Neurexin and neuroligin mediate retrograde synaptic inhibition in C. elegans

Abstract

The synaptic adhesion molecules neurexin and neuroligin alter the development and function of synapses and are linked to autism in humans. Here, we found that Caenorhabditis elegans neurexin (NRX-1) and neuroligin (NLG-1) mediated a retrograde synaptic signal that inhibited neurotransmitter release at neuromuscular junctions. Retrograde signaling was induced in mutants lacking a muscle microRNA (miR-1) and was blocked in mutants lacking NLG-1 or NRX-1. Release was rapid and abbreviated when the retrograde signal was on, whereas release was slow and prolonged when retrograde signaling was blocked. The retrograde signal adjusted release kinetics by inhibiting exocytosis of synaptic vesicles (SVs) that are distal to the site of calcium entry. Inhibition of release was mediated by increased presynaptic levels of tomosyn, an inhibitor of SV fusion.

Figure 1. The retrograde signal was induced in mir-1 mutants and inactivated in nlg-1 mutants

Endogenous (A-B) and evoked EPSCs (C-E) were recorded from adult body wall muscles. Representative traces of endogenous EPSCs (A), averaged evoked responses (C-D), and summary data (B,E) are shown. The effects of miR-1 on EPSC rate and quantal content were eliminated in mir-1; nlg-1 double mutants. (B,E) Retrograde inhibition was re-instated in mir-1; nlg-1 double mutants by nlg-1 transgenes expressed in cholinergic motor neurons (ACh rescue) but not by those expressed in body muscles (muscle rescue). (F-H) Expression of GFP-tagged NRX-1 in body muscles was analyzed. NRX-1 puncta fluorescence in the dorsal cord was significantly increased in mir-1 mutants. This effect was eliminated in mir-1 mef-2 double mutants. Representative images (F), mean (G), and cumulative probability distributions (H) of puncta intensity are shown. Values that differ significantly are indicated (***, p <0.001; **, p <0.01; ns, not significant). The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Scale bar indicates 5 µm.

Figure 2. Activating and inactivating the retrograde signal had opposite effects on the kinetics of evoked responses

Evoked EPSCs were compared for the indicated genotypes. For each genotype, averaged evoked responses (A,E), decay kinetics (B,F), cumulative transfer of quantal charge (C,G), and charge transfer kinetics (D,H) are shown. Rescue refers to transgenic animals expressing NLG-1 in cholinergic neurons of nlg-1 single mutants (B,D) and in nlg-1; mir-1 double mutants (E-H). Values that differ significantly from wild type controls are indicated (***, p <0.001; **, p <0.01; *, p <0.05). The number of animals analyzed is indicated for each genotype. Error bars indicate SEM.

Figure 3. The effects of the retrograde signal on evoked responses are eliminated by EGTA

(A) Averaged evoked responses are compared for wild type, mir-1, and nlg-1 mutants with and without EGTA-AM treatment. (B) Summary data are shown comparing evoked EPSC amplitude, decay kinetics, and quantal content. The numbers of animals analyzed were: wild type (14 control, 12 + EGTA); mir-1 (25 control, 11 + EGTA); and nlg-1 (13 control, 8 + EGTA). Error bars indicate SEM. The amplitude, quantal content, and decay kinetics of evoked EPSCs were not significantly different in EGTA treated wild type, mir-1, and nlg-1 animals: amplitude (WT vs. mir-1, p = 0.976; WT vs. nlg-1, p = 0.972); quantal content (WT vs. mir-1, p = 0.999; WT vs. nlg-1, p = 0.993); and decay tau (WT vs. mir-1, p = 0.321; WT vs. nlg-1, p = 0.118).

Figure 4. TOM-1/Tomosyn is required for the retrograde signal

(A-B) GFP-tagged TOM-1 was expressed in DA and DB motor neurons and puncta fluorescence was analyzed in the dorsal nerve cord. Representative images (A), mean and cumulative probability distributions of puncta intensity (B) are shown. TOM-1 puncta intensity was significantly increased in mir-1 mutants. This effect was eliminated in mir-1; nlg-1 double mutants. Scale bar indicates 5 µm. (C-F) Endogenous (C-D) and evoked EPSCs (E-F) were recorded from adult body wall muscles. Representative traces of endogenous EPSCs (C), averaged evoked responses (E), and summary data (D,F) are shown. The effects of miR-1 on EPSC rate, quantal content, and evoked decay kinetics were eliminated in tom-1 mir-1 double mutants. Values that differ significantly from wild type controls are indicated (***, p <0.001; **, p <0.01; ns, not significant). The number of animals analyzed is indicated for each genotype. Error bars indicate SEM.