Dopamine signaling in C. elegans is mediated in part by HLH-17-dependent regulation of extracellular dopamine levels

Abstract

In Caenorhabditis elegans, the dopamine transporter DAT-1 regulates synaptic dopamine (DA) signaling by controlling extracellular DA levels. In dat-1(ok157) animals, DA is not taken back up presynaptically but instead reaches extrasynpatic sites, where it activates the dopamine receptor DOP-3 on choligeneric motor neurons and causes animals to become paralyzed in water. This phenotype is called swimming-induced paralysis (SWIP) and is dependent on dat-1 and dop-3. Upstream regulators of dat-1 and dop-3 have yet to be described in C. elegans. In our previous studies, we defined a role for HLH-17 during dopamine response through its regulation of the dopamine receptors. Here we continue our characterization of the effects of HLH-17 on dopamine signaling. Our results suggest that HLH-17 acts downstream of dopamine synthesis to regulate the expression of dop-3 and dat-1. First, we show that hlh-17 animals display a SWIP phenotype that is consistent with its regulation of dop-3 and dat-1. Second, we show that this behavior is enhanced by treatment with the dopamine reuptake inhibitor, bupropion, in both hlh-17 and dat-1 animals, a result suggesting that SWIP behavior is regulated via a mechanism that is both dependent on and independent of DAT-1. Third, and finally, we show that although the SWIP phenotype of hlh-17 animals is unresponsive to the dopamine agonist, reserpine, and to the antidepressant, fluoxetine, hlh-17 animals are not defective in acetylcholine signaling. Taken together, our work suggests that HLH-17 is required to maintain normal levels of dopamine in the synaptic cleft through its regulation of dop-3 and dat-1.

Keywords: acetylcholine signaling; bupropion; dopamine receptor; fluoxetine; reserpine.

Figure 1

HLH-17 functions upstream of dop-3 to regulate dopamine signaling. (A) DA-induced paralysis: hlh-17(ns204), dop-3 (vs106), and hlh-17(ns204); dop-3 (vs106) animals are less sensitive than wild-type animals to 10 mM DA. Transgenic expression of HLH-17::GFP in hlh-17(ns204) animals rescues the DA-induced paralysis phenotype. The bar for hlh-17R represents the average measurements from three biological replicas of three independent lines. *Statistical significance when compared to wild-type, n = 10 animals/strain/rep for three biological replicas. (B) Basal slowing response: Well-fed wild-type animals, but not hlh-17(ns204), dop-3 (vs106), or hlh-17(ns204); dop-3 (vs106) animals, move significantly slower in the presence of food (white bars) than in the absence of food (gray bars). (C) Transgenic expression of HLH-17::GFP rescues the basal slowing response of hlh-17(ns204) animals. Three independent lines, 15.3, 15-1, and 3.1, were assayed. In (B) and (C), five animals/rep/strain for a total of three biological replicas were assayed. Each animal was analyzed for three separate 20-sec intervals, so that the total number of observations was 15 observations/rep/strain. *P < 0.05; **P < 0.005; ***P < 0.0005.

Figure 2

Loss-of hlh-17 affects extracellular DA levels. (A) mRNA levels in L4-stage hlh-17(ns204) animals when normalized against mRNA levels in age-matched wild-type animals. Light gray shading represents wild-type range of expression (1.0 ± 0.115). The levels of cat-1 and mod-5 mRNA are not significantly affected in hlh-17(ns204) animals. (B) hlh-17(ns204) animals demonstrate SWIP behavior that is an intermediate of the behavior in N2 and dat-1(ok157) animals, and that is rescued by transgenic expression of HLH-17::GFP. The bar for hlh-17R represents the average measurements from three biological replicas of three independent lines. For all strains except hlh-17R, n = 30 animals/rep/strain. For hlh-17R, n was equal to an average of at least 15 animals/line/biological rep (range, 12–26) because of differences in transmission frequency of the transgene. (C) SWIP phenotype in double mutant hlh-17(ns204); dat-1(ok157) and hlh-17(ns204); dop-3(vs106) animals is more similar to the phenotype in dat-1(ok157) and dop-3(vs106) animals, respectively, than in wild-type animals. The SWIP phenotype of hlh-17(ns204); dat-1(ok157); dop-3(vs106) animals is not significantly different from the SWIP phenotypes of dop-3 or hlh-17(ns204); dop-3(vs106) animals. n = 30 animals/rep/strain for three biological replicas. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Figure 3

The hlh-17 animals respond selectively to reuptake inhibitors. (A) Pretreatment with the DAT reuptake inhibitor, bupropion, increases the SWIP phenotype of N2, hlh-17(ns204), dat-1(ok157), and hlh-17(ns204); dop-3(vs106) animals. The ability of bupropion to enhance SWIP behavior is not dependent on DOP-3. The SWIP phenotype in hlh-17(ns204) animals is unaffected by pretreatment with reserpine (B) or fluoxetine (C). In all panels, n = 30 animals/rep/strain; dark bars = minus inhibitor; and light bars = plus inhibitor. *P < 0.05; **P < 0.005; ***P < 0.0005.

Figure 4

The hlh-17 animals do not have reduced acetylcholine signaling. (A) hlh-17(ns204) animals are more susceptible to aldicarb-induced paralysis than wild-type (P = 0.0428) and dat-1(ok157) animals (P = 0.1319). (B) The hlh-17(ns204) animals are more susceptible to levamisole-induced paralysis than wild-type (P = 0.0002) and dat-1(ok157) animals (P = 0.0002). In all panels, n = 30 animals/rep/strain.