SWIP assays were performed essentially as described previously (McDonald et al., 2007). Briefly, worms cultured on NGM plates at 20°C were synchronized using a freshly made alkaline sodium hypochlorite solution (0.25 M KOH, 1.25% NaOCl). L4 stage hermaphrodites from the wild type and mutant strains were tested blindly by the investigators (RF and KDR); ~8 L4 animals were placed into 50μL of 1xM9 solution in a single well of Pyrex spot plate. The number of worms paralyzed was counted and documented at each minute and scored throughout 20-minute duration of the assay. Two-way ANOVA with Bonferroni multiple comparisons post hoc test was made for comparison of the paralysis rate at each time point.

Results and Discussion

Results from FRAP measurements at individual synaptic puncta from live, anesthetized animals surrogating for constitutive synaptic vesicle fusion rates are presented and discussed here. Fluorescence recovery at dopaminergic synapses labelled with SNB-1::SEpHluorin in dat-1 (ok157) deletion mutants is faster, as compared to wild type animals (Figure-1). The faster rate of vesicle fusion in dat-1 deletion mutants suggests that these mutants are impaired in a mechanism that down-regulates the release of dopamine at synapses. In other words, removal of DAT-1 function in dat-1 deletion mutants function frees its inhibitory effect leading to faster recovery. To our knowledge, these results provide the first direct evidence of a feed-back role for the dopamine transporter that has been suggested previously (Stagkourakis et al., 2016). Notably, the FRAP recovery of dat-1 deletion mutants is qualitatively similar to that observed for another negative modulator (D2 auto-receptor) as reported in animals deleted for dop-2 (Formisano et al., 2020).

Figure 1:

(a) Schematic of the FRAP strategy and a qualitative representation of the raw confocal images of a bleached synapse and its recovery. (b) The dopamine membrane transporter (DAT-1) mediates negative feedback on synaptic vesicle fusion. Mutants carrying a deletion in the dopamine membrane transporter gene dat-1 (red curve) display significantly faster fluorescence recovery after-photobleaching (FRAP) at dopaminergic synapses labelled with SNB-1::SEpHluorin as compared to wild-type N2 animals (green curve). Previously reported FRAP recovery rates of mutants carrying a deletion in the dopamine auto-receptor gene (dop-2) also correspond to negative feed-back modulation (orange curve). However, the kinetics of feedback inhibition in the fast (τ_F) and slow (τ_S) recovery phases are distinct in the two deletion mutants. Fitting the data to our mechanistic model explains the biphasic FRAP recovery, we obtained the fast timescale related to fluorescence decay to be τ_F = 2 sec, and slow timescale of recovery related to vesicle fusion being 17 seconds and 84 seconds for dat-1 and dop-2 deletion mutants, respectively as compared to 155 seconds for wildtype. FRAP data from the three strains were fitted to the equation $\mathit{F}\left(\mathit{t}\right)={\mathit{F}}_{\mathit{s}}\left(1-\mathit{f}\mathit{r}\mathit{a}\mathit{c}{\mathit{e}}^{-\mathit{d}\mathit{t}}-\left(1-\mathit{f}\mathit{r}\mathit{a}\mathit{c}\right){\mathit{e}}^{-\left(\mathit{f}+\mathit{\lambda }\right)\mathit{t}}\right)$ (equation-1, detailed in Box-1). Bootstrap statistical sampling was performed on fitted data to obtain values for λ, the refilling rate per empty docking site, plus f, the constitutive rate of vesicle fusion (n ≥ 10 synapses for each strain; 95% confidence intervals).

Closer examination of recovery kinetics indicates biphasic nature of recovery: a rapid initial increase in fluorescence recovery (τ_F), followed by a slower recovery (τ_S) in all 3 strains: the dat-1 deletion, the dop-2 deletion and wild-type animals. We reasoned that the fluorescence recovery at a constitutively active synapse is dependent upon the vesicle fusion rate as well as decay rate of the fluorescence signal to diffusion outside the FRAP region of interest (ROI). In order to unravel the dynamic contributions of vesicle fusion and decay rates, we develop a mechanistic model that attributes the double-exponential recovery observed in Figure 1 to a slow recovery time-scale (τ_S) that correlates with the docking rate of synaptic vesicles, and a fast recovery time-scale(τ_F) that is related to the diffusion rate of the fluorescent reporter to outside the experimental region of interest (Box-1). This mechanistic model when applied to recovery kinetics at steady state values, reveals that in comparison to wild-type animals the overall vesicle fusion rate is nine-fold higher in dat-1 deletion mutants, and two-fold higher in dop-2 deletion mutants (Figure 2a and 2b).

Figure-2:

Quantitative comparison of the biphasic recovery dynamics of the two negative modulators of dopamine based on the mechanistic two-phase exponential function explained in Box-1. (a) Differential analyses of the slow and fast recovery phases of our data sets revealed that d≈0.5 sec^-1, frac≈0.30, F_s≈85% with the slow time-scale of recovery related to vesicle fusion being 17 seconds, 155 seconds and 84 seconds for dat-1 deletion, wildtype, and dop-2 deletion, respectively. (b) The overall vesicle fusion rate which is inverse of the slow time-scale of recovery is nine-fold higher in dat-1 deletion mutants, and two-fold higher in dop-2 deletion mutants in comparison to wild-type animals. Error bars denote 95% confidence intervals estimated using bootstrapping.

In order to further investigate results from our synaptic vesicle fusion experiments, we employed the swimming-induced paralysis (SWIP) assay, a behavioral tool to study dopaminergic synapse regulation. Worms tend to thrash rapidly when placed in water; however, excessive extracellular dopamine results in decreased locomotion, or paralysis, producing a quantifiable SWIP phenotype (McDonald et al., 2007). Our SWIP results show that both dat-1 deletion mutants as well as dop-2 deletion mutants display significantly greater paralyses as compared to wild type animals (Figure-3), and our observations are consistent with the previously reported results for dat-1 (McDonald et al., 2007; Hardaway 2015). We found that 64% of dat-1 deletion mutants and 44% of dop-2 deletion mutants are paralyzed at the end of the 20 minute SWIP assay, as compared to only 14% of wild type animals. The increased paralysis behavior observed for both mutants correlates with potentially negative feedback roles of their protein products. A disruption of either of the feedback switches will lead towards increased extracellular dopamine, hence leading to paralysis. Interestingly, the SWIP behavior of the two mutants mirror our FRAP results in that we observe a stronger feedback effect as well as a stronger SWIP phenotype in the dat-1 deletion mutants as compared to the dop-2 deletion mutants. It has been suggested that DAT blockage increases dopamine release by mobilizing dopamine from the synaptic vesicle reserve pool through interactions with the vesicle-associated synapsin proteins (Venton et al., 2006; Sulzer et al., 2016). Cocaine and other pharmacological inhibitors of DAT can increase dopamine release in mice, suggesting a role for DAT in restricting vesicle pool mobility and/or release (Jones et al., 1998). Our FRAP results confirm that there is a higher fraction of mobile synaptic vesicles in dat-1 mutants, supporting the concept that the broader function of DAT-1 in modulation of dopamine release by influencing synaptic vesicle fusion, in addition to its canonical role of neurotransmitter clearance from the synaptic cleft through reuptake. Functional consequences of the excess extracellular dopamine manifests in increased paralysis behavior as observed in our SWIP assay results, and as reported and interpreted in previous investigations (McDonald et al., 2007).

Figure-3:

Swimming induced paralysis (SWIP) behavioral assays reveal that both dat-1 deletion mutants and dop-2 deletion mutants show greater paralysis, as compared to wild type animals. Comparison of the dat-1 deletion mutant with wild type animals for SWIP behavior phenotype at each time point reveals that the mutant displays significant paralysis from minute 3 till the end of the assay at 20 minutes. Likewise, dop-2 deletion mutants show significant increase in paralysis from 7–20 minutes, as compared to wild type animals. In order to compare the interaction of two nominal variables in our assay [(i) time, and (ii) wild-type or mutant strains] with one measurement variable [number of worms paralyzed], a two-away ANOVA was used, which was followed by Bonferroni post hoc test for false discovery rate correction [(F-distribution (40, 1300) =16.86, p values ≤ 0.05, n ≥ 150 animals (dat-1Δ=157, dop-2Δ=157, N2 wild-type=175)].

Taken together, results from our FRAP experiments reveal that DAT-1 acts similar to DOP-2, a known negative modulator. Interestingly, based on effective recovery phase dynamics, DAT-1 exerts a remarkably dominant effect on the feedback signal than DOP-2 does on the rate of constitutive synaptic vesicle fusion. Likewise, increased paralysis observed in the dat-1 deletion as compared to the dop-2 deletion may be due to DAT-1 providing a stronger feedback signal as compared to DOP-2. Alternately, since dat-1 deletion mutants also lack the ability to reuptake extracellular dopamine in addition to the negative feedback function, this could bolster further increase in extracellular dopamine. Our results from quantitative visualization of neurotransmitter vesicle fusion rates in individual synapses in a living, intact organism, provide in vivo evidence for the participation of the DAT-1 transporter in modulation of synaptic dopamine levels. While it has been previously suggested and is reasonable to expect, that accumulation of extracellular synaptic dopamine in dat-1 mutants may necessitate a need for lowering the rate of dopamine release; however, evidence implicating the dopamine membrane transporter in such a role had been elusive (Ford, 2014; Gowrishanker et al., 2014). To our knowledge, the results presented here are the first to reveal a role for DAT-1 in modulating dopamine release. Furthermore, analyses of our FRAP recovery kinetic characteristics indicates that the negative feedback loop provided by DAT-1 is distinct from the negative feedback loop of DOP-2. Alternately, DAT-1 may mediate multiple negative feedback mechanisms, one of which may coupe with the auto-receptor (DOP-2) mediated modulation. Additionally, we utilized our numerical data to develop a mechanistic model that takes into consideration the dynamic contributions of synaptic vesicle fusion and signal decay rates in the FRAP process, thereby, allowing the dissection of the biphasic or double-exponential nature of observed fluorescence recovery.

Based on behavioral and genetic evidence, it has been suggested that DAT-1 and DOP-2 auto-receptors may influence synaptic vesicular fusion through functional interactions (Bermingham et al., 2016). The DAT symporter drives the neuronal membrane towards depolarization by coupling dopamine re-uptake with the intracellular translocation of one Cl^- and two Na⁺ ions. Membrane depolarization associated with DAT activity is likely to cause increased activation of presynaptic auto-receptors coupled to a G_αi subunit of an inhibitory G-protein, which would reduce vesicle fusion and dopamine release. In vitro experiments also suggest that the dopamine transporter along with dopamine auto-receptors may form part of a larger signaling complex, termed as the ‘D2 signalplex’, that appears well placed to exhibit tight control over synaptic dopamine levels (Lee et al., 2007; Vaughan and Foster, 2013;). However, there is very limited information on the functional activity of the complex. Results from our experimental approach pave the path towards in vivo testing of DAT interactions as well as efficacy of pharmacological agents (such as nisoxetine, azaperone and methylphenidate) that have been reported to affect modulation of synaptic dopamine (Bermingham et al., 2016; Refai and Blakely, 2019; Stagkourakis et al., 2019).

In conclusion, given the highly-conserved nature of molecular pathways underlying dopaminergic neurotransmission across phyla, our results with the worm model provide a foundational understanding on similar mechanisms that may modulate synaptic vesicle fusion in vertebrates (Ford, 2014). We remain mindful that we have measured synaptic vesicle fusion as an indirect surrogate for dopamine levels. Direct measurement of synaptic dopamine levels is not feasible with currently available technology. Advances in synthetic compounds such as false fluorescent neurotransmitters that can mimic dopamine and can be packaged into vesicles by the vesicular monoamine transporter (Pereira et al., 2016; Black et al., 2020) hold potential for direct measurements of vesicular contents in the future. Surrogate tools such as genetically encoded or chemical indicators for calcium to monitor Ca2+ influx (Tian et al., 2009) have the potential to bolster conclusions derived from the FRAP and behavioral approaches reported in the current study.

To explain the biphasic recovery of the FRAP data our mechanistic model considers a two-phase exponential function capturing a fast time-scale τ_F corresponding to decay of the fluorescence signal and a slow time-scale τ_S corresponding to synaptic vesicle fusion. We considered the following model for vesicle recruitment to the active zone of the synapse:

$$\frac{\mathit{d}\mathit{V}}{\mathit{d}\mathit{t}}=\mathit{\lambda }\left(\mathit{M}-\mathit{V}\right)-\mathit{f}\mathit{V}\mathit{,}$$

where V(t) is the number of docked vesicles, M is the total number of docking sites, λ is the refilling rate per empty docking site, f is the constitutive rate of vesicle fusion (Brill et al., 2019). Assuming each vesicle contributes to k fluorescence units upon fusing, the dynamics of the fluorescence signal F(t) is given by:

$$\frac{\mathit{d}\mathit{F}}{\mathit{d}\mathit{t}}=\mathit{k}\mathit{f}\mathit{V}-\mathit{d}\mathit{F}\mathit{,}$$

where d is the decay rate of the fluorescence signal that is related to the diffusion of the fluorescent reporter from the FRAP region.

At steady-state, the number of docked vesicles V_s and the measured fluorescence signal F_s is given by:

$${\mathit{V}}_{\mathit{s}}≔\underset{\mathit{t}\to \mathrm{\infty }}{\mathbf{lim}}\mathit{V}\left(\mathit{t}\right)=\frac{\mathit{\lambda }\mathit{M}}{(\mathit{f}+\mathit{\lambda })}\mathrm{and}{\mathit{F}}_{\mathit{s}}≔\underset{\mathit{t}\to \mathrm{\infty }}{\mathbf{lim}}\mathit{F}\left(\mathit{t}\right)=\frac{\mathit{f}\mathit{k}\mathit{\lambda }\mathit{M}}{\mathit{d}(\mathit{f}+\mathit{\lambda })},$$

respectively. Considering a scenario where after photo-bleaching only a fraction (frac) of vesicles remain docked, followed by solving the above differential equations with initial conditions V(0) = frac × V_s and F(0) = 0 results in the double-exponential recovery of the fluorescence signal:

$$\frac{\mathit{F}\left(\mathit{t}\right)}{{\mathit{F}}_{\mathit{s}}}=1-\frac{\mathit{d}\mathrm{ }\mathit{f}\mathit{r}\mathit{a}\mathit{c}-\mathit{f}-\mathit{\lambda }}{\mathrm{ }\mathit{d}-\mathit{f}-\mathit{\lambda }}{\mathit{e}}^{-\mathit{d}\mathit{t}}-\frac{\mathit{d}-\mathit{d}\mathrm{ }\mathit{f}\mathit{r}\mathit{a}\mathit{c}}{\mathrm{ }\mathit{d}-\mathit{f}-\mathit{\lambda }}{\mathit{e}}^{-(\mathit{f}+\mathit{\lambda })\mathit{t}}$$

This signal exhibits two timescales: a fast recovery timescale (τ_F) represented by the rate of fluorescence decay (e^-dt) and a slow recovery timescale (τ_S) represented by the rate of vesicle recruitment and fusion (e^(f+λ)t. Given that molecular diffusion occurs at a much faster time scale than the time scale of constitutive vesicle fusion and vesicle recruitment, we assume that d frac ≫ f+λ (which also implies that d ≫ f+λ) the above fluorescence recovery reduces to:

$$\mathit{F}\left(\mathit{t}\right)={\mathit{F}}_{\mathit{s}}(1-\mathit{f}\mathit{r}\mathit{a}\mathit{c}{\mathit{e}}^{-\mathit{d}\mathit{t}}-(1-\mathit{f}\mathit{r}\mathit{a}\mathit{c}\left){\mathit{e}}^{-\left(\mathit{f}+\mathit{\lambda }\right)\mathit{t}}\right)\mathbf{.}$$ (1)

We fit this equation to data from N2, and the two deletion mutants (dat-1 and dop-2) assuming the exact same values for frac, d, and F_s but different values for the rate of vesicle fusion (f+λ) across different strains. Differential analyses of the slow and fast recovery phases was carried out using the above two-phase exponential function.

SIGNIFICANCE STATEMENT.

Dopamine is an important neurotransmitter involved in several behaviors. Optimal functioning of dopaminergic synapses depends upon precise regulation of dopamine release as well as its clearance. Dopamine imbalances are implicated in several neuro-pathologies, whose underlying physiological mechanisms remain unclear, as does the fundamental biology that underlies dopamine modulation in healthy individuals. We have used the simple-yet-powerful C. elegans in vivo model to uncover a novel function of the dopamine membrane transporter (DAT) in modulating synaptic vesicle release. Considering the similarities of the mammalian and C. elegans dopaminergic systems, results from our experiments will provide a viable platform for investigating mammalian DAT and its interactions with other known dopamine modulators.