The Role of Ion Channels in Coordinating Neural Circuit Activity in Caenorhabditis elegans: A Dissertation

Despite the current understanding that sensorimotor circuits function through the action of transmitters and modulators, we have a limited understanding of how the nervous system directs the flow of information necessary to orchestrate complex behaviors. In this dissertation, I aimed to uncover how the nervous system coordinates these behaviors using the escape response of the soil nematode, Caenorhabditis elegans, as a paradigm. C. elegans exhibits a robust escape behavior in response to touch. The worm typically moves forward in a sinusoidal pattern, which is accompanied by exploratory head movements. During escape, the worm quickly retreats by moving backward from the point of stimulus while suppressing its head movements. It was previously shown that the biogenic amine tyramine played an important role in modulating the suppression of these head movmemetns in response to touch. We identified a novel tyramine-gated chloride channel, LGC-55, whose activation by tyramine coordinates motor programs essential for escape. Furthermore, we found that changing the electrical nature of a synapse within the neural circuit for escape behavior can reverse its behavioral output, indicating that the C. elegans connectome is established independent of the nature of synaptic activity or behavioral output. Finally, we characterized a unique mutant, zf35 , which is hyperactive in reversal behavior. This mutant was identified as a gain of function allele of the C. elegans P/Q/N-type voltage-gated calcium channel, UNC-2. Taken together, this work defines tyramine as a genuine neurotransmitter and completes the neural circuit that controls the initial phases of the C. elegans escape response. Additionally, this research further advances the understanding of how the interactions between transmitters and ion channels can precisely regulate neural circuit activity in the execution of a complex behavior

Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in C. elegans

Mutations in pre-synaptic voltage gated calcium channels can lead to familial hemiplegic migraine type 1 (FHM1). While mammalian studies indicate that the migraine brain is hyperexcitable due to enhanced excitation or reduced inhibition, the molecular and cellular mechanisms underlying this excitatory/inhibitory (E/I) imbalance are poorly understood. We identified a gain-of-function (gf) mutation in the Caenorhabditis elegans CaV2 channel α1 subunit, UNC-2, which leads to increased calcium currents. unc-2(zf35gf) mutants exhibit hyperactivity and seizure-like motor behaviors. Expression of the unc-2 gene with FHM1 substitutions R192Q and S218L leads to hyperactivity similar to that of unc-2(zf35gf) mutants. unc-2(zf35gf) mutants display increased cholinergic-and decreased GABAergic-transmission. Moreover, increased cholinergic transmission in unc-2(zf35gf) mutants leads to an increase of cholinergic synapses and a TAX-6/calcineurin dependent reduction of GABA synapses. Our studies reveal mechanisms through which CaV2 gain-of-function mutations disrupt excitation-inhibition balance in the nervous system.Fil: Huang, Yung Chi. University of Massachussets; Estados UnidosFil: Pirri, Jennifer K.. University of Massachussets; Estados UnidosFil: Rayes, Diego Hernán. University of Massachussets; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Gao, Shangbang. Mount Sinai Hospital; Estados UnidosFil: Mulcahy, Ben. Mount Sinai Hospital; Estados UnidosFil: Grant, Jeff. University of Massachussets; Estados UnidosFil: Saheki, Yasunori. The Rockefeller University; Estados UnidosFil: Francis, Michael M.. University of Massachussets; Estados UnidosFil: Zhen, Mei. University of Toronto; Canadá. Mount Sinai Hospital; Estados UnidosFil: Alkema, Mark J.. University of Massachussets; Estados Unido

Intercellular calcium signaling in a gap junction-coupled cell network establishes asymmetric neuronal fates in C. elegans

The C. elegans left and right AWC olfactory neurons specify asymmetric subtypes, one default AWC(OFF) and one induced AWC(ON), through a stochastic, coordinated cell signaling event. Intercellular communication between AWCs and non-AWC neurons via a NSY-5 gap junction network coordinates AWC asymmetry. However, the nature of intercellular signaling across the network and how individual non-AWC cells in the network influence AWC asymmetry is not known. Here, we demonstrate that intercellular calcium signaling through the NSY-5 gap junction neural network coordinates a precise 1AWC(ON)/1AWC(OFF) decision. We show that NSY-5 gap junctions in C. elegans cells mediate small molecule passage. We expressed vertebrate calcium-buffer proteins in groups of cells in the network to reduce intracellular calcium levels, thereby disrupting intercellular communication. We find that calcium in non-AWC cells of the network promotes the AWC(ON) fate, in contrast to the autonomous role of calcium in AWCs to promote the AWC(OFF) fate. In addition, calcium in specific non-AWCs promotes AWC(ON) side biases through NSY-5 gap junctions. Our results suggest a novel model in which calcium has dual roles within the NSY-5 network: autonomously promoting AWC(OFF) and non-autonomously promoting AWC(ON)

Monoaminergic Orchestration of Motor Programs in a Complex C. elegans Behavior

Monoamines provide chemical codes of behavioral states. However, the neural mechanisms of monoaminergic orchestration of behavior are poorly understood. Touch elicits an escape response in Caenorhabditis elegans where the animal moves backward and turns to change its direction of locomotion. We show that the tyramine receptor SER-2 acts through a $G\alpha_o$ pathway to inhibit neurotransmitter release from GABAergic motor neurons that synapse onto ventral body wall muscles. Extrasynaptic activation of SER-2 facilitates ventral body wall muscle contraction, contributing to the tight ventral turn that allows the animal to navigate away from a threatening stimulus. Tyramine temporally coordinates the different phases of the escape response through the synaptic activation of the fast-acting ionotropic receptor, LGC-55, and extrasynaptic activation of the slow-acting metabotropic receptor, SER-2. Our studies show, at the level of single cells, how a sensory input recruits the action of a monoamine to change neural circuit properties and orchestrate a compound motor sequence.Physic

The neuroethology of C. elegans escape

Escape behaviors are crucial to survive predator encounters. Touch to the head of Caenorhabditis elegans induces an escape response where the animal rapidly backs away from the stimulus and suppresses foraging head movements. The coordination of head and body movements facilitates escape from predacious fungi that cohabitate with nematodes in organic debris. An appreciation of the natural habitat of laboratory organisms, like C. elegans, enables a comprehensive neuroethological analysis of behavior. In this review we discuss the neuronal mechanisms and the ecological significance of the C. elegans touch response

A switch in LGC-55 ion selectivity reverses behavioral output.

<p>(A) Touch induces neck relaxation in LGC-55 anion and contraction in LGC-55 cation transgenic animals. Still images of the animal’s head before (top) and after (bottom) touch stimulus. Scale bar, 0.1 mm. Arrow indicates neck length. (B) As measured in A, neck length from posterior of the pharynx to the tip of the nose before (light gray bars) and after (dark gray bars) anterior touch of wild type (<i>n</i> = 39); <i>lgc-55(tm2913)</i> (<i>n</i> = 32); <i>Plgc-55</i>::LGC-55 cation-I (<i>lgc-55(tm2913); zfEx8)</i>, <i>n</i> = 32; <i>Plgc-55</i>::LGC-55 cation-II (<i>lgc-55(tm2913)</i>; <i>zfEx40)</i>, <i>n</i> = 26. Analyses were performed in an <i>unc-3</i> mutant background to prevent backward locomotion in response to touch and to maintain the animal in the field of view at high magnification. Error bars represent SEM. Statistical difference as indicated, ** <i>p</i> < 0.001, *** <i>p</i> < 0.0001, two-tailed Student’s <i>t</i> test. (C) LGC-55 cation animals fail to execute a long reversal in response to touch. Shown is the average number of backward body bends in response to anterior touch of wild type <i>n</i> = 100; <i>Plgc-55</i>::<i>lgc-55 (lgc-55(tm2913); zfEx2</i>), <i>n</i> = 100, <i>lgc-55(tm2913)</i>, <i>n</i> = 100; <i>Plgc-55</i>:: LGC-55 cation-I (<i>lgc-55(tm2913); zfEx8)</i>, <i>n</i> = 100; <i>Plgc-55</i>::LGC-55 cation-II (<i>lgc-55(tm2913)</i>; <i>zfEx40)</i>, <i>n</i> = 100. Error bars represent SEM. Statistical difference from anion, * <i>p</i> < 0.01, *** <i>p</i> < 0.0001, two-tailed Student’s <i>t</i> test. (D) Tyramine release from the RIM activates the LGC-55 cation channel. Shown is the length of the neck before (light gray bars) and after (dark grey bars) exposure to blue light in retinal fed animals expressing the light-activated cation channel, ChannelRhodopsin 2 (ChR2), in the RIM in a wild-type background (P<i>tdc-1</i>::ChR2(<i>zfIs9</i>), <i>n</i> = 28); TA deficient (<i>tdc-1(n3420); Ptdc-1</i>::ChR2(<i>zfIs9</i>), <i>n</i> = 25); receptor deficient (<i>lgc-55(tm2913); Ptdc-1</i>::ChR2<i>(zfIs9)</i>, <i>n</i> = 28; LGC-55 cation-II (<i>lgc-55(tm2913);</i> P<i>lgc-55</i>::LGC-55 cation-II; <i>Ptdc-1</i>::ChR2 (<i>zfEx213)</i>, <i>n</i> = 20); TA deficient; LGC-55 cation-II (<i>tdc-1(n3420); lgc-55(tm2913);</i> P<i>lgc-55</i>::LGC-55 cation-II; <i>Ptdc-1</i>::ChR2(<i>zfEx275</i>), <i>n</i> = 16) animals. Analyses were performed in an <i>unc-3</i> mutant background. Blue light causes activation of the RIM and release of tyramine. Tyraminergic activation of the LGC-55 anion causes a relaxation of the neck muscles, while activation of LGC-55 cation-II causes a hypercontraction of the neck muscles. There is no response in animals that are raised on plates without all-<i>trans</i> retinal. Error bars represent SEM. Statistical difference as indicated, ** <i>p</i> < 0.001, *** <i>p</i> < 0.0001, two-tailed Student’s <i>t</i> test.</p

LGC-55 cation channel mutants gate sodium.

<p>(A) Cys-loop LGICs are homopentameric channels, each subunit containing four transmembrane domains. Depicted is a schematic representation of an LGIC with transmembrane domains 1 and 2 (M1, M2) in light gray. In blue is the intracellular loop that links M1 and M2, which determines the ion selectivity of the channel. (B) Alignment of M1–M2 loop region of LGC-55 with structurally related Cys-loop LGICs. Identities are shaded in dark gray, while similarities are light gray. The blue boxes indicate residues that determine selectivity of anions, while red boxes indicate those for cation selectivity. The engineered LGC-55 cation-I and cation-II channels contain the M1 loop of the cationic 5HT3a receptor. The LGC-55 cation-II channel also contains an additional mutation at the 20ʹ residue, which is predicted to enhance cation selectivity (see text for details). (C) Ion selectivity of the LGC-55 anion (left) and LGC-55 cation-II (right) receptor in cultured <i>C</i>. <i>elegans</i> muscle cells. Tyramine (TA) evoked (0.5 mM, 250 ms) currents were recorded at the holding potentials shown. Black circles: ES1 (standard solution: 150 mM Na⁺, 165 mM Cl^-), LGC-55 anion: E_rev = -26.8 ± 3.1mV (<i>n</i> = 4), LGC-55 cation-II: E_rev = 2.4 ± 1.2 mV (<i>n</i> = 5); red squares: ES2 (low Na⁺: 15 mM Na⁺, 165 mM Cl^-), LGC-55 anion: = -24.3 ± 1.6 mV (<i>n</i> = 4), LGC-55 cation-II: -21.9 ± 2.6 mV (<i>n</i> = 5); blue triangles: ES3 (low Cl^-: 150 mM Na⁺, 30 mM Cl^-), LGC-55 anion: -1.9 ± 2.3 mV (<i>n</i> = 4) LGC-55 cation-II: 1.7 ± 0.9 mV (<i>n</i> = 5). The insets show representative macrocurrents of LGC-55 anion (left) and LGC-55 cation-II (right) elicited after perfusion of 0.5 mM tyramine at membrane holding potentials ranging from -60 to +60 mV in 20 mV steps in the standard solution.</p

Engineered LGC-55 cation channels are functional in vivo.

<p>(A) Still images of wild-type and transgenic animals expressing LGC-55 anion or cation-II ectopically in all body wall muscle cells, on exogenous tyramine. LGC-55 anion animals paralyze in relaxed extended posture, while LGC-55 cation-II animals are hypercontracted. Scale bar = 0.25 mm. (B) Quantification of body length on exogenous tyramine (wild type, <i>n</i> = 57, P<i>myo-3</i>::LGC-55 anion(<i>zfEx31</i>), <i>n</i> = 53; P<i>myo-3</i>::LGC-55 cation-I<i>(zfEx120)</i>, <i>n</i> = 59; P<i>myo-3</i>::LGC-55 cation-II<i>(zfEx41)</i>, <i>n</i> = 55). Error bars represent the standard error of the mean (SEM). Statistical significance as indicated, *** <i>p</i> < 0.0001.</p

Phylogenetic comparisons of ion channel domains of members of the Cys-loop family of LGICs.

<p>LGIC phylogenetic comparison was performed on the ion channel domains of human and invertebrate LGICs. The neurotransmitter identities are indicated on the right. Blue shading indicates anionic channels, while red shading indicates cationic channels. <i>Ce</i>, <i>C</i>. <i>elegans</i>; <i>Ls</i>, <i>Lymnaea stagnalis</i>; <i>Hs</i>, <i>Homo sapiens</i>. Protein alignments were performed with ClustalW [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002238#pbio.1002238.ref040" target="_blank">40</a>]. Phylogenetic analysis was performed using the neighbor joining method and midpoint rooted. Alignments and phylogenetic analyses were carried out using MacVector Software (Accelrys). GenBank accession number for the sequences used are as follows: LGC-55 <i>Ce</i>, NM_075469; ACC-1 <i>Ce</i>, NM_069314; ACC-3 <i>Ce</i>, NM_076409; LGC-53 <i>Ce</i>, NM_171813; MOD-1 <i>Ce</i>, N_741580; UNC-49 <i>Ce</i>, NM_001027610; EXP-1 <i>Ce</i>, NP_495229; LGC-35 <i>Ce</i>, NM_001027268; ACR-16 <i>Ce</i>, NM_001028676; UNC-29 <i>Ce</i>, NM_09998; UNC-38 <i>Ce</i>, NM_059071; GABAα <i>Ls</i>, X58638; GABAζ <i>Ls</i>, X71357; AchF <i>Ls</i>, DQ167349; AchI <i>Ls</i>, DQ167352; AchB <i>Ls</i>, DQ167345; AchK <i>Ls</i>, DQ167353; AchH <i>Ls</i>, DQ167351; AchG <i>Ls</i>, DQ167350; AchD <i>Ls</i>, DQ167347; AchA <i>Ls</i>, DQ167344; AchJ <i>Ls</i>, DQ167348; AchC <i>Ls</i>, DQ167344; AchE <i>Ls</i>, DQ167348; GABAθ <i>Hs</i>, NP_061028; GABAγ <i>Hs</i>, NP_775807; GABAα1 <i>Hs</i>, <i>NP_000797</i>; GABAρ <i>Hs</i>, NP_002033; GABAβ <i>Hs</i>, NP_000803; ACHα9 <i>Hs</i>, NP_060051; ACHα7 <i>Hs</i>, P36544; ACHδ <i>Hs</i>, NP_000742; ACHβ <i>Hs</i>, NP_000738; 5HT3A <i>Hs</i>, AAH04453; 5HT3B <i>Hs</i>, AAH46990.</p

Model of the neural circuit for tyraminergic signaling in the neural escape response circuit that controls the coordination of head movements and locomotion in response to gentle anterior touch.

<p>Tyramine release from the RIM (blue) activates LGC-55 anion channel, which is expressed in the neck muscles, RMD/SMD motor neurons, and the AVB forward premotor interneuron (purple). Hyperpolarization of the neck muscles and RMD/SMD motor neurons induces neck relaxation and the suppression of head movements; hyperpolarization of the AVB forward premotor interneuron promotes backward locomotion. Tyramine signaling is induced through activation of the anterior touch sensory neurons (ALM/AVM), which activate premotor interneurons (AVD/AVA) that drive backward locomotion and are electrically coupled to the RIM (AVA-RIM). Sensory neurons are shown as triangles, premotor interneurons required for locomotion as hexagons, motor neurons as circles, and muscles as an oval.</p