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 C. 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.

Synaptic vesicle fusion is modulated through feedback inhibition by dopamine auto‐receptors

“…In summary, 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 D2 auto‐receptors in modulation of synaptic dopamine levels. Behavioral and genetic evidence suggests that DOP‐2 influences synaptic vesicular fusion through functional interactions with dopamine modulators including the membrane dopamine transporter (DAT; Bermingham et al, ). DAT works as a symporter by coupling dopamine uptake along with intracellular translocation of one Cl − and two Na + ions which tends to depolarize the neuronal membrane.…”

Blockade and reversal of swimming-induced paralysis in C. elegans by the antipsychotic and D2-type dopamine receptor antagonist azaperone

“…The previous studies that examined the ability of azaperone to reduce phenotypes associated with tau overexpression (McCormick, Wheeler, et al 2013) provided evidence that azaperone effects were mediated as a result of antagonism of both DOP-2 and DOP-3. As a dop-2 loss of function mutation does not attenuate the Swip exhibited by dat-1 mutants (Bermingham et al 2016), we suspected that the actions of azaperone were likely to be solely mediated by DOP-3 antagonism. To test this hypothesis, we tested double mutants bearing both dat-1(ok157) and either dop-2(vs105) or dop-3(vs106) loss of function mutations (Figure 3D).…”

“…To diminish this concern, we tested the ability of azaperone to suppress Swip when paralysis is triggered acutely by pharmacological blockade of DAT-1. Previously, we showed that the mammalian norepinephrine transporter antagonist nisoxetine is a highaffinity antagonist of C. elegans DAT-1 (Jayanthi et al, 1998) and that acute application of the drug produces DA-dependent Swip in wildtype animals, effects lost in a dop-3 mutant (Bermingham et al 2016). As previously demonstrated, transfer of wildtype worms into a solution containing 20 μM nisoxetine resulted in highly penetrant Swip within the 10 min time period of the swimming assay, as compared to animals tested in water alone (Figure 3A,B).…”

“…when azaperone was added to animals that had already paralyzed in water), azaperone stock solution was added to the liquid surface, or well's edge, to reach the desired final concentration, without disturbing paralyzed worms. Nisoxetine hydrochloride (Sigma-Aldrich, St. Louis, MO) was used to induce Swip at 20 μM concentration as described in our prior studies (Bermingham et al 2016) and prepared as a 100 μM stock solution in distilled water. Studies of the ability of azaperone to suppress nisoxetine-induced Swip were performed in solutions of both 20 μM nisoxetine and 250 μM azaperone.…”

“…As this effect is in the opposite direction as the suppression of Swip seen when dat-1 animals are exposed to azaperone, this suggests that the drug may antagonize a D2-type receptor that promotes Swip. We have previously presented evidence that DOP-2 receptors may act as presynaptic DA autoreceptors, providing for feedback inhibition of DA release (Bermingham et al 2016). Azaperone antagonism of these receptors would be predicted to increase DA release and promote Swip.…”