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 Caenorhabditis 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 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.

Keywords: Caenorhabditis elegans; DAT; DAT-1; FRAP; dopamine; dopamine transporter; neurotransmitter; synaptic modulation.

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).

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.

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)].