A new brain dopamine-deficient Drosophila and its pharmacological and genetic rescue

Abstract

Dopamine (DA) is a neurotransmitter with conserved behavioral roles between invertebrate and vertebrate animals. In addition to its neural functions, in insects DA is a critical substrate for cuticle pigmentation and hardening. Drosophila tyrosine hydroxylase (DTH) is the rate limiting enzyme for DA biosynthesis. Viable brain DA-deficient flies were previously generated using tissue-selective GAL4-UAS binary expression rescue of a DTH null mutation and these flies show specific behavioral impairments. To circumvent the limitations of rescue via binary expression, here we achieve rescue utilizing genomically integrated mutant DTH. As expected, our DA-deficient flies have no detectable DTH or DA in the brain, and show reduced locomotor activity. This deficit can be rescued by l-DOPA/carbidopa feeding, similar to human Parkinson's disease treatment. Genetic rescue via GAL4/UAS-DTH was also successful, although this required the generation of a new UAS-DTH1 transgene devoid of most untranslated regions, as existing UAS-DTH transgenes express in the brain without a Gal4 driver via endogenous regulatory elements. A surprising finding of our newly constructed UAS-DTH1m is that it expresses DTH at an undetectable level when regulated by dopaminergic GAL4 drivers even when fully rescuing DA, indicating that DTH immunostaining is not necessarily a valid marker for DA expression. This finding necessitated optimizing DA immunohistochemistry, showing details of DA innervation to the mushroom body and the central complex. When DA rescue is limited to specific DA neurons, DA does not diffuse beyond the DTH-expressing terminals, such that DA signaling can be limited to very specific brain regions.

Keywords: Carbiodopa; Dopamine deficiency; Drosophila; Drosophila behavior; Parkinson's disease; central complex; dopamine rescue; genetic model of Parkinson's disease; l-DOPA; mushroom body.

Fig. 1

DTH and DA in w¹¹¹⁸, DTH ple², and DA-deficient flies. (A) DTH and DA Immunoreactivity (IR) in adult brain. DTH ple² flies show normal patterns of DTH and DA IR, whereas DA-deficient flies show no detectable signals. DA IR shows strong innervation of mushroom body subregions and central complex. The few remaining TH positive cells in the DA-deficient brain are superficially located, and most likely represent antibody cross-reactivity because they do not show DA IR. (B) Analysis of brain DA by HPLC in adult brain extracts. DTH ple² brains show near-normal levels of DA, whereas DA-deficient brains show <5% normal levels of DA, with the residual peak most likely caused by an unknown electroactive compound that can comigrate with DA (Riemensperger et al, 2011). [***P<1E-10, *P<0.01, one-way ANOVA (Bonferroni-corrected), error bars represent SEM, n=6 to 10].

Fig. 2

DA IR (A) and TH IR (B) reveals details of mushroom body (MB) and central complex (CC) DA innervation. TH localizes primarily to cell bodies. DA localizes to cell bodies and to synaptic projections innervating mushroom body and central complex, showing discrete localization to MB subregions. Images are maximum intensity projections of selected brain Z-projection regions.

Fig. 3

Pharmacological or genetic modulation of DA biosynthesis affects spontaneous locomotor activity. (A) DA biosynthesis pathway with inhibitors used in this study. (B) Locomotion of DTH ple² flies (black bars) is inhibited by the DTH inhibitor 3-IY. DA-deficient flies (gray) are hypoactive, similar to DTH ple² flies treated with 3IY. Their locomotor activity is not further reduced by 3-IY, indicating that residual activity is not affected by trace levels of DA synthesis [***P<0.001, one-way ANOVA (Bonferroni-corrected), error bars represent SEM, n=16]. (C) L-DOPA/Carbidopa feeding rescues (C) behavioral hypoactivity and (D) brain DA of DA-deficient flies in a dose dependent manner. Drugs were included in fly food and present throughout the experiment. Locomotor activity averaged over 5 days of feeding [***P<5E-04, one-way ANOVA (Bonferroni-corrected), error bars represent SEM, n=32]. HPLC [***P<1E-03, one-way ANOVA (Bonferroni-corrected), error bars represent SEM, n=5]

Fig. 4

Neuron selective rescue of DA biosynthesis. (A) UAS constructs tested for neuron selective restoration of DTH expression and DA. (B) Undriven UAS-DTHg, UAS-DTH1, and UAS-DTH2 rescue DA biosynthesis in the DA-deficient background without a Gal4 driver. Only UAS-DTH1m does not rescue DA biosynthesis [*P<7E-03, one-way ANOVA (Bonferroni-corrected), error bars represent SEM], n=3]. (C) TH-Gal4 and/or DDC-Gal4 driven UAS-DTH1m rescues normal locomotor activity [***P<1E-6, *P<0.006, one-way ANOVA (Bonferroni-corrected), error bars represent SEM], and (D) brain DA [a vs b P<1E-5, a vs c and b vs c P<1E-3, one-way ANOVA (Bonferroni-corrected), error bars represent SEM, n=32]. (E) TH-and/or DDC-GAL4 driven UAS-DTH1m rescues normal pattern of adult brain DA, in spite of the fact that the DTH1 protein is almost undetectable by TH immunohistochemistry (F).

Fig. 5

DA does not diffuse nor is recycled in distant neurons and its biosynthesis and signaling can be limited to specific MB neurons. Patterns of Split-Gal4 DA neuron subsets mapped with UAS-GFP (green) on a DTH/DA-competent background coimmunostained for TH (red); maximum intensity projections of whole brain. DA-rescue in subsets of DA-neurons using Split-Gal4 system on a DA-deficient background show innervation limited to specific MB neurons; DA immunohistochemistry (grey); maximum intensity projections of MB planes. Even though the Split-Gal4 expression system is highly specific, we found some off-target neurons expressing UAS-GFP that do not overlap with TH IR and do not show DA IR in rescued brains (Fig. 5). We also noticed a surprising coverage of the PPL1 DA neurons by the DDC-DBD driver (Table 1), which was absent from the DDC-Gal4 pattern (Aso et al., 2012).