Wnt signaling regulates acetylcholine receptor translocation and synaptic plasticity in the adult nervous system

Abstract

The adult nervous system is plastic, allowing us to learn, remember, and forget. Experience-dependent plasticity occurs at synapses--the specialized points of contact between neurons where signaling occurs. However, the mechanisms that regulate the strength of synaptic signaling are not well understood. Here, we define a Wnt-signaling pathway that modifies synaptic strength in the adult nervous system by regulating the translocation of one class of acetylcholine receptors (AChRs) to synapses. In Caenorhabditis elegans, we show that mutations in CWN-2 (Wnt ligand), LIN-17 (Frizzled), CAM-1 (Ror receptor tyrosine kinase), or the downstream effector DSH-1 (disheveled) result in similar subsynaptic accumulations of ACR-16/α7 AChRs, a consequent reduction in synaptic current, and predictable behavioral defects. Photoconversion experiments revealed defective translocation of ACR-16/α7 to synapses in Wnt-signaling mutants. Using optogenetic nerve stimulation, we demonstrate activity-dependent synaptic plasticity and its dependence on ACR-16/α7 translocation mediated by Wnt signaling via LIN-17/CAM-1 heteromeric receptors.

Figure 1

Wnt and CAM-1/Ror signaling proteins contribute to synaptic signaling. (A) Schematic of the NMJ showing the three classes of ligand-gated receptors and deletion variants of the CAM-1/Ror RTK. The orange oblong represents the serine/threonine rich domain. (B) Currents recorded in muscle cells in response to pressure application of 100 uM ACh in wild-type worms, cam-1(ak37) mutants, and transgenic mutants that expressed either CAM-1(ΔECD)∷GFP or CAM-1(ΔTKD)∷GFP in muscles or neurons, respectively. (C) Normalized peak ACh-gated current, n≥4 for all genotypes. (D) Thrashing behavior of Wnt-signaling mutants in the unc-29(x29) mutant background. For all genotypes, n≥10. (E) ACh-gated currents in the muscle cells of wild-type (WT) and mutant worms. (F) Peak ACh-gated current normalized to wild-type values. For all genotypes, n≥5. *, p<0.05; **, p<0.01. Error bars indicate SEM. See also Figure S1, S2 and S3.

Figure 2

ACR-16/α7-dependent behavior requires neuronal CWN-2 and Fzd/Ror/Dvl in muscles. (A) Thrashing behavior normalized to unc-29 single mutants. For all genotypes, n≥7. (B) Thrashing behavior normalized to wild-type (WT) worms (left) or unc-29 mutants (right) in transgenic worms in which cwn-2 was knocked down [cwn-2(RNAi)] in either neurons or muscles. For WT background, n≥5; and unc-29 mutant background, n≥10. Gray bars indicate unc-29 mutant background. (C) ACh-gated currents in unc-29 mutants with or without cwn-2 knock down. (D) Peak ACh-gated current normalized to unc-29 mutants. For all genotypes, n≥4. (E and F) Thrashing behavior showing muscle specific rescue of lin-17 (E) and dsh-1 (F) mutants. For all genotypes, n≥9. (G and I) Currents evoked in muscle cells by pressure application of 100 μM ACh. (H and J) Average peak current amplitude of ACh-gated currents. For all genotypes, n≥4. *, p<0.05; **, p<0.01. Error bars indicate SEM. See also Figure S3.

Figure 3

Muscle LIN-17/CAM-1 heteromeric receptors regulate ACR-16 localization. (A) Single plane confocal images showing expression of CAM-1∷GFP and LIN-17∷mCherry in the tips of muscle arms. Box indicates the region shown in the images on the right. Scale bars, 2 μm. (B–E) Images of the tips of muscle arms in transgenic worms that expressed split-YFP fused to LIN-17 and either full-length CAM-1 (B), one of two CAM-1 deletion variants (C and D), or CAM-1(ΔECD)∷GFP (E). Scale bars, 1 μm. (F) Image of the ventral nerve chord in a transgenic worm that expressed ACR-16∷GFP in muscle cells and ChR2∷mCherry in motor neurons. Scale bar, 5 μm. (G) ACR-16∷GFP and SYD-R∷RFP expression in the muscle arm of a transgenic wild-type (WT) worm or cwn-2 mutant. Scale bar, 2 μm. (H) Images of muscle arms in transgenic wild-type and mutant worms that expressed ACR-16∷GFP. The red, dashed lines outline the muscle arms. Scale bar, 1 μm. (I) Intensity of ACR-16∷GFP fluorescence in muscle arms normalized to wild type. For all genotypes, n≥8. (J) Images of ACR-16∷GFP expression in muscle arms. Scale bar, 1 μm. (K) Intensity of ACR-16∷GFP fluorescence in muscle arms relative to wild-type. For all genotypes, n≥11. (L) ACR-16∷GFP expression in muscle arms of wild-type worms with or without cwn-2 knock down. Scale bar, 2 μm. (M) ACR-16∷GFP fluorescence intensity in muscle arms relative to wild type, n≥11. *, p<0.05; **, p<0.01. Error bars indicate SEM. See also Figure S4.

Figure 4

ACR-16∷GFP is mislocalized and its mobility reduced in Wnt-signaling mutants. (A) Images of the tips of muscle arms in transgenic wild-type (WT) and mutant worms injected with fluorescently labeled α-BgTx. These worms also expressed soluble mCherry in muscle cells to identify muscle arms. Scale bar, 1 μm. (B) Quantification of α-BgTx fluorescence. *, p<0.05; **, p<0.01. For all genotypes, n≥12. (C) Images of muscle arms in a wild-type transgenic worm that expressed ACR-16∷EosFP both before and after photoconversion from green to red. The white, dashed lines outline individual muscle arms. The circles show the converted EosFP puncta. Scale bar, 5 μm. (D) Loss of fluorescence observed with photoconversion-chase strategy. Shown are examples of the loss of ACR-16∷EosFP fluorescence in muscle arms following photoconversion from green to red. Scale bar, 1 μm. (E) Quantification of EosFP after photoconversion in wild-type worms and lin-17 mutants (n≥11). lin-17 mutants are significantly different from WT, p<0.001. Error bars represent SEM. See also Figure S5.

Figure 5

LIN-17 expression in adult mutants rescues ACR-16-mediated behavior and receptor localization. (A) Thrashing behavior in unc-29 single mutants, lin-17; unc-29 double mutants and transgenic double mutants that expressed Phsp∷lin-17. n≥10 for each strain and condition. Shown is behavior 10 and 24 hrs following heat shock. (B) Images of ACR-16∷GFP expression in the muscle arms of wild-type (WT) worms, lin-17 mutants and transgenic mutants following heat shock (HS). Scale bar, 1 μm. (C) Intensity of ACR-16∷GFP fluorescence relative to wild-type worms. n≥9 for each strain and condition. *, significantly different from lin-17, heat shock (−), p<0.05. Error bars indicate SEM. See also Figure S5.

Figure 6

Chronic stimulation of the NMJ modifies ACR-16 localization and ACh-gated current. (A) ACR-16∷GFP in muscle arms of transgenic wild type (WT) and cwn-2 mutant worms that expressed ChR2 with or without light stimulation and in the presence or absence of retinal. Scale bar, 1 μm. (B) Quantification of GFP fluorescence in the worms shown in (A). Normalized to WT, retinal (−) (black bars) or cwn-2, retinal (−) (gray bars). For all strains and conditions, n≥12. (C) ACh-gated currents in the muscle cells of light stimulated, transgenic worms that expressed ChR2 in motor neurons. (D) Peak ACh-gated current amplitude normalized to WT, retinal (−) (black bars) or cwn-2, retinal (−) (gray bars). For each genotype and condition, n≥6. *, p<0.05. (E) ACR-16∷GFP expression in muscle arms of cwn-2 mutants with or without a wild-type cwn-2 transgene expressed in muscles or neurons. Scale bar, 2 μm. (F) Quantification of ACR-16∷GFP fluorescence normalized to transgene (−) controls. For all genotypes and conditions, n≥18. (G) ACR-16∷GFP fluorescence in muscle arms of light-stimulated, transgenic wild-type worms with cwn-2 knock down in either muscles or neurons. Scale bar, 1 μm. (H) Quantification of ACR-16∷GFP fluorescence in muscle arms. For all genotypes and conditions, n≥15. *, p<0.05. Error bars indicate SEM. See also Figure S6.

Figure 7

Translocation of ACR-16/α7 is independent of protein synthesis and rapidly induced by recombinant CWN-2. (A) ACR-16∷GFP expression in muscle arms of transgenic wild-type worms that expressed ChR2 in motor neurons. Scale bar, 2 μm. (B) Quantification of ACR-16∷GFP fluorescence intensity normalized to retinal (−), light (−), CHX (−) control. For all conditions, n≥9. (C) CWN-2∷GFP expression in motor neuron cell bodies in transgenic wild-type (WT) and mig-14 mutant worms. Scale bars, 4 μm. (D) Quantification of CWN-2∷GFP fluorescence intensity normalized to either WT, retinal (−) (black bars) or mig-14, retinal (−) (gray bars). For all genotypes and conditions, n≥16. (E) ACh-gated currents in cwn-2 mutants with or without the application of recombinant CWN-2. (F) Quantification of peak ACh-gated current normalized to cwn-2 control. For all conditions, n≥4. *,p<0.05, **p<0.01. Error bars indicate SEM. See also Figure S7.