Acute blockade of the Caenorhabditis elegans dopamine transporter DAT-1 by the mammalian norepinephrine transporter inhibitor nisoxetine reveals the influence of genetic modifications of dopamine signaling in vivo

Abstract

Modulation of neurotransmission by the catecholamine dopamine (DA) is conserved across phylogeny. In the nematode Caenorhabditis elegans, excess DA signaling triggers Swimming-Induced Paralysis (Swip), a phenotype first described in animals with loss of function mutations in the presynaptic DA transporter (dat-1). Swip has proven to be a phenotype suitable for the identification of novel dat-1 mutations as well as the identification of novel genes that impact DA signaling. Pharmacological manipulations can also induce Swip, though the reagents employed to date lack specificity and potency, limiting their use in evaluation of dat-1 expression and function. Our lab previously established the mammalian norepinephrine transporter (NET) inhibitor nisoxetine to be a potent antagonist of DA uptake conferred by DAT-1 following heterologous expression. Here we demonstrate the ability of low (μM) concentrations of nisoxetine to trigger Swip within minutes of incubation, with paralysis dependent on DA release and signaling, and non-additive with Swip triggered by dat-1 deletion. Using nisoxetine in combination with genetic mutations that impact DA release, we further demonstrate the utility of the drug for demonstrating contributions of presynaptic DA receptors and ion channels to Swip. Together, these findings reveal nisoxetine as a powerful reagent for monitoring multiple dimensions of DA signaling in vivo, thus providing a new resource that can be used to evaluate contributions of dat-1 and other genes linked to DA signaling without the potential for compensations that attend constitutive genetic mutations.

Keywords: Caenorhabditiselegans; Dopamine; Nematode; Nisoxetine; Transporter.

Figure 1

NIS induces DA-dependent Swip. (A) NIS-treated N2 animals show dose-dependent Swip with a curve fit yielding an EC50 = 5.80 ± 0.12 μM. Neither dop-3(vs106) nor cat-2(e1112) animals demonstrate Swip at any NIS doses whereas dat-1(ok157) animals paralyze similarly at all doses of NIS. (B) Heat maps display thrashing frequencies of individual animals over time, with red designating high frequency values, and green designating low values. This analysis shows a high degree of Swip in N2 animals treated with 5 μM NIS, with very little paralysis observed in cat-2(e1112) and dop-3(vs106) animals treated with the same dose of NIS. n ≥ 40 for each condition. (C) N2 animals treated with MPH show dose-dependent increases in paralysis, though cat-2(e1112) animals show paralysis comparable to N2 at all doses, with no significant difference (p>.05) between genotypes at any dose of MPH. dat-1(ok157) animals paralyzed in all doses of MPH. Significance was calculated using a two-way ANOVA with selected Bonferroni posttests comparing genotypes at each dose of MPH.

Figure 2

NIS does not enhance Swip in dat-1 mutants. (A) N2 animals treated with 10 μM NIS show robust Swip that is suppressed by increasing osmolarity. dat-1(ok157) animals display Swip suppression in higher osmolarity solutions, though dat-1(ok157) animals treated with 10 μM NIS exhibit no significant increase in Swip compared to untreated dat-1(ok157) animals at any osmolarity (p>.05). Data were analyzed using a two-way ANOVA with selected Bonferroni posttests comparing genotypes/treatments at each osmolarity. (B) Heat map analysis shows no significant difference between untreated dat-1(ok157) animals and those treated with 5 μM NIS. This similarity between treatments was reflected in percent paralysis (96.6% untreated vs. 90.2% treated), latency to paralyze (205 ± 11.5 sec untreated vs. 197 ± 16.8 sec treated, p<.0001 two-tailed Student’s t-test), and percent reversions to swimming (33.6% untreated vs. 34.8% treated). n ≥ 50 for each condition

Figure 3

NIS-induced Swip is suppressed by loss of asic-1. (A) asic-1(ok415) animals show no basal Swip in water, but display a significant reduction in Swip in 10 μM NIS compared to N2 animals. Data were analyzed using a two-way ANOVA with selected Bonferroni post-tests comparing genotypes at each dose of NIS. **** p<.0001 (B) Heat map analysis shows asic-1(ok157) animals treated with 5 μM NIS have a suppression of Swip compared to N2 animals, with fewer asic-1(ok157) animals paralyzing (48.0% vs. 68.2% for N2). (C) Graphing of average thrashing behavior over time (mean ± SE) reveals a significant genotype difference in thrashing rates between asic-1(ok415); dat-1(ok157) double mutants and dat-1(ok157) animals (p<.0001). (D) Thrashing plots show asic-1(ok415) animals treated with 5 μM NIS have increased average thrashing rates compared to N2 animals treated with 5 μM NIS, with both significant genotype (p<.0001) and time x genotype interaction effects (p<.0001). n ≥ 34 for each condition

Figure 4

Enhancement of NIS-induced Swip by dop-2 mutation supports negative presynaptic regulation of DA signaling (A) Average thrashing plots show enhanced paralysis in dat-1(ok157); dop-2(vs105) double mutants compared to dat-1(ok157) mutants. DA neuron expression of dop-2 with the plasmid Pdat-1::dop-2 suppressed this enhancement. (B) dat-1(ok157); dop-2(vs105) double mutants have a significantly decreased average latency to paralysis compared to dat-1(ok157) mutants, and DA neuron expression of dop-2 rescues this effect up to dat-1(ok157) mutant levels. No significant difference was observed between dat-1(ok157) and dat-1(ok157); dop-2(vs105) (pdat-1::dop-2) transgenic animals. Data was analyzed using individual two-tailed Student’s t-tests. ****p<.0001. n ≥ 25 for each condition. (C) dop-2(vs105) animals have subtle, yet significant increase in Swip compared to N2 in water, and a greater enhancement in 1 μM and 10 μM NIS. cat-2(e1112) animals have significantly less paralysis than N2 in both doses of NIS. Data was analyzed by two-way ANOVA with Bonferroni posttests comparing genotypes at each dose of NIS. **p<.01, ****p<.0001. (D) Heat map analysis of animals in 5 μM NIS further demonstrates the increase in Swip in dop-2(vs105) mutants vs. N2. dop-2(vs105) showed a higher percentage of paralyzed animals (96.0% vs. 68.2% for N2), lower percentage of animals reverting to swimming (14.6% vs. 33.3% for N2) and significantly fewer average reversion events per paralyzed animal (2.14 ± 0.15 events vs. 4.40 ± 0.33 events for N2, p<.0001 two-tailed Student’s t-test). n ≥ 44 for each condition.