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. 2016 Apr 25;4:e1971.
doi: 10.7717/peerj.1971. eCollection 2016.

PKC in Motorneurons Underlies Self-Learning, a Form of Motor Learning in Drosophila

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Free PMC article

PKC in Motorneurons Underlies Self-Learning, a Form of Motor Learning in Drosophila

Julien Colomb et al. PeerJ. .
Free PMC article

Abstract

Tethering a fly for stationary flight allows for exquisite control of its sensory input, such as visual or olfactory stimuli or a punishing infrared laser beam. A torque meter measures the turning attempts of the tethered fly around its vertical body axis. By punishing, say, left turning attempts (in a homogeneous environment), one can train a fly to restrict its behaviour to right turning attempts. It was recently discovered that this form of operant conditioning (called operant self-learning), may constitute a form of motor learning in Drosophila. Previous work had shown that Protein Kinase C (PKC) and the transcription factor dFoxP were specifically involved in self-learning, but not in other forms of learning. These molecules are specifically involved in various forms of motor learning in other animals, such as compulsive biting in Aplysia, song-learning in birds, procedural learning in mice or language acquisition in humans. Here we describe our efforts to decipher which PKC gene is involved in self-learning in Drosophila. We also provide evidence that motorneurons may be one part of the neuronal network modified during self-learning experiments. The collected evidence is reminiscent of one of the simplest, clinically relevant forms of motor learning in humans, operant reflex conditioning, which also relies on motorneuron plasticity.

Keywords: Egocentric strategy; FoxP; Habit; Motor adaptation; Motor learning; Operant learning; Procedural memory; Skill learning.

Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Failed attempts to identify the PKC gene involved in operant self-learning.
Performance indices (PI) during a test period following an 8 min training session is reported. LEFT: Flies putatively mutant for PKC genes (PKC-53e, PKC-delta and PCK-InaC) performed well in the self-learning assay. RIGHT: Flies with RNAi constructs targeting PKC53e and PKC InaC were crossed to elav-Gal4;tub-Gal80ts or to CS females. RNAi was induced for two days before the experiment via a 32°heat-shock. While the construct targeting PKC InaC had no effect, the construct for PKC53e prevented self-learning formation even in absence of Gal4 driven expression, such that no firm conclusions can be drawn. Full genotypes of the flies tested are indicated below. CS × 53eRi_V: ;;UAS_PKC53eRNAi_27696/+. elavG4;tG80 × PKC53eRi_V: elavGal4/+;tubGal80ts/+;UAS_PKC53eRNAI_27696/+. elavG4;tG80 x PKCInacRi : elavGal4/+;tubGal80ts/+;UAS_PKCInacRNAI_2895/+. Data is shown as Tukey’s boxplots (median is the line surrounded by boxes representing quartiles) with a superposed violinplot. Asterisks indicate significant differences of the scores against 0, using a non-parametrical Wilcox test.
Figure 2
Figure 2. PKC inhibition (achieved by using an effective heat-shock protocol) in neurons, but not central brain regions, prevents self-learning formation.
Driving Gal4 in all neurons using the elav-Gal4 driver while inactivating its ubiquitously expressed inhibitor Gal80 with a heat-shock induces the expression of the PKC inhibitor. While test flies are still performing well after a mild heat-shock (A, data pooled from different protocols: 33 °for 15 h, 36 °for 2 h, and 37 °for 1 h), a strong heat-shock prevents learning in control flies (B, 37 °for 2 h). After a 4-hour heat-shock at 35 °C, test but not control flies were unable to form self-learning (C). Using this latter protocol, we restricted the expression of Gal4 in central brain regions using different drivers targeting central brain regions (D), which were all ineffective in preventing self-learning. Full genotypes of the flies tested are indicated below. elavG4 xCS : elav-Gal4/+. elavG4 × T_PKCi : elav-Gal4/+;tubGal80ts/+; UAS-PKCi/+. CS × T_PKCi : tubGal80ts/+; UAS-PKCi/+. 7y;c819G4 × T_PKCi : tubGal80ts/+; UAS-PKCi/+__ H24-Gal4. c232G4 × T_PKCi : tubGal80ts/+; UAS-PKCi/7y_Gal4,c819-Gal4 . H24G4 × T_PKCi : tubGal80ts/+; UAS-PKCi/c232-Gal4. Data is shown as Tukey’s boxplots (median is the line surrounded by boxes representing quartiles) with a superposed violinplot. Asterisks indicate significant differences of the scores against 0, using a non-parametrical Wilcox test.
Figure 3
Figure 3. Flies with PKCi expression in motorneurons, are impaired in self-learning.
(A) Using OK371-Gal4 (expression in most glutamatergic neurons) or d42-Gal4 to drive PKCi expression was effective in preventing self-learning, while the control flies seem to learn, although the score of the d42Gal4 × CS control line was not statistically significantly positive. (B) Both D42-Gal4 and c380-GAL4 driving PKCi expression overlappingly in motorneurons, yield comparable inhibition of self-learning. (C) The use of the d42Gal4,chaGal80 double construct as a driver was effective in preventing self-learning, while the controls performed well. Heat-shock protocol was a 4-hour heat-shock at 35 °C. Full genotypes of the flies tested are indicated below. CS × T_PKCi: tubGal80ts/+; UAS-PKCi/+. d42G4 × T_PKCi : tubGal80ts/+; UAS-PKCi/d42-Gal4 . OK371G4 × T_PKCi : tubGal80ts/+; UAS-PKCi/+;OK371/+. d42G4 × CS : d42Gal4/+. OK371 × CS : OK371/+.c380G4 × T_PKCi : c380-Gal4/+. c380G4 × CS : c380Gal4/+; tubGal80ts/+; UAS-PKCi/+. d42G4,chaG80 × CS : d42-Gal4, cha-Gal80/+. d42G4,chaG80 × T_PKCi : tubGal80ts/+; UAS-PKCi/d42-Gal4, cha-Gal80 . CS × T_PKCi : tubGal80ts/+; UAS-PKCi/+. Data is shown as Tukey’s boxplots (median is the line surrounded by boxes representing quartiles) with a superposed violinplot. Asterisks indicate significant differences of the scores against 0, using a non-parametrical Wilcox test.

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