Table 5 Author statements collection B

A

Bretscher et al. [13]

‘‘When placed in a 0–5% CO₂ gradient, C. elegans migrate away from high CO₂ (Fig. 1A, B) [12]. We used this assay to identify potential CO₂-sensing neurons….. We next attempted to rescue the tax-2(p694) defect by expressing tax-2 cDNA from neuron-specific promoters, confirming appropriate expression by polycistronic constructs that coexpress tax-2 and gfp [24]. Expressing tax-2 cDNA in the AFD thermosensory neurons strongly rescued CO₂ avoidance, both on and off food (Fig. 1D). In contrast, restoring tax-2 to the BAG O2-sensing neurons rescued CO₂ avoidance on food, as shown previously [35], but not off food. Expressing tax-2 cDNA in the ASE taste neurons or in the AQR, PQR, and URX O2-sensing neurons also partially rescued CO₂ avoidance, both on food and off food (Fig. 1D). These data implicate functionally diverse sensory neurons in CO₂ avoidance.’’

B

Kopchock et al. [42]

‘‘Optogenetic stimulation of the vulval muscles triggered an immediate rise in vulval muscle cytosolic Ca2 + , tonic contraction of the vulval muscles, vulval opening, and egg release (Fig. 7B, C). Although optogenetic stimulation resulted in sustained vulval muscle Ca2 + activity and contraction, vulval opening and egg release remained rhythmic and phased with locomotion, as previously observed in WT animals [3, 25]. Simultaneous brightfield recordings showed the vulva only opened for egg release when the adjacent ventral body wall muscles were in a relaxed phase (Movie 5). We have previously shown that eggs are preferentially released when the vulva is at a particular phase of the body bend, typically as the ventral body wall muscles anterior to the vulva go into a more relaxed state (Collins and Koelle, 2013; [25]. We now interpret this phasing of egg release with locomotion as evidence that vulval muscle Ca2 + activity drives contraction, but the vulva only opens for successful egg release when contraction is initiated during relaxation of the adjacent body wall muscles. Together, these results show that optogenetic stimulation of the vulval muscles is sufficient to induce vulval muscle Ca2 + activity for egg release in a locomotion phase-dependent manner.’’

C

Branicky et al. [11]

‘‘Because clh-3 encodes chloride channels, we reasoned that it might affect HSN activity by affecting HSN excitability. To test this, we crossed the clh-3 mutants with an integrated transgenic line that expresses Channelrhodopsin-2 (ChR2), the blue-light-activated cation channel, in the HSNs (wzIs6 [pegl-6::ChR2]; [27, 46]. In wild-type worms, egg laying is robustly stimulated by ChR2 activation [46], Fig. 7). The magnitude of the response, as indicated by both the percentage of stimulations resulting in egg-laying events and the number of eggs laid per stimulation, is dependent on both the strength and duration of the light stimulus (Fig. 7A). The response is also completely dependent on the addition of all-trans retinal, the cofactor for ChR2, to the plates (Fig. 7B), as well as the presence of the HSNs (Fig. 7D). We observed that the clh-3(n995gf) mutant laid significantly fewer eggs per stimulation than the wild type and blue light stimulation elicited an egg-laying event significantly less frequently in mutant animals than in wild type. Conversely, the clh-3(ok768 and ok763) mutants laid significantly more eggs than the wild-type and blue light stimulation elicited egg-laying events, including the laying of multiple eggs, more frequently than for the wild type (Fig. 7C, D). Together, these data support a role for the clh-3-encoded channels in inhibiting HSN excitability: increased channel activity inhibits HSN excitability, whereas loss of the channel promotes HSN excitability.’’

D

Emtage et al. [27]

‘‘Having established a method for exciting the HSN neurons in freely behaving animals, we next tested whether Go signaling controls the sensitivity of the HSNs to ChR2-mediated stimulation. egl-10 encodes an RGS family GTPase-activating protein (GAP) that accelerates hydrolysis of GTP by Goα and thereby antagonizes Go signaling [40]. egl-10 mutants carrying a Promegl-6::ChR2 transgene did not lay eggs in response to a photostimulus that reliably evoked egg-laying behavior when applied to wild-type transgenic animals (Fig. 6E), indicating that globally increasing Go signaling reduced the excitability of the HSN neurons. We next measured the effect of activating Go signaling downstream of the EGL-6 GPCR by testing the behavioral responses of transgenic egl-6(gf) mutants to photostimulation. Like egl-10 mutants, transgenic egl-6(gf) mutants had reduced behavioral responses to photostimulation of HSN neurons (Fig. 6F). Deletion of irk-1 significantly restored the response of egl-6(gf) mutants to excitatory input (Fig. 6F)’’

E

Collins et al. [25]

‘‘We have previously shown that two Cl-extruding transporters, KCC-2 and ABTS-1, are expressed in the HSNs, where they promote the development of inhibitory ligand-gated Cl-channel signaling [10, 68]. These data suggest that tyramine signaling through LGC-55 would hyperpolarize the HSN and inhibit activity. To test this directly, we compared HSN activity in wild-type and lgc-55 mutant animals. We observed a significant increase in the frequency of Ca2 + transients in HSNs of lgc-55 mutant animals (Fig. 6E, F) in both the inactive and active states of egg-laying behavior. Mean HSN inter-transient intervals in wild-type animals were 41 ± 5 s in the inactive state and 17 ± 2 s during the active state, while intervals in lgc-55 mutants were reduced to 22 ± 2 s in the inactive state and 13 ± 1 s during the active state. Thus, the absence of inhibitory feedback by tyramine signaling onto the HSNs leads to increased activity in both the active and inactive egg-laying behavior states.’’

F

Bretscher et al. [13]

‘‘The timing of CO2-evoked Ca2 + responses in both AFD and BAG correlated with peaks in locomotory activity (Fig. 6A). We investigated these correlations directly by ablating AFD and/or BAG and examining behavioral responses (Fig. 6B). For statistical comparison, we chose time intervals before and after gas switches according to the occurrence of peaks in wild-type behavioral rates. In the absence of food, neither AFD nor BAG ablation abolished modulation of speed across shifts in CO2 (Fig. 6B and Additional file 1: Fig. S4). Stronger phenotypes were observed for reversal and omega rates (Fig. 6B). Unexpectedly, ablation of AFD increased reversal and omega rates following a sharp CO2 rise (ttx-1, Fig. 6B, 7B, C, H, and I) and reduced suppression of omega turns following a CO2 fall (ttx-1, Figs. 6B,7 K,7 K, and L), suggesting that AFD acts to suppress reversals and omega turns at these two timepoints. Ablation of BAG abolished reversal and omega responses to a rise in CO2 (pBAG::egl-1, Figs. 6B, 7B, C, H, I) and reduced the suppression of omega turns following a CO2 fall (pBAG::egl-1, Figs. 6B, 7 K,  K, and L), consistent with BAG excitation promoting reversals and omega turns. Coablation of AFD and BAG abolished the suppression of reversals and omega turns following a fall in CO2 (ttx-1; pBAG::egl-1, Fig. 7F, L). This effect was due to reduced reversal and omega rates under prolonged high CO2 (ttx-1; pBAG::egl-1, red bars, Fig. 7E, K). These data suggest that together BAG and AFD act to suppress reversals and omega turns when CO2 decreases.’’

G

Shyn et al. [62]

‘‘Behavioral data implicated serotonin, a neuromodulator released from the HSN egg-laying motorneurons, in the control of egg-laying behavior 2, 3, 4. When we treated animals with exogenous 5HT, we observed a significant increase in the frequency of Ca2 + events from a baseline of 5.63 min −1 to a rate of 35.01 min  − 1 (p < 0.001, Kolmogorov–Smirnov test). Thus, exogenous serotonin appeared to modulate the functional state of the vulval muscles, switching them from a pattern of sporadic Ca2 + activity to a pattern of continual Ca2 + activity. In principle, serotonin could exert its effects directly on the vulval muscles, or it could act indirectly by altering the activity of the egg-laying motorneurons. To resolve this issue, we ablated the egg-laying motorneurons and assayed the effect of serotonin on vulval muscle Ca2 + transients. We found that ablated animals exhibited a continuous train of Ca2 + transients on serotonin essentially identical to that exhibited by unablated wild-type animals (Fig. 2, Table 1). Thus, the ability of serotonin to increase the frequency of Ca2 + events was not markedly affected by the absence of the egg-laying motorneurons, indicating that serotonin directly stimulates the activity of the vulval muscles.’’