Dop1R1, a type 1 dopaminergic receptor expressed in Mushroom Bodies, modulates Drosophila larval locomotion

Abstract

As in vertebrates, dopaminergic neural systems are key regulators of motor programs in insects, including the fly Drosophila melanogaster. Dopaminergic systems innervate the Mushroom Bodies (MB), an important association area in the insect brain primarily associated to olfactory learning and memory, but that has been also implicated with the execution of motor programs. The main objectives of this work is to assess the idea that dopaminergic systems contribute to the execution of motor programs in Drosophila larvae, and then, to evaluate the contribution of specific dopaminergic receptors expressed in MB to these programs. Our results show that animals bearing a mutation in the dopamine transporter show reduced locomotion, while mutants for the dopaminergic biosynthetic enzymes or the dopamine receptor Dop1R1 exhibit increased locomotion. Pan-neuronal expression of an RNAi for the Dop1R1 confirmed these results. Further studies show that animals expressing the RNAi for Dop1R1 in the entire MB neuronal population or only in the MB γ-lobe forming neurons, exhibit an increased motor output, as well. Interestingly, our results also suggest that other dopaminergic receptors do not contribute to larval motor behavior. Thus, our data support the proposition that CNS dopamine systems innervating MB neurons modulate larval locomotion and that Dop1R1 mediates this effect.

Fig 1. Larvae bearing mutations affecting the dopaminergic system show altered locomotion.

A. Motor behavior (distance covered by larvae over 140 sec) was assessed in mutants for the enzymes responsible for dopamine biosynthesis (TH and Ddc), in animals bearing a mutation in the dopamine plasma membrane transporter (DAT), and in larvae mutant for the cAMP-phosphodiesterase (dnc¹). In the upper insert is indicated the dopamine biosynthetic pathway. B. Motor behavior was assessed in animals containing a mutation for three DA receptors Dop1R1, Dop1R2 and Dop2R. Data shown as a box and whiskers plot from 30 or more animals per genotype. *P<0,05; ***P<0,005, one-way ANOVA followed by Tukey post-hoc test, as compared to controls. Genetic background does not affect motor output in our setup.

Fig 2. Dop1R1 receptor expressed in MB modulates larval locomotion.

A. Pan-neuronal expression of an RNAi against Dop1R1 (elav>RNAi^Dop1R1) results in increased motor behavior as compared to genetic controls. B. Expression of RNAi^Dop1R1 in the entire MB neuronal population (OK107>RNAi^Dop1R1) or C. in the γ-lobe forming neurons (201y>RNAi^Dop1R1), induces an increase in larval locomotion. D. No effect on locomotion is observed when the RNAi is expressed in the α′β′ MB forming neurons (c305a>RNAi^Dop1R1). In each case, genetic controls are animals bearing one copy of the Gal4 driver or the undriven UAS-RNAi Dop1R1 (in white and gray bars respectively). Data represent results obtained from at least 34 different larvae per experimental condition. * and *** indicates p<0.05 and p<0.005 compared to genetic controls; one-way ANOVA followed by Tukey post-hoc test.

Fig 3. Dop1R2 does not affect larval locomotion.

A. Expression of RNAi directed to Dop1R2 transcript (RNAi^Dop1R2) in the entire CNS (elav>RNAi^Dop1R2); B. in the MB (OK107>RNAi^Dop1R2); C. in the MB γ-lobe (201y>RNAi^Dop1R2); or α′β′ (c305a>RNAi^Dop1R2) neuronal populations do not affect larval locomotion as compared to genetic controls. Results from 34 larvae or more, per experimental condition. One-way ANOVA followed by Tukey post-hoc test indicated no statistical differences between experimental groups and genetic controls (Gal4/+, white bars; UAS-RNAi^Dop1R2/+, gray bars), except when indicated (*, p<0.05).

Fig 4. Dop2R does not affect larval locomotion.

Expression of an RNAi against Dop2R (RNAi^Dop2R) A. in the entire CNS (elav>RNAi^Dop2R); B. in the whole MB (OK107>RNAi^Dop1R2); or in the neuronal populations forming the C. γ lobe (201y>RNAi^Dop1R2) or D. α′β′ lobe (c305a>RNAi^Dop1R2) does not affect locomotion, when compared to genetic controls (Gal4/+, white bars; UAS-RNAi^Dop2R/+, gray bars). Data obtained from at least 34 larvae per experimental condition. One-way ANOVA followed by Tukey post-hoc test indicated no statistical differences between experimental groups.

Fig 5. Expression of RNAi^Dop1R1 in the γ-lobe MB neurons affects motor output in Drosophila larvae.

Expression of RNAi for Dop1R1, Dop1R2 and Dop2R receptors in MB γ-lobe neurons using a different Gal4 driver line (MB247-Gal4) induces a differential response on motor output. The different RNAi were expressed under the control of this driver that only labels neurons in flies at the larval stage. It is only observed an effect on motor behavior after expression of the RNAi^Dop1R1 (left panel, MB247>RNAi^Dop1R1) while no effect is observed after expression of the other RNAis (MB247>RNAi^Dop1R2 and MB247>RNAi^Dop2R; center and right panel, respectively) in this neuronal population. These data further support the proposition that the Dop1R1 receptor expressed in the MB γ-lobe is responsible for the effects on locomotion. Data obtained from at least 30 different animals. *, ** indicates p<0.05 and p<0.01, respectively; one-way ANOVA followed by Tukey post-test, when experimental group is compared to respective controls (Gal4/+, in white; undriven UAS-RNAi/+, in gray).

Fig 6. A model to explain the contribution of Dop1R1 expressed in MB to Drosophila larval locomotion.

This model proposes, that the type 1 Dop1R1 receptor is expressed in MB and activated by dopaminergic neurons arriving to this brain area. The activation of the dopamine receptor induces an increase in intracellular cAMP levels, which results in decreased motor output. DAT, the dopamine plasma membrane transporter, regulates the availability of dopamine in the synaptic cleft, so that when it is not present or not functional, increases dopaminergic signaling, which results in reduced motor output.