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• W1636429246 abstract "•Three neural circuits modulate and alter C. elegans pharyngeal pumping•One intrinsic circuit inhibits pumping, whereas another promotes spitting•A third circuit relays a signal from outside its organ, like the autonomic system•Functional synapses can exist despite the absence of a synapse in the connectome Neural circuits have long been known to modulate myogenic muscles such as the heart, yet a mechanistic understanding at the cellular and molecular levels remains limited. We studied how light inhibits pumping of the Caenorhabditis elegans pharynx, a myogenic muscular pump for feeding, and found three neural circuits that alter pumping. First, light inhibits pumping via the I2 neuron monosynaptic circuit. Our electron microscopic reconstruction of the anterior pharynx revealed evidence for synapses from I2 onto muscle that were missing from the published connectome, and we show that these “missed synapses” are likely functional. Second, light inhibits pumping through the RIP-I1-MC neuron polysynaptic circuit, in which an inhibitory signal is likely transmitted from outside the pharynx into the pharynx in a manner analogous to how the mammalian autonomic nervous system controls the heart. Third, light causes a novel pharyngeal behavior, reversal of flow or “spitting,” which is induced by the M1 neuron. These three neural circuits show that neurons can control a myogenic muscle organ not only by changing the contraction rate but also by altering the functional consequences of the contraction itself, transforming swallowing into spitting. Our observations also illustrate why connectome builders and users should be cognizant that functional synaptic connections might exist despite the absence of a declared synapse in the connectome. Neural circuits have long been known to modulate myogenic muscles such as the heart, yet a mechanistic understanding at the cellular and molecular levels remains limited. We studied how light inhibits pumping of the Caenorhabditis elegans pharynx, a myogenic muscular pump for feeding, and found three neural circuits that alter pumping. First, light inhibits pumping via the I2 neuron monosynaptic circuit. Our electron microscopic reconstruction of the anterior pharynx revealed evidence for synapses from I2 onto muscle that were missing from the published connectome, and we show that these “missed synapses” are likely functional. Second, light inhibits pumping through the RIP-I1-MC neuron polysynaptic circuit, in which an inhibitory signal is likely transmitted from outside the pharynx into the pharynx in a manner analogous to how the mammalian autonomic nervous system controls the heart. Third, light causes a novel pharyngeal behavior, reversal of flow or “spitting,” which is induced by the M1 neuron. These three neural circuits show that neurons can control a myogenic muscle organ not only by changing the contraction rate but also by altering the functional consequences of the contraction itself, transforming swallowing into spitting. Our observations also illustrate why connectome builders and users should be cognizant that functional synaptic connections might exist despite the absence of a declared synapse in the connectome. Animals rely on muscles for functions critical to their lives, from the execution of behavior to internal processes such as digestion and circulation. In general, animals have two kinds of muscles. The first requires neural activity to contract, such as skeletal muscle. The second, myogenic muscle, does not require neural activity to contract, and neural activity instead serves a modulatory role. Cardiac muscle, including cardiomyocytes, and some enteric muscles are myogenic [1Irisawa H. Brown H.F. Giles W. Cardiac pacemaking in the sinoatrial node.Physiol. Rev. 1993; 73: 197-227Crossref PubMed Scopus (581) Google Scholar, 2Donnelly G. Jackson T.D. Ambrous K. Ye J. Safdar A. Farraway L. Huizinga J.D. The myogenic component in distention-induced peristalsis in the guinea pig small intestine.Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 280: G491-G500PubMed Google Scholar], and input from the autonomic nervous system plays a modulatory role, such as altering heart rate [3Palma J.-A. Benarroch E.E. Neural control of the heart: recent concepts and clinical correlations.Neurology. 2014; 83: 261-271Crossref PubMed Scopus (120) Google Scholar]. Understanding the logic of such modulatory neural circuits requires an understanding at the cellular level, which can be difficult to achieve in vertebrates. We sought to investigate neural control of a myogenic muscle organ in an organism readily amenable to cellular and molecular analyses. Neural circuits in invertebrates can be understood in a “gap-free” manner, meaning that the function of each individual neuron that contributes to a larger neural circuit can be identified [4Chalfie M. Sulston J.E. White J.G. Southgate E. Thomson J.N. Brenner S. The neural circuit for touch sensitivity in Caenorhabditis elegans.J. Neurosci. 1985; 5: 956-964Crossref PubMed Google Scholar, 5Chalasani S.H. Chronis N. Tsunozaki M. Gray J.M. Ramot D. Goodman M.B. Bargmann C.I. Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans.Nature. 2007; 450: 63-70Crossref PubMed Scopus (425) Google Scholar, 6Olsen S.R. Wilson R.I. Cracking neural circuits in a tiny brain: new approaches for understanding the neural circuitry of Drosophila.Trends Neurosci. 2008; 31: 512-520Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar]. We selected the nematode Caenorhabditis elegans to study neural control of myogenic muscles because (1) its nervous system has only 302 neurons, (2) its connectome (the putatively complete set of all anatomical synapses among all neurons) has been described [7Albertson D.G. Thomson J.N. The pharynx of Caenorhabditis elegans.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1976; 275: 299-325Crossref PubMed Scopus (505) Google Scholar, 8White J.G. Southgate E. Thomson J.N. Brenner S. The structure of the nervous system of the nematode Caenorhabditis elegans.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1986; 314: 1-340Crossref PubMed Google Scholar] and is easily accessed [9Bhatla, N. (2011). WormWeb.org: C. elegans interactive neural network. http://wormweb.org/neuralnet.Google Scholar], (3) neural circuits can be examined at the cellular level in vivo, and (4) neural circuits can be analyzed at the molecular level using genetic methods. The C. elegans pharynx is a myogenic muscle group that functions as the worm’s feeding organ, pumping bacteria into the intestine [10Fang-Yen C. Avery L. Samuel A.D.T. Two size-selective mechanisms specifically trap bacteria-sized food particles in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2009; 106: 20093-20096Crossref PubMed Scopus (74) Google Scholar]. The pharyngeal nervous system consists of 20 neurons of 14 classes, and, as with the heart, neural innervation serves a modulatory rather than necessary role for pumping [11Avery L. Horvitz H.R. Pharyngeal pumping continues after laser killing of the pharyngeal nervous system of C. elegans.Neuron. 1989; 3: 473-485Abstract Full Text PDF PubMed Scopus (281) Google Scholar]. Physiological or behavioral functions have been described for nine neuron classes (MC, M2, M3, M4, I1, I2, I4, I5, and NSM) [11Avery L. Horvitz H.R. Pharyngeal pumping continues after laser killing of the pharyngeal nervous system of C. elegans.Neuron. 1989; 3: 473-485Abstract Full Text PDF PubMed Scopus (281) Google Scholar, 12Avery L. Horvitz H.R. A cell that dies during wild-type C. elegans development can function as a neuron in a ced-3 mutant.Cell. 1987; 51: 1071-1078Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 13Avery L. Motor neuron M3 controls pharyngeal muscle relaxation timing in Caenorhabditis elegans.J. Exp. Biol. 1993; 175: 283-297PubMed Google Scholar, 14Dent J.A. Smith M.M. Vassilatis D.K. Avery L. The genetics of ivermectin resistance in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2000; 97: 2674-2679Crossref PubMed Scopus (311) Google Scholar, 15Trojanowski N.F. Padovan-Merhar O. Raizen D.M. Fang-Yen C. Neural and genetic degeneracy underlies Caenorhabditis elegans feeding behavior.J. Neurophysiol. 2014; 112: 951-961Crossref PubMed Scopus (37) Google Scholar, 16Bhatla N. Horvitz H.R. Light and hydrogen peroxide inhibit C. elegans Feeding through gustatory receptor orthologs and pharyngeal neurons.Neuron. 2015; 85: 804-818Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Raizen D.M. Lee R.Y. Avery L. Interacting genes required for pharyngeal excitation by motor neuron MC in Caenorhabditis elegans.Genetics. 1995; 141: 1365-1382Crossref PubMed Google Scholar]. We previously reported that short wavelength light (violet and UV) interrupts the pumping rhythm of the pharynx and suggested that light generates hydrogen peroxide or another reactive oxygen species that is toxic to the worm [16Bhatla N. Horvitz H.R. Light and hydrogen peroxide inhibit C. elegans Feeding through gustatory receptor orthologs and pharyngeal neurons.Neuron. 2015; 85: 804-818Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar]. In an effort to reduce exposure to the toxic effects of light, the worm inhibits feeding and avoids the light [18Edwards S.L. Charlie N.K. Milfort M.C. Brown B.S. Gravlin C.N. Knecht J.E. Miller K.G. A novel molecular solution for ultraviolet light detection in Caenorhabditis elegans.PLoS Biol. 2008; 6: e198Crossref PubMed Scopus (202) Google Scholar, 19Ward A. Liu J. Feng Z. Xu X.Z.S. Light-sensitive neurons and channels mediate phototaxis in C. elegans.Nat. Neurosci. 2008; 11: 916-922Crossref PubMed Scopus (207) Google Scholar, 20Liu J. Ward A. Gao J. Dong Y. Nishio N. Inada H. Kang L. Yu Y. Ma D. Xu T. et al.C. elegans phototransduction requires a G protein-dependent cGMP pathway and a taste receptor homolog.Nat. Neurosci. 2010; 13: 715-722Crossref PubMed Scopus (136) Google Scholar]. Here, we use the inhibition of C. elegans pumping in response to light as a tool to analyze how neurons control the worm’s myogenic muscular pump, the pharynx. By studying this behavioral response using cellular and molecular methods, we identify three neural circuits that control this myogenic muscle organ. In the presence of food, the C. elegans pharynx pumps rapidly (4 to 5 Hz). Short-wavelength light (436 nm; 13 mW/mm2) alters pumping in three distinct phases, as previously reported [16Bhatla N. Horvitz H.R. Light and hydrogen peroxide inhibit C. elegans Feeding through gustatory receptor orthologs and pharyngeal neurons.Neuron. 2015; 85: 804-818Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar]. First, pumping rapidly stops in response to light (the “acute” response; 0–5 s after light onset). Second, pumping subsequently increases in the continued presence of light (the “burst” response; 5–10 s after light onset). Third, pumping slowly begins to recover after light is removed (the “recovery” response; 0–10 s after light removal; Figure 1A). Previously, we showed that loss of the I2 pharyngeal neuron pair (Figure 1B) causes a partial defect in the acute response to light (Figures 1C and 1D) [16Bhatla N. Horvitz H.R. Light and hydrogen peroxide inhibit C. elegans Feeding through gustatory receptor orthologs and pharyngeal neurons.Neuron. 2015; 85: 804-818Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar]. In addition, light causes an increase in I2 calcium [16Bhatla N. Horvitz H.R. Light and hydrogen peroxide inhibit C. elegans Feeding through gustatory receptor orthologs and pharyngeal neurons.Neuron. 2015; 85: 804-818Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar]. We concluded that I2 executes part of the acute response to light. The increase in I2 calcium caused by light could result either from I2’s receiving a signal from another cell or from I2’s directly sensing light without a cellular intermediary. If I2 receives a signal from another cell, that signal could be communicated either via a direct synapse or humorally. To identify candidate neurons that directly synapse onto I2, we examined the connectome of the pharynx [7Albertson D.G. Thomson J.N. The pharynx of Caenorhabditis elegans.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1976; 275: 299-325Crossref PubMed Scopus (505) Google Scholar]. The I1 neuron pair and the M1 neuron were reported to form gap junctions with or provide chemical synaptic input to I2. Our connectome analysis (see below) suggested that the original connectome might be missing functional synapses, so we used a laser to ablate all pharyngeal neurons that would not affect the worm’s growth or health; specifically, the MC neuron pair, which promotes pumping, and the M4 neuron, which promotes peristalsis, were not killed [11Avery L. Horvitz H.R. Pharyngeal pumping continues after laser killing of the pharyngeal nervous system of C. elegans.Neuron. 1989; 3: 473-485Abstract Full Text PDF PubMed Scopus (281) Google Scholar, 12Avery L. Horvitz H.R. A cell that dies during wild-type C. elegans development can function as a neuron in a ced-3 mutant.Cell. 1987; 51: 1071-1078Abstract Full Text PDF PubMed Scopus (224) Google Scholar]. Altogether, 15 of 20 pharyngeal neurons were killed in each worm. Ablated worms exhibited an I2 response at 56% of the level of mock-ablated worms (Figures 1E and 1F). This result shows that I2 can respond to light in the absence of 11 of 13 pharyngeal neuron classes, although part of the I2 response depends on other pharyngeal neurons. We next sought to determine whether the I2 response to light requires a humoral signal. Humoral signaling is partially mediated by dense-core vesicle release, which requires UNC-31, the worm ortholog of human CADPS/CAPS [21Speese S. Petrie M. Schuske K. Ailion M. Ann K. Iwasaki K. Jorgensen E.M. Martin T.F.J. UNC-31 (CAPS) is required for dense-core vesicle but not synaptic vesicle exocytosis in Caenorhabditis elegans.J. Neurosci. 2007; 27: 6150-6162Crossref PubMed Scopus (194) Google Scholar]. Putative null mutants of unc-31(u280) showed an I2 response similar to that of wild-type (Figures 1G and 1H), suggesting that I2 does not receive a dense-core vesicle-mediated signal to trigger its response to light. Because neither direct synaptic input nor dense-core vesicle input appears to be necessary for I2 to respond to light, I2 likely senses light without a neuronal intermediary. A third reason to think that I2 functions as a cellular sensor for light is that it expresses a putative molecular sensor for a light-generated molecule, hydrogen peroxide. The molecular receptor GUR-3 functions in I2 to detect hydrogen peroxide and increase calcium [16Bhatla N. Horvitz H.R. Light and hydrogen peroxide inhibit C. elegans Feeding through gustatory receptor orthologs and pharyngeal neurons.Neuron. 2015; 85: 804-818Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar]. Taking these results together, we conclude that the I2s can function as sensory neurons, although the I2 response to light is also modulated by other pharyngeal neurons. To identify regions of I2 that when exposed to light are sufficient to induce the cellular response, we restricted light to the anterior neurite, the soma, or the posterior neurite (Figure S1A). We found that illumination of the posterior neurite caused a large increase in fluorescence throughout I2, whereas anterior neurite or soma illumination caused little increase in fluorescence (Figures S1B–S1D). To determine which of the two neurites is necessary for the light-induced response, we cut the anterior or posterior neurite using a laser, killing the severed neurite, and then exposed the worm’s head to light. We found that I2 responded in the absence of either the anterior or the posterior neurite, although the response in the anterior neurite after the posterior neurite was cut was significantly smaller than the response in the anterior neurite in the intact control (Figures S1E–S1H). Overall, these results suggest that, whereas the posterior neurite is the most light-sensitive compartment, the soma and anterior neurite are also light sensitive and can be sufficient to trigger an influx of calcium into I2. To determine the molecular source of the increase in I2 calcium in response to light, we tested mutants disrupted in calcium influx. Although most of these mutants had a normal I2 response (Figures S2A–S2G), we found that mutants carrying nonsense alleles of unc-2(e55) (α1 subunit; N/P/Q-type voltage-gated calcium channel [VGCC]) [22Mathews E.A. García E. Santi C.M. Mullen G.P. Thacker C. Moerman D.G. Snutch T.P. Critical residues of the Caenorhabditis elegans unc-2 voltage-gated calcium channel that affect behavioral and physiological properties.J. Neurosci. 2003; 23: 6537-6545Crossref PubMed Google Scholar] or unc-36(e251) (α2δ subunit; VGCC) [23Schafer W.R. Sanchez B.M. Kenyon C.J. Genes affecting sensitivity to serotonin in Caenorhabditis elegans.Genetics. 1996; 143: 1219-1230PubMed Google Scholar] exhibited a partial defect: the calcium response latency was approximately doubled and the peak amplitude of the response was approximately halved (Figures 2A, 2B, 2D, and 2E ). The unc-36; unc-2 mutant was no more defective in the I2 response than the unc-2 mutant (Figures 2C–2E), suggesting that unc-2 and unc-36 function in the same pathway. We next assayed the pumping response of calcium channel mutants to determine whether any mutant exhibited an acute response defect similar to I2-ablated animals. Whereas several mutants showed differences from wild-type in the burst and recovery responses (Figures 2F, 2G, and S2H–S2N), only unc-36 mutants exhibited a small but statistically significant defect in the latency of the acute response to light (Figures 2G, 2I, and 2J). unc-36; unc-2 double mutants had a latency defect similar to that of unc-36 mutants (Figures 2H–2J). In addition, the remaining double mutants among unc-36, unc-2, egl-19, and cca-1 were either wild-type or no more defective than unc-36 or unc-2 mutants in the I2 response (Figures S2O–S2S). Finally, the unc-36; egl-19; cca-1 triple mutant was only modestly more defective than the unc-36 single mutant in the I2 response (Figure S2T). We conclude that unc-2 and unc-36 are partially required for the I2 calcium response to light and likely function in the same pathway. To identify cells downstream of I2, we took a molecular approach and sought to determine the neurotransmitter(s) that I2 secretes, the receptor(s) functioning downstream, and the cells in which the receptor(s) functions. Whereas most neurotransmitter mutants showed a normal acute pumping response to light (Figures S3A–S3H), mutants defective in glutamate neurotransmission because of a deletion in eat-4 (allele ky5), a vesicular glutamate transporter (VGLUT) [24Lee R.Y.N. Sawin E.R. Chalfie M. Horvitz H.R. Avery L. EAT-4, a homolog of a mammalian sodium-dependent inorganic phosphate cotransporter, is necessary for glutamatergic neurotransmission in Caenorhabditis elegans.J. Neurosci. 1999; 19: 159-167Crossref PubMed Google Scholar], exhibited a severe defect in the latency and amplitude of the acute response (Figures 3A, 3E, and 3F ). Multiple alleles of eat-4 (n2458, n2474, ad819, ad572, and ok2233) exhibited a similar defect (data not shown), and a genomic eat-4 transgene (njEx378) [25Ohnishi N. Kuhara A. Nakamura F. Okochi Y. Mori I. Bidirectional regulation of thermotaxis by glutamate transmissions in Caenorhabditis elegans.EMBO J. 2011; 30: 1376-1388Crossref PubMed Scopus (61) Google Scholar] fully rescued the defect of eat-4(ky5) mutants (Figures 3B, 3E, and 3F). Thus, mutation of eat-4 causes the acute response defect of eat-4 strains. We next sought to determine whether eat-4 is expressed in I2 by examining transgenic worms carrying the njEx378[eat-4::gfp] transgene. We observed fluorescence in I2 (Figure 3C), indicating that I2 is likely glutamatergic. A recent study also observed eat-4 expression in I2 [26Serrano-Saiz E. Poole R.J. Felton T. Zhang F. De La Cruz E.D. Hobert O. Modular control of glutamatergic neuronal identity in C. elegans by distinct homeodomain proteins.Cell. 2013; 155: 659-673Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar]. To determine whether eat-4 functions in I2 for the pumping response to light, we expressed eat-4 cDNA using an I2-specific promoter [27Kim K. Li C. Expression and regulation of an FMRFamide-related neuropeptide gene family in Caenorhabditis elegans.J. Comp. Neurol. 2004; 475: 540-550Crossref PubMed Scopus (227) Google Scholar] (flp-15prom::eat-4 cDNA::gfp). I2-specific expression of eat-4 partially rescued the acute latency and amplitude defects of eat-4 mutants (Figures 3D–3F), whereas eat-4 expression in I1, AWC, and AWB (gcy-10prom::eat-4 cDNA::gfp) had no effect (Figure S3I). We previously showed that mutants of lite-1, which encodes a gustatory receptor ortholog critical for light avoidance [18Edwards S.L. Charlie N.K. Milfort M.C. Brown B.S. Gravlin C.N. Knecht J.E. Miller K.G. A novel molecular solution for ultraviolet light detection in Caenorhabditis elegans.PLoS Biol. 2008; 6: e198Crossref PubMed Scopus (202) Google Scholar, 20Liu J. Ward A. Gao J. Dong Y. Nishio N. Inada H. Kang L. Yu Y. Ma D. Xu T. et al.C. elegans phototransduction requires a G protein-dependent cGMP pathway and a taste receptor homolog.Nat. Neurosci. 2010; 13: 715-722Crossref PubMed Scopus (136) Google Scholar], exhibit a partially defective acute response and a completely defective recovery response [16Bhatla N. Horvitz H.R. Light and hydrogen peroxide inhibit C. elegans Feeding through gustatory receptor orthologs and pharyngeal neurons.Neuron. 2015; 85: 804-818Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar]. Strikingly, eat-4; lite-1 double mutants were nearly completely defective in the pumping response to light (Figures 3G, 3I, and 3J). I2-specific eat-4 expression partially rescued the acute pumping response defect of eat-4; lite-1 mutants (Figures 3H–3J). Together, these I2-specific expression experiments suggest that I2 secretes glutamate in response to light. The partial nature of these rescues, as well as the fact that eat-4 mutants had a more-severe acute response defect than I2-ablated worms, suggests that glutamatergic neurons in addition to I2 function in the acute response to light. I2 also expresses several neuropeptide genes: flp-5; flp-15; nlp-3; and nlp-8 [27Kim K. Li C. Expression and regulation of an FMRFamide-related neuropeptide gene family in Caenorhabditis elegans.J. Comp. Neurol. 2004; 475: 540-550Crossref PubMed Scopus (227) Google Scholar, 28Nathoo A.N. Moeller R.A. Westlund B.A. Hart A.C. Identification of neuropeptide-like protein gene families in Caenorhabditiselegans and other species.Proc. Natl. Acad. Sci. USA. 2001; 98: 14000-14005Crossref PubMed Scopus (264) Google Scholar]. Mutants defective in these neuropeptide genes had a normal acute pumping response to light (Figures S4A–S4E). Moreover, mutants defective in the neuropeptide-processing enzymes egl-3 (PC2) and egl-21 (CPE) also had a normal acute pumping response to light (Figures S4F–S4H). unc-31 mutants defective in dense-core vesicle release exhibited a normal acute latency in response to light, although they did exhibit a small but statistically significant defect in acute amplitude (Figure S4I). Finally, genetic ablation of I2 did not further enhance the acute defect of eat-4 mutants, suggesting that the entirety of I2’s function in acute pumping is mediated by eat-4 (Figure S4J). Altogether, it appears unlikely that neuropeptide signaling from I2 plays a critical role in the I2-mediated acute response to light. Next, we sought to identify the glutamate receptor(s) that functions downstream of I2 for the acute response to light. We tested mutants of all 18 glutamate receptors and found defective acute responses in avr-15(ad1051), avr-14(ad1032), glc-2(gk179), and glc-4(ok212) (Figures 3K and S5). Here, we report our detailed analysis of avr-15, because these mutants exhibited a defect in the latency of the acute response similar to that of I2-ablated worms (Figures 3K and 3M). avr-15 is expressed in pharyngeal muscle [29Dent J.A. Davis M.W. Avery L. avr-15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans.EMBO J. 1997; 16: 5867-5879Crossref PubMed Scopus (275) Google Scholar], and we found that pharyngeal-muscle-specific expression of avr-15 (myo-2prom::avr-15 cDNA) fully rescued the latency defect of avr-15 mutants (Figures 3L and 3M). This result indicates that the AVR-15 glutamate receptor functions in pharyngeal muscle to reduce the latency of the acute response to light. Our findings that I2 secretes glutamate and that the AVR-15 glutamate receptor functions in muscle suggest that I2 signals directly to muscle after being activated by light. However, the described connectome does not include any synapses from I2 to muscle but rather identifies synapses from I2 to five neuron classes (NSM, I4, I6, M1, and MC) and gap junctions with two neuron classes (M1 and I1; Figure 4A, derived from [7Albertson D.G. Thomson J.N. The pharynx of Caenorhabditis elegans.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1976; 275: 299-325Crossref PubMed Scopus (505) Google Scholar]). If some or all of these neurons function together as relay stations between I2 and muscle, ablating them together would be expected to cause a defect at least as severe as that caused by I2 ablation. Worms lacking all pharyngeal neurons except I2, M4, and MC (15 neurons killed per animal) did not exhibit a defect in the acute response to light (Figures 4B–4D), consistent with the hypothesis that I2 signals directly to muscle. To further explore this possibility, we searched for additional synapses from I2 by examining the pharynx using transmission electron micrographs of serial sections (ssTEM). We identified an area as a synapse if it contained two or more synaptic or dense-core vesicles near the plasma membrane or a clearly visible presynaptic dense projection (DP) [30Stigloher C. Zhan H. Zhen M. Richmond J. Bessereau J.-L. The presynaptic dense projection of the Caenorhabditis elegans cholinergic neuromuscular junction localizes synaptic vesicles at the active zone through SYD-2/liprin and UNC-10/RIM-dependent interactions.J. Neurosci. 2011; 31: 4388-4396Crossref PubMed Scopus (83) Google Scholar]. We confirmed the previous finding of synapses from I2 onto NSM, I4, and I6 and found that these synapses had an average dense projection volume of nearly 1,500,000 nm3 (Figure S6). Additionally, we found 13 to 14 synapses from each I2 neuron onto pharyngeal muscle 3 (PM3) and a smaller number of synapses onto pharyngeal muscles 1 (PM1), 4 (PM4), and 5 (PM5) (Figures 4E–4H and S6). These neuromuscular synapses localized primarily to the anterior neurite of I2 and had much-smaller dense projections (average of ∼80,000 nm3). The presence of synapses from I2 to PM3 was confirmed in a second worm (worm no. 5; data not shown). Furthermore, when we re-examined the imagery used to generate the published connectome (the N2T series) with our criterion for identifying a synapse, we found six synapses from I2R onto PM3 and 15 synapses from I2L onto PM3. These synapses from I2 directly onto pharyngeal muscle could be the sites of I2 neurotransmission in response to light. Together, these data suggest that the I2 neurons function not only as sensory neurons but also as motor neurons to inhibit the pumping rhythm by directly silencing muscle. We next sought to identify additional neural circuits that could account for the acute response that remained after I2 ablation. We used laser microsurgery to kill each of the remaining 13 neuron classes in the pharynx. Although ablation of most neurons had minimal to no effect on the pumping response to light (Figure S7), we found that ablation of the I1, MC, and M1 neurons had effects. Ablation of the I1 neuron pair impaired the acute response, as measured by both response latency and amplitude (Figures 5A, 5G, and 5H ). This result indicates that I1 promotes the acute response to light. To determine whether I1 functions in the same neural pathway as I2, we conducted double ablations with the goal of interpreting our results as double mutants are interpreted in genetic studies [31Avery L. Wasserman S. Ordering gene function: the interpretation of epistasis in regulatory hierarchies.Trends Genet. 1992; 8: 312-316Abstract Full Text PDF PubMed Scopus (178) Google Scholar]. Double ablation of I1 and I2 caused a defect in the acute response that was more severe than either single ablation (Figures 5B, 5G, and 5H), suggesting that I1 and I2 function in parallel. To assess whether other pharyngeal neurons might also be involved, we ablated all pharyngeal neurons except I1, I2, M4, and MC. These animals retained a normal acute response to light (Figures 5C, 5G, and 5H), making it unlikely that any other pharyngeal neurons beyond I1, I2, M4, and MC play a critical role in the acute response. We next sought neurons that might function upstream of I1. The pharyngeal nervous system is anatomically connected to the main nervous system through gap junctions between the I1 and RIP neurons [7Albertson D.G. Thomson J.N. The pharynx of Caenorhabditis elegans.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1976; 275: 299-325Crossref PubMed Scopus (505) Google Scholar]. Ablation of the RIP neuron pair did not affect the acute response (Figures 5D, 5G, and 5H). Because I2 ablation enhanced the defect of I1 ablation, we suspected that I2 ablation might serve as a sensitized background with which to observe more-subtle functions for neurons in the" @default.
• W1636429246 created "2016-06-24" @default.
• W1636429246 creator A5005616363 @default.
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• W1636429246 date "2015-08-01" @default.
• W1636429246 modified "2023-10-13" @default.
• W1636429246 title "Distinct Neural Circuits Control Rhythm Inhibition and Spitting by the Myogenic Pharynx of C. elegans" @default.
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