Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling

Abstract

Adrenergic receptors and their ligands are important regulators of synaptic plasticity and metaplasticity, but the exact mechanisms underlying their action are still poorly understood. Octopamine, the invertebrate homolog of mammalian adrenaline or noradrenaline, plays important roles in modulating behavior and synaptic functions. We previously uncovered an octopaminergic positive-feedback mechanism to regulate structural synaptic plasticity during development and in response to starvation. Under this mechanism, activation of Octß2R autoreceptors by octopamine at octopaminergic neurons initiated a cAMP-dependent cascade that stimulated the development of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). However, the regulatory mechanisms that served to brake such positive feedback were not known. Here, we report the presence of an alternative octopamine autoreceptor, Octß1R, with antagonistic functions on synaptic growth. Mutations in octß1r result in the overgrowth of both glutamatergic and octopaminergic NMJs, suggesting that Octß1R is a negative regulator of synaptic expansion. As Octß2R, Octß1R functioned in a cell-autonomous manner at presynaptic motorneurons. However, unlike Octß2R, which activated a cAMP pathway, Octß1R likely inhibited cAMP production through inhibitory Goα. Despite its inhibitory role, Octß1R was required for acute changes in synaptic structure in response to octopamine and for starvation-induced increase in locomotor speed. These results demonstrate the dual action of octopamine on synaptic growth and behavioral plasticity, and highlight the important role of inhibitory influences for normal responses to physiological stimuli.

Figure 1.

Expression of Octß1R-Gal4 transcriptional reporters at the NMJ. A–D, NMJs at muscle 12 of third-instar larvae expressing mCD8-GFP using four different Octß1R-Gal4 strains generated from different intronic regions of the octß1r locus (Pfeiffer et al., 2008). NMJs were double labeled with anti-GFP (green) and anti-HRP (red). The panels represent confocal Z-stack projections. As shown by the white arrowheads, reporter expression is found in the following synaptic endings: 19H07-Gal4: type Ib and type II (A1–3); 21E03-Gal4: type Is and type II (B1–3); 20C11-Gal4: type III (C1–3); 20E11-Gal4: type Ib and type Is (D1–3). E, F, Reporter gene expression in the CNS and imaginal discs of 19H07-Gal4, showing reporter expression in the CNS and proximal band of retinal cells in the optic disc (E); 21E03-Gal4, showing signal in the CNS and edge of imaginal discs (F); 20C11-Gal4: CNS, showing GFP signal in differentiated retinal cells at the optic disc, one to two cells in other imaginal discs, and neurons innervating the pharyngeal muscles (inset) (G); 20E11-Gal4, showing Gal4 expression in the CNS (H). Scale bar: A–D, 20 μm; E–H, 240 μm; G, inset, 70 μm. I, Schematic representation of the octß1r genomic region, showing predicted alternatively spliced isoforms A–C, and the approximate locations (brackets) of different intronic regions used to generate the Gal4 transcriptional reporters (Pfeiffer et al., 2008). The approximate location of the two PBac insertions used to generate the octß1r mutant is shown with blue arrowheads. Orange boxes, Coding region within exons. Gray boxes, Noncoding regions within exons (UTRs). Black lines, Introns. J, RT-PCR from wild-type and octß1r mutant RNA. Virtually no expression of the remaining fragment in octß1r mutants was observed, indicating that the mutant is likely a null mutant. +RT, Reverse transcription reactions with reverse transcriptase added; −RT, reverse transcriptase absent.

Figure 2.

octß1r mutants display an overgrowth of octopaminergic endings at the NMJ. A, B, Confocal Z-stack projections of type II arbors at muscle 12 in larvae expressing mCD8-GFP in octopaminergic neurons of control (A) and octß1r mutant (B), showing a marked increase in the number of natural synaptopods (white arrows). C, Quantification of the number of natural synaptopods per 100 μm of type II arbor in octß2r mutants, octß1r mutants, and animals expressing Octß1R-RNAi in type II motorneurons, showing increased natural synaptopods in octß1r mutants and Octß1R-RNAi animals [N (left to right) = 175, 13, 21, 10, 11, 25, 17 NMJs]. D, E, Third-instar larval NMJs at muscles 12 of wild-type (D) and octß1r mutant (E), showing a marked increase in the number of type II boutons (shown by TBH labeling). NMJs were double labeled with anti-TBH (green) and anti-HRP (red). The panels represent confocal Z-stack projections. F, Quantification of the number of type II boutons at muscle 12 in octß2r mutants, octß1r mutants, and animals expressing Octß1R-RNAi in type II motorneurons, showing increased type II boutons in octß1r mutants and Octß1R-RNAi animals [N (left to right) = 22, 17, 15, 11, 16, 20, 11 NMJs]. Animals used in RNAi experiments were reared at 29°C to increase knockdown efficiency. Scale bar: A, B, 8 μm; D, E, 20 μm. Error bars indicate SEM. ***p < = 0.0001; **p < = 0.001; *p < 0.05.

Figure 3.

Disruption of Goα phenocopies synaptic overgrowth of octß1r. A, B, Confocal Z-stack projections of type II arbors at muscle 12 in larvae expressing mCD8-GFP in octopaminergic neurons before and after bath application of octopamine. A, Bath application of octopamine increases the number of synaptopods on type II arbors (white arrowheads). B, Bath application of octopamine in the presence of PTX results in significantly larger increase of synaptopods than octopamine alone (white arrowheads). C, Quantification of the net increase of synaptopods in A and B per 100 μm of type II arbor, showing that octopamine induces a larger increase in synaptopods in larval preparations with 2 h PTX incubation than preparations without PTX treatment [N (left to right) = 18, 13, 16 NMJs]. D–F, Confocal Z-stack projections of type II arbors at muscle 12 in larvae expressing mCD8-GFP in octopaminergic neurons of control (D), animals expressing PTX in type II (E), and animals expressing Goα-RNAi1 in type II (F), showing a marked increase in the number of natural synaptopods (white arrowheads). G, Quantification of natural synaptopods per 100 μm of type II arbor in animals expressing PTX, Goα-RNAi, or Giα-RNAi in type II, showing that that the disruption of Goα function but not Giα results in increased number of natural synaptopods [N (left to right) = 175, 12, 25, 19, 10, 11, 10 NMJs]. Animals used in RNAi experiments were reared at 29°C to increase knockdown efficiency. H, Quantification of type II boutons at muscle 12 in animals expressing PTX, Goα-RNAi, or Giα-RNAi in type II motorneurons alone or type I+II motorneurons, showing that that the disruption of Goα function but not Giα results in increased number of type II boutons. [N (left to right) = 22, 16, 12, 20, 24, 16, 16, 10, 16 NMJs]. I, Quantification of natural synaptopods per 100 μm of type II arbor in octß1r mutants, octß1r/+ heterozygotes, goα⁰⁰⁷/+ heterozygotes, and goα⁰⁰⁷/+; octß1r/+ transheterozygotes, showing that goα⁰⁰⁷/+; octß1r/+ transheterozygotes have increased number of natural synaptopods, indicating a genetic interaction [N (left to right) = 175, 21, 11, 10, 11 NMJs]. J, Quantification of type II boutons at muscle 12 in animals of the same genotypes in I, showing that octß1r/+; goα⁰⁰⁷/+ transheterozygotes have increased number of type II boutons, indicating a genetic interaction [N (left to right) = 22, 15, 16, 12, 16 NMJs]. Scale bar, 12 μm. Error bars indicate SEM. ***p < = 0.0001; **p < = 0.001; *p < 0.05.

Figure 4.

Suppression of the overgrowth phenotype of octß1r by decreasing cAMP. A, B, Quantification of natural synaptopods per 100 μm of type II arbor (A) and type II boutons at muscle 12 (B) in octß1r mutants, rut mutants, animals overexpressing Dnc in type II and combinations of these genetic manipulations, showing that rut and overexpressing Dnc fully suppress the synaptic overgrowth phenotype of octß1r, suggesting that Rut and Dnc are downstream components of the Octß1R signaling pathway [N (left to right) = 175, 21, 24, 13, 14, 13 NMJs in A; N (left to right) = 22, 15, 18, 18, 12, 12 NMJs in B]. C, D, Quantification of natural synaptopods per 100 μm of type II arbor (C) and type II boutons at muscle 12 (D) in octß2r mutants, octß1r mutants, and octß1r, octß2r double mutants, showing that Octß1R and Octß2R function partially independently [N (left to right) = 175, 13, 21, 12 NMJs in C; N (left to right) = 22, 17, 15, 16 NMJs in D]. Error bars indicate SEM. ***p < = 0.0001; **p < = 0.001; *p < 0.05.

Figure 5.

octß1r mutation or disruption of Goα function likely results in saturating levels of cAMP. A, Quantification of the net increase of synaptopods per 100 μm of type II arbor in response to exogenous octopamine application in octß1r mutants, dnc mutants, and animals expressing PTX, Goα-RNAi, or Giα-RNAi in type II, showing that bath application of octopamine increases synaptopods in control and Giα-RNAi animals, but not in animals with disrupted Octß1R or Goα pathway, which have increased natural synaptopods [N (left to right) = 14, 13, 12, 10, 10, 11, 12, 11, 19, 10 NMJs]. Animals used in RNAi experiments were reared at 29°C to increase knockdown efficiency. B, Quantification of the net increase of synaptopods per 100 μm of type II arbor in response to exogenous octopamine or forskolin application in octß1r mutants and octß2r mutants, showing that bath application of octopamine fails to increase synaptopods in both octß1r and octß2r, whereas bath application of forskolin increases synaptopods in octß2r but not in octß1r. This indicates that the lack of response to octopamine in octß1r is likely due to saturating levels of cAMP [N (left to right) = 14, 13, 11, 10, 12, 13, 11, 13, 10 NMJs]. Error bars indicate SEM. ***p < = 0.0001; **p < = 0.001; *p < 0.05.

Figure 6.

Octß1R is required for starvation-induced locomotor increase. A, Quantification of the percentage increase of larval crawling speed in response to 2 h starvation in octß1r mutants, octß2r mutants, octß1r, octß2r double mutants, and animals expressing PTX, Goα-RNAi, or Giα-RNAi in octopaminergic neurons, showing that the disruption of the Octß1R pathway results in defects in the increase of locomotor activity in response to starvation [N (left to right) = 26, 16, 16, 15, 16, 16, 16, 16, 17, 13, 19, 16, 16, 17 animals]. B, Quantification of the basal crawling speed of the same genotypes in A, showing that the defect starvation response of PTX-type II and Goα-RNAi1-type II in A is not due to a defect in basal locomotor speed [N (left to right) = 31, 16, 18, 29, 16, 18, 20, 14, 17, 15, 20, 17, 16, 20 animals]. Animals used in RNAi experiments were reared at 29°C to increase knockdown efficiency. Error bars indicate SEM. ***p < = 0.0001; **p < = 0.001; *p < 0.05.

Figure 7.

Octß1R negatively regulates type I synaptic growth in a cell-autonomous manner. A–D, Third-instar larval NMJs at muscles 6/7 of wild type (A), octß1r mutant (B), Goα-RNAi1/+ control (reared at 29°C) (C), and Goα-RNAi1-type I+II (reared at 29°C) (D), showing a marked increase in the number of type I boutons. NMJs were labeled with anti-HRP. The panels represent confocal Z-stack projections. E, Quantification of type I boutons at muscle 6/7 in octß1r mutants, octß1r/+ heterozygotes, goα⁰⁰⁷/+ heterozygotes, and goα⁰⁰⁷/+; octß1r/+ transheterozygotes, showing that octß1r mutants and goα⁰⁰⁷/+; octß1r/+ transheterozygotes have increased number of type I boutons [N (left to right) = 18, 15, 16, 16, 12, 18 NMJs]. F–H, Quantification of type I boutons at muscle 6/7 in animals expressing PTX, Octß1R-RNAi, Goα-RNAi, or Giα-RNAi in type I and type II (F), type I (G), and type II (H), showing that the disruption Octß1R or Goα in type I and type II simultaneously or type I alone increase type I boutons [N (left to right) = 18, 14, 14, 16, 15, 12, 14, 12, 12, 16, 12, 16, 14, 16 NMJs in F; N (left to right) = 18, 12, 16, 15, 13, 18, 12, 10, 12, 13 NMJs in G; N (left to right) = 18, 12, 14, 15, 11, 12, 16, 16, 10, 16 NMJs]. Animals used in RNAi experiments were reared at 29°C to increase knockdown efficiency. Scale bar: A–D, 10 μm. Error bars indicate SEM. ***p < = 0.0001; **p < = 0.001; *p < 0.05.