Table 4 Author statements collection A

A

Waggoner et al. [72]

‘‘The roles of individual neurons in controlling the timing of egg-laying events can be determined with high precision by eliminating specific neurons by laser ablation and assaying the effect of the ablation on behavior. We, therefore, eliminated the neurons with prominent synaptic input to the egg-laying muscles to determine how their absence affected the timing of egg-laying events. We first investigated the involvement of the HSNs, a pair of serotonergic motor neurons that are required for efficient egg laying. By tracking the behavior of animals lacking both HSNs, we found that elimination of the HSNs did not qualitatively alter the pattern of egg laying: eggs were still laid in clusters, and the intervals between clusters and between egg-laying events within a cluster were still exponentially distributed. However, HSN ablation did cause a substantial lengthening of the inactive phase, which led to a slower overall rate of egg laying (Fig. 2A). Since loss of the HSNs decreased the frequency of egg-laying clusters (i.e., λ2 was decreased; Table 1) but did not slow the egg-laying rate within these clusters (λ1 was actually increased), these results suggest that the HSNs stimulate egg laying by inducing the active state.’’

B

Bany et al. [5]

‘‘Because the VC neurons appear to inhibit egg laying and are cholinergic, we tested whether the VCs release acetylcholine to inhibit egg laying. The VCs are the only cells of the egg-laying system that express the UNC-4 complex, CHA-1, and UNC-17 [48]; however, because unc-4, cha-1, and unc-17 are each expressed in other neurons, it was necessary to determine whether mutations in these genes cause hyperactive egg laying specifically attributable to their effects on the VC neurons. For this purpose, we expressed the unc-4, cha-1,or unc-17 cDNAs in the VC neurons and determined whether this rescued the hyperactive egg-laying defects of the corresponding mutants. To direct VC expression, we used a modified lin-11 promoter similar to that used to express GFP in Fig. 3A (see Materials and Methods). Expression of the unc-4 cDNA using this promoter rescued the hyperactive egg-laying defect of unc-4 mutants, returning the percentage of early stage eggs laid to near-wild-type levels (Fig. 4A). Furthermore, expressing the cha-1 cDNA in the VC neurons of cha-1 mutants also rescued their hyperactive egg-laying phenotype (Fig. 4B). Similar experiments with unc-17 gave analogous results (data not shown). Restoring the inhibition of egg laying by restoring the ability of the VC neurons to signal with acetylcholine provides our most compelling evidence that it is the VC neurons that inhibit egg laying.’’

C

Banerjee et al. [4]

‘‘We next sought to determine whether uv1 activation is sufficient to inhibit egg-laying. To address this question, we expressed channel rhodopsin (ChR2) in uv1 cells using the regulatory regions of ocr-2 as above. Light stimulation immediately following the initial egg-laying event of an active phase (see Methods) significantly delays subsequent egg-laying events, and also significantly reduces the total number of egg-laying events within an active phase (Fig. 2) (S1 Movie). For example, under control conditions a majority (~ 80%) of animals show a delay between the first and second egg-laying events within an active phase of < 20 s (light stimulation, -ATR) (Fig. 2B). This proportion is reduced dramatically (to around 10%) when uv1 cells are activated (light stimulation, + ATR)…Taken together, our findings provide evidence that uv1-mediated inhibition of egg-laying promotes periods of quiescence in the egg-laying program and plays a key role in setting their duration.’’

D

Carnell et al. [15]

‘‘The expression of gfp in the vulval muscles suggests that ser-1 may be acting in vulval muscles to mediate the stimulatory effect of 5-HT on egg laying. To test this hypothesis, we expressed the ser-1 cDNA using the muscle-specific myo-3 promoter [54] to determine whether it could rescue 5-HT-induced egg laying. Consistent with this hypothesis, we found the Pmyo-3::ser-1( +) transgene partially restored 5-HT-dependent egg laying to ser-1(ok345) animals (Fig. 2A). A wild-type ser-1 transgene with the same 3.4 kB promoter that failed to express gfp in the vulval muscles also failed to rescue the egg-laying defects of the ser-1 mutant animals (Fig. 2A). These results indicate that ser-1 expression in muscle can restore egg laying. Previous studies have indicated that 5-HT acts on vulval muscle to stimulate egg laying [14, 71, 72], Bastiani et al. 2003; [62]. Our results indicate that ser-1 mediates this response.’’

E

Carillo et al. [16]

‘‘NPR-1 is not expressed in BAG neurons but is expressed in a number of other sensory neurons as well as some interneurons [50]. To identify the site of action for the regulation of CO₂ response by npr-1, we introduced the N2 allele of npr-1 into npr-1(lf) mutants in different subsets of neurons and assayed CO2 response. We found that expressing npr-1 in neuronal subsets that included the O2-sensing URX neurons [20, 32] restored CO₂ response (Fig. 3A). These results suggest that NPR-1 activity in URX neurons is sufficient to enable CO2 avoidance. However, we cannot exclude the possibility that NPR-1 function in other neurons also contributes to CO₂ avoidance.’’

F

Bretscher et al. [13]

‘‘Strikingly, AFD also responded to removal of CO₂ with a fast Ca₂ + spike that peaked within 10 s (“CO₂-OFF” response…CO₂-evoked activity in AFD could be due to synaptic input to AFD. To test this, we imaged CO₂ responses in unc-13 mutants, which have severe defects in synaptic release [58]. The AFD CO₂ responses of unc-13 animals were indistinguishable from wild type (Figs. 2H and S1C). These data suggest that, as well as being a thermosensory neuron [23, 39, 53], AFD is a CO₂ sensor with both ON and OFF responses.’’

G

Collins et al. [25]

‘‘To directly test how neurotransmitter signaling from the HSNs regulates egg-laying circuit activity, we used the egl-6 promoter to express Channelrhodopsin-2 in the HSNs [27], allowing us to drive neurotransmitter release specifically from the HSNs with blue light. …We found that activation of HSNs resulted in circuit activity reminiscent of a spontaneous active state, including rhythmic Ca2 + activity of both VCs and vulval muscles, and egg-laying events that accompanied a subset of these Ca2 + transients… These results suggest that the high level of HSN activity after optogenetic activation induces strong coupling of VC and vulval muscle excitation.’’

H

Kopchock et al. [42]

‘‘To determine whether VC synaptic transmission regulates egg laying via HSN, we recorded HSN Ca2 + activity in WT and transgenic animals expressing TeTx in the VCs (Fig. 6A). During the egg-laying active state, the HSNs drive egg laying during periods of increased Ca2 + transient frequency in the form of burst firing (Fig. 6B), [25, 56]. We observed a significant increase in HSN Ca2 + transient frequency when VC synaptic transmission was blocked compared with nontransgenic control animals (Fig. 6C). WT animals spent ∼11% of their time exhibiting high-frequency burst activity in the HSN neurons, whereas transgenic animals expressing TeTx in the VC neurons spent ∼21% of their time exhibiting HSN burst firing activity (Fig. 6D). These results are consistent with the interpretation that VC neurotransmission is inhibitory toward the HSNs, such as proposed in previous studies [5, 74].”

J

Choi et al. [21]

‘‘VGLUTs are members of a family of anion transporters that move diverse solutes, including inorganic phosphate, acidic sugars, negatively charged amino acids, and phosphorylated adenosine nucleotides33. As a member of the SLC17 family of transporters, VST-1 is likely an anion transporter and there are different ways an anion transporter in the synaptic vesicle membrane could limit glutamate uptake. …we used synaptopHluorin to measure vesicular pH in wild-type and vst-1 BAG neurons. Measurements of total and surface-accessible pHluorin (Fig. 3g) allow computation of vesicular pH42…Importantly, we found that loss of VST-1 caused a measurable increase in vesicular pH (Fig. 3h), consistent with a model in which VST-1 supports anion influx into synaptic vesicles. We also measured vesicular pH in BAG neurons lacking EAT-4/VGLUT (Fig. 3h). Unlike loss of VST-1, loss of EAT-4/VGLUT did not cause a measurable change in vesicular pH. The effect of VST-1 mutation on vesicular pH provides additional evidence that VST-1 functions in the synaptic vesicle membrane. These data are also consistent with a model in which VST-1 is an anion transporter that competes with EAT-4/VGLUT for the electrochemical gradient required for glutamate uptake into synaptic vesicles. However, some SLC17 family transporters can cotransport cations, such as Na + and H + 33, and we cannot rule out the possibility that cation efflux (rather than anion influx) contributes to the effect of VST-1 on vesicular pH.’’

'K

Choi et al. [21]

‘‘We further tested whether the effects of vst-1 mutation on RIA activation by BAGs require GLR-1 glutamate receptors, as predicted by our model. In mutants lacking GLR-1, there was no clear effect of vst-1 mutation (Fig. 6d, e), indicating that the increased activation of RIAs observed in vst-1 mutants requires signaling through GLR-1.’’