Neurexin controls plasticity of a mature, sexually dimorphic neuron

Abstract

During development and adulthood, brain plasticity is evident at several levels, from synaptic structure and function to the outgrowth of dendrites and axons. Whether and how sex impinges on neuronal plasticity is poorly understood. Here we show that the sex-shared GABA (γ-aminobutyric acid)-releasing DVB neuron in Caenorhabditis elegans displays experience-dependent and sexually dimorphic morphological plasticity, characterized by the stochastic and dynamic addition of multiple neurites in adult males. These added neurites enable synaptic rewiring of the DVB neuron and instruct a functional switch of the neuron that directly modifies a step of male mating behaviour. Both DVB neuron function and male mating behaviour can be altered by experience and by manipulation of postsynaptic activity. The outgrowth of DVB neurites is promoted by presynaptic neurexin and antagonized by postsynaptic neuroligin, revealing a non-conventional activity and mode of interaction of these conserved, human-disease-relevant factors.

Extended Data Fig. 1. Progressive neurite outgrowth in DVB in adulthood

(a) DVB neuron visualized with lim-6^int4::gfp at days 1, 3, and 5 in adult males and quantified by (b) total neurite length and (c) number of neurite junctions (dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD). (d) DVB neurite outgrowth visualized with flp-10::gfp in males at days 1, 3, and 5 of adulthood (n>10). (e) Tracing reconstruction of male DVB from EM sections compiled by wormwiring.org showing DVB neurites. (f) Inset of DVB neurites showing pre-synaptic specializations identified in EM sections shown in pink. EM section showing DVB pseudo-colored yellow with pre-synaptic specialization indicated with red ‘x’ with (g) SPCR (Image Right1200, Section 14871) and (h) spicule sheath (Image N2YDRG1175, Section 14816), shown in white in inset panel.

Extended Data Fig. 2. DVB neurite outgrowth in adult male C. elegans is stochastic and other neurons in the male tail do not show progressive neurite outgrowth in adulthood

DVB neurites at day 5 visualized with (a) lim-6^int4::wCherry or (b) lim-6^int4::gfp (n>10 for each). DVB posterior neurites were traced through confocal stacks using Simple Neurite Tracer plugin. (c) DVA neuron visualized with ser-2(prom-2)::gfp (n=5)(red dashed line indicates axon of relevant neuron), (d) DVC neuron visualized with inx-18p::gfp (n=5), (e) CP6 neuron visualized with flp-13::gfp (cell soma not shown) (n=5), (f) ray neurons visualized with dat-1::gfp (ventral view)(n=5). PVT neuron visualized with srz-102p::gfp (n=5)(g) and srg-4p::gfp (n=5)(h) at day 1 and day 5. Axons of indicated neurons highlighted by red dashed line.

Extended Data Fig. 3. DVB inhibits expulsion-associated spicule protraction at day 3. Laser ablation of DVB and Channelrhodopsin expression in DVB and spicule protraction circuit

(a) Confocal images of male worm with lim-6^int4::wCherry and lim-6^int4::HisCl1::gfp at day 3 and (b) quantification of the percentage of expulsion steps with spicule protraction for day 1 control, day 3 control, day 3 control + histamine, and day 3 lim-6^int4::HisCl1::gfp + histamine males. (c) Quantification of time between consecutive expulsion steps for day 1 control, day 3 control, day 3 control + histamine, and day 3 lim-6^int4::HisCl1::gfp + histamine males (+ histamine = 10mM histamine plates)(dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD). (d) Confocal images of male worms with or without laser ablation of DVB at day 1–2, visualized with lim-6^int4::gfp. (e) Confocal images of DVB (lim-6^int4::wCherry) expressing channelrhodopsin at day 1 and 5, Ex[lim-6^int4::ChR2::yfp]. (f) Confocal images of DVB (lim-6^int4::wCherry) and spicule circuit expressing channelrhodopsin at day 1 and 5, Ex[gar-3b::ChR2::yfp]. (n>10 for d, e, and f).

Extended Data Fig. 4. DVB neurite outgrowth in unc-49, pkd-2 and unc-97 mutant males. flp-13p::gfp labels CP6 and spicule retractor muscles

(a) Confocal images and quantification of (b) total neurite outgrowth and (c) number of neurite junctions in control and unc-49(e407) males at days 3 and 5. (d) Time to spicule protraction on aldicarb at day 5 for control and unc-49(e407) males. (e) Confocal images and quantification of (f) total neurite outgrowth and (g) number of neurite junctions in control, pkd-2(pt8), and unc-97(su110) males at day 3. (h) Confocal images of male worms with lim-6^int4::wCherry, flp-10p::gfp, and DIC at day 1 in ventral and lateral views. Inset showing DVB and CP6 axons, with schematic of axons demonstrating lack of contact (red is DVB axon, green is CP6 axon, blue dashed lines are spicule retractor muscles). Asterisks in flp-13::gfp panel mark spicule retractor muscles. (dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD).

Extended Data Fig. 5. Day 1 male mating defects involving spicule coordination, spicule circuit activation in unc-25, unc-97, and nrx-1 mutant males, and spicule neuron or muscle activation induces DVB neurite outgrowth

(a) Quantification of percentage of average mating success (sperm transfer) for day 1 and 3 males during 5-minute timed mating assays with 15 unc-31(e928) hermaphrodites (number of worms indicated with n=, data points represent average percentage for each replicate of multiple males). (b) Quantification of attempts at spicule prodding during 5-minute timed mating assay for day 1 and 3 males. (c) Ratio of protraction/prodding attempts during 5-minute timed mating assay for males at day 1 and 3. (d) Confocal images of lim-6^int4::wCherry, (e) total neurite length, and (f) number or neurite junctions of unc-25(e156), unc-25(e156); Ex[gar-3b::ChR2::yfp], unc-97(su110), unc-97(su110); Ex[gar-3b::ChR2::yfp], nrx-1(wy778), and nrx-1(wy778); Ex[gar-3b::ChR2::yfp] males following activation at day 1 (488 nm light for 3×15s every 45 mins for 4.5h). (g) Confocal images and quantification of (h) total neurite outgrowth and (i) number of neurite junctions in control, Ex[unc-103E::ChR2::yfp], and Ex[unc-103F⁴::ChR2::yfp] after activation at day 1 with retinal (488 nm light for 3×15s every 45 mins for 4.5h). Quantification of (j) total neurite outgrowth and (k) number of neurite junctions at day 1 in control, Ex[lim-6^int4::ChR2::yfp](DVB), Ex[unc-103E::ChR2::yfp](neuron-specific), and Ex[unc-103F⁴::ChR2::yfp](muscle-specific) males after activation but in absence of retinal. (l) Time to protraction of control and Ex[lim-6^int4::ChR2::yfp] males after day 1 activation in absence of retinal. (dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD).

Extended Data Fig. 6. Exposure to exogenous GABA or silencing spicule protraction circuit activity overnight reduces DVB neurites on day 5

(a) Confocal images of lim-6^int4::wCherry, (b) total neurite length, and (c) number or neurite junctions of males exposed overnight to 30mM GABA at days 3 and 5. (d) Confocal images of lim-6^int4::wCherry, (e) total neurite length, and (f) number or neurite junctions at day 5 of control +/− overnight 10mM histamine, and gar-3b::HisCl1::gfp +/− overnight 10mM histamine. (dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD).

Extended Data Fig. 7. NLG-1 expression in multiple male sex muscles rescues nlg-1 mutant DVB neurite phenotype. Silencing spicule circuit or exposure to exogenous GABA does not reduce DVB neurites in nlg-1 mutant males

(a) Confocal images of DVB (lim-6^int4::wCherry), and quantification of (b) total neurite outgrowth and (c) number of neurite junctions in control, nlg-1(ok259), and nlg-1(ok259); nlg-1p::nlg-1::gfp, and nlg-1p::nlg-1::gfp day 3 males. Quantification of (d) total neurite outgrowth and (e) number of neurite junctions in control or nlg-1(ok259) mutant males with or without NLG-1 tissue-specific expression. Expression patterns for rescue promoters - lim-6^int4 – DVB, gar-3b – SPC, spicule protractor muscles; unc-103F – SPC, PCA, PCB, other neurons; unc-103E – male sex muscles; flp-13 – spicule retractor muscles, CP6. (f) Confocal images of lim-6^int4::wCherry and Ex[gar-3b::HisCl1::gfp], (g) total neurite length, and (h) number of neurite junctions of nlg-1(ok259) +/− 10mM histamine overnight, nlg-1(ok259); gar-3b::HisCl1::gfp +/− 10mM histamine overnight, and nlg-1(ok259) + 30mM GABA overnight in day 5 males. (dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD).

Extended Data Fig. 8. NLG-1 expression decreases from day 1 to day 3

(a) Confocal images of nlg-1p::nlg-1::gfp in males at day 1, 3, and 5. Example regions of interest for measurements taken from single planes – blue – dorsal spicule muscles, red – pre-anal ganglion, magenta – DVB. Quantification of fluorescence intensity of nlg-1p::nlg-1::gfp in males at day 1, 3, and 5 reported as a ratio of mean fluorescence in (b) dorsal spicule muscles or (c) pre-anal ganglion normalized to background of DVB, which has little to undetectable expression. Dorsal spicule muscles refer to the gubernacular retractor, gubernacular erector, anterior oblique, anal depressor. (d) Confocal images of nlg-1p::nlg-1::gfp in control, nlg-1(ok259), nlg-1(ok259) with overnight GABA exposure, nlg-1(ok259) with 3 day GABA exposure, and nrx-1(wy778) males at day 3. Quantification of fluorescence intensity of nlg-1p::nlg-1::gfp in day 1 and 3 control, nlg-1(ok259), and nrx-1(wy778) males and day 3 nlg-1(ok259) with overnight GABA exposure and nlg-1(ok259) with 3 day GABA exposure, as a ratio of mean fluorescence in (e) dorsal spicule muscles or (f) pre-anal ganglion normalized to background of DVB. (dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD).

Extended Data Fig. 9. NRX-1 long isoform functions in DVB to control DVB neurite outgrowth and NRX-1 expression in DVB controls neurite outgrowth of nlg-1 mutants

(a) Genetic loci of nrx-1 showing long and short isoforms, PDZ domain, and location of point mutation gk246237, and deletions ok1649 and wy778. Quantification of (b) total neurite length and (c) number of neurite junctions in controls and long-isoform specific mutants nrx-1(ok1649) and nrx-1(gk246237) at day 3. Quantification of (d) total neurite outgrowth and (e) number of neurite junctions at day 3 in control, Ex[lim-6^int4::birA::nrx-1^LONG], nrx-1(wy778), nrx-1(wy778); Ex[lim-6^int4::birA::nrx-1^LONG], nrx-1(wy778); Ex[lim-6^int4::birA::nrx-1^SHORT], nrx-1(wy778); Ex[lim-6^int4::birA::nrx-1^noPDZ]. (f) Time to spicule protraction at day 3 in control, nrx-1(wy778), nrx-1(wy778); Ex[lim-6^int4::birA::nrx-1^LONG], and Ex[lim-6^int4::birA::nrx-1^LONG]. (g) Confocal images of lim-6^int4::wCherry expression, and quantification of (h) total neurite length and (i) number of neurite junctions of day 3 nlg-1(ok259), nlg-1(ok259); Ex[lim-6^int4::birA::nrx-1^LONG], nrx-1(wy778), nrx-1(wy778); nlg-1(ok259), nrx-1(wy778); nlg-1(ok259); Ex[lim-6^int4::birA::nrx-1^LONG] males. (j) Confocal images of lim-6^int4::wCherry and Ex[lim-6^int4::gfp::nrx-1^LONG] in control, nrx-1(wy778), and nlg-1(ok259) males at day 1 and 3. (dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD).

Extended Data Fig. 10. DVB in hermaphrodites does not show neurite branching upon gar-3b::ChR2::yfp activation or NRX-1/NLG-1 manipulations

(a) Confocal images of lim-6^int4::wCherry and Ex[gar-3b::ChR2::yfp] expression in day 1 hermaphrodites showing DVB axon projection after activation on retinal (488 nm light for 3×15s every 45 mins for 4.5h). (b) Confocal images of lim-6^int4::wCherry or lim-6^int4::gfp in control, nrx-1(wy778), nlg-1(ok259), and Ex[lim-6^int4::gfp::nrx-1^LONG] hermaphrodites at day 3. (c) Quantification of the percentage of hermaphrodites with DVB axon abnormalities, neurites (in almost all cases, a single neurite off axon just posterior of the pre-anal ganglion) in day 1 control and Ex[gar-3b::ChR2::yfp] with activation, day 3 control, nrx-1(wy778), nlg-1(ok259), and Ex[lim-6^int4::gfp::nrx-1^LONG]. (number of worms indicated with n=, data points represent average percentage for each replicate of multiple hermaphrodites). (dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD).

Fig. 1. Progressive neurite outgrowth of the GABAergic DVB neuron in adult males

(a) DVB neuron schematic. (b) DVB visualized with lim-6^int4::wCherry during adulthood in males and hermaphrodites (asterisk=PVT neuron, arrowheads=DVB neurites, scale bar=10 μm(true for all subsequence figures), n same as (c)). Presynaptic boutons visualized with presynaptic marker lim-6^int4::gfp::rab-3. DVB neurite outgrowth quantified by (c) total neurite length and (d) number of neurite junctions (dot=one animal, magenta bar=median, and boxes=quartiles. Comparison using one-way ANOVA and post-hoc Tukey HSD, p-values and n shown). (e) Schematic of DVB and post-synaptic spicule-associated neurons and muscles in male tail. (f) Example demonstrating males with normal or protracted spicules (red line indicates spicule(n>10)). (g) Connectivity of DVB at adult stage inferred from electron micrographs^,. Chemical synapses depicted as arrows, black arrows and neurons are sex-shared, red are hermaphrodite-specific, blue are male-specific. Behavioral output indicated for each sex.

Fig. 2. DVB neuron undergoes a functional switch in adulthood resulting in dynamic behavioral output

(a) Percent of mock or DVB-ablated males with chronically protracted spicules 20 hours after ablation at day indicated (mean +/− S.E.M., two-tailed Student’s t-test). (b) Percent of worms responding to 488nm light with movement of spicules for control, Ex[lim-6^int4::ChR2::yfp], and Ex[gar-3b::ChR2::yfp](mean +/− S.E.M., one-way ANOVA and post-hoc Tukey HSD). (c) Percent of worms with or without Ex[lim-6^int4::ChR2::yfp] responding to blue light with spicule movement at day 1 in control, unc-49(e407), and unc-25(e156) males (mean +/− S.E.M., one-way ANOVA and post-hoc Tukey HSD). (d) Diagram of GABA and acetylcholine input onto spicule muscles showing site of aldicarb action. (e) Males on 5mM aldicarb media timed for spicule protraction >5s (dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD (true for f, h, and k)). (f) Control, mock-, DVB-ablated, or lim-6(nr2073) mutant males timed for aldicarb-induced spicule protraction 12 hours after ablation. (g) Diagram of gar-3b GRASP. (h) Quantification and (i) confocal images of gar-3b GRASP puncta. (j) Diagram of flp-13p GRASP. (k) Quantification and (l) confocal images of flp-13p GRASP puncta.

Fig. 3. DVB neurite outgrowth is experience-dependent, can be driven by circuit activity, and impacts behavior

(a) Confocal images of lim-6^int4::wCherry, (b) total neurite length, and (c) number or neurite junctions of males housed by themselves (single), with hermaphrodites (mated), unc-97 mutant males housed by themselves (single), or with hermaphrodites (‘mated’) after 48h. Controls and males expressing channelrhodopsin (Ex[gar-3b::ChR2::yfp]) activated at day 1 (488 nm light for 3×15s every 45 mins for 4.5h) or recovered for 20h (day 2). (d) Confocal images of DVB (lim-6^int4::wCherry), and quantification of (e) total neurite outgrowth, (f) number of neurite junctions, and (g) time to spicule protraction on aldicarb. (dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD).

Fig. 4. Neuroligin and neurexin impact DVB neurite outgrowth and spicule protraction behavior

(a) Confocal images of DVB (lim-6^int4::wCherry) in nlg-1(ok259) mutant and control and quantification of (b) total neurite outgrowth and (c) number of neurite junctions in nlg-1(ok259) and control. (d) Time to aldicarb-induced spicule protraction in control and nlg-1(ok259) males. (e) Confocal images of DVB (lim-6^int4::gfp) in nrx-1(wy778) mutant and control. Quantification of (f) total neurite outgrowth and (g) number of neurite junctions in nrx-1(wy778) and control. (h) Time to aldicarb-induced spicule protraction in control and nrx-1(wy778). (dot=one animal, magenta bar=median, and boxes=quartiles, one-way ANOVA and post-hoc Tukey HSD).