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Electrical Coupling in Caenorhabditis elegans Mechanosensory Circuits
CHAPTER 1
Electrical Coupling in Caenorhabditis elegans Mechanosensory Circuits I. Rabinowitch1, W.R. Schafer2 1
Fred Hutchinson Cancer Research Center, Seattle, WA, United States; 2MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
1. INTRODUCTION The nematode Caenorhabditis elegans is in many ways an ideal organism to investigate microcircuits and their roles in behavior. It is currently the only organism with a complete physical connectome; each of its 302 neurons has been individually identified and its synaptic and gap junctional connections mapped at the level of electron microscopy. It is also highly accessible to genetic manipulation, with a sequenced genome, a short generation time, and amenability to transgenesis and gene replacement. Moreover, its transparency and compactness have made it well suited for optogenetic manipulation and recording of neural activity in behaving animals. Together, these tools make it possible to dissect how the interactions between defined neurons generate the functional properties of microcircuits, and how those properties relate to whole animal behavior. Gap junctions form an important component of the C. elegans connectome. The published C. elegans “wiring diagram” includes approximately 900 gap junctions along with 8000 chemical synapses (White et al., 1986). Analyses using modern machine vision methods (Xu et al., 2013) suggest this is an underestimate, with over 4000 gap junctions reported in data published online (wormwiring.org). Like other invertebrates, C. elegans gap junctions are formed from innexins rather than connexins (Altun et al., 2009; Simonsen et al., 2014). The C. elegans genome contains 25 innexin genes, 20 of which are neuronally expressed (Altun et al., 2009). These show varying patterns of expression, some expressed widely and others in only a few neurons. A few have been shown to have behavioral phenotypes; for example, loss-of-function mutations in unc-7 and unc-9, which are expressed in motorneurons and premotor interneurons, result in strongly uncoordinated movement (Kawano et al., 2011). The pattern of gap junction connections in the worm nervous system has been analyzed with the goal of identifying motifs of potential functional importance. In particular, the frequencies of all possible three-neuron and four-neuron connectivity patterns have been determined and compared with their expected frequencies in a random network (Varshney et al., 2011). Overrepresented patterns, such as a triangular connection of three neurons, might represent microcircuit elements with a conserved function in computation. One Network Functions and Plasticity ISBN 978-0-12-803471-2, http://dx.doi.org/10.1016/B978-0-12-803471-2.00001-1
© 2017 Elsevier Inc. All rights reserved.
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overrepresented motif observed in the four-neuron analysis is the hub and spoke, in which a single hub neuron is connected to each of the other three neurons (“spokes”). Another overrepresented four-neuron motif is the “diamond” motif whereby all neuron pairs except for one are connected by gap junctions (Varshney et al., 2011). The hub-and-spoke architecture, whereby multiple neurons are connected to one central neuron, is a recurring circuit motif in the C. elegans connectome (Varshney et al., 2011); indeed, larger hub-andspoke circuits, with a single hub receiving gap junctions from a large number of “spoke” inputs, are not uncommon. For example, the hub-and-spoke circuit in which many sensory neurons of varying modalities are connected by gap junctions to interneurons called RMG has been shown to control aggregation behavior, and to modulate responses to nematode pheromones (Macosko et al., 2009; Jang et al., 2012). We have undertaken an analysis of a simpler hub-and-spoke network involved in nose touch (Chatzigeorgiou and Schafer, 2011; Rabinowitch et al., 2013). This circuit involves a small number of input neurons of a single (mechanosensory) modality, and the behavioral output, an escape response called a reversal, is robust and easily correlated with the activity of the hub neuron. From these studies, we have aimed to uncover general principles of how hub-and-spoke circuits process information and control behavior.
2. THE NOSE TOUCH CIRCUIT The natural habitat of C. elegans consists of soil and rotting fruit. With no sense of vision, it relies heavily on a range of mechanosensory cues to navigate, locate food, interact with conspecifics, and avoid threats. Such complex interaction with the environment presents several challenges to the worm’s mechanosensory system. It must be able to discriminate between different textures and patterns, distinguishing, for example, between food (bacteria), soil particles, a mating partner, and a predator. In addition, its dynamic range must be extensive enough to detect both the gentlest and harshest mechanical inputs. Gap junctions might be useful building blocks for neural circuits that implement these features. We demonstrate this in the nose touch circuit, one of several neural circuits involved in mechanosensation in C. elegans. Other circuits include the polymodal nociceptive circuit involving the ASH neurons (Kaplan and Horvitz, 1993; Hart et al., 1995), the gentle body touch circuit (Chalfie et al., 1985), and the harsh body touch circuit (Way and Chalfie, 1989). The nose touch circuit is important for the transduction and processing of mechanosensory information sensed by the nose, often the first body part to come into contact with the changing texture that the worm encounters as it navigates through its surroundings. The circuit comprises several classes of mechanosensory neurons. The neurons in each class share a distinct morphology, are equipped with specific mechanoreceptors, and are linked to separate downstream circuits (Fig. 1.1): Four CEP neurons extend their dendrites to the tip of the nose and require the transient receptor potential N
Caenorhabditis elegans Mechanosensory Circuits
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Figure 1.1 The C. elegans nose touch circuit. The C. elegans nose touch circuit consists of the CEP, OLQ, and FLP mechanosensory neurons (rectangles), each expressing specific mechanoreceptors (indicated by different colors), each contributing to a specific circuit (indicated by downward pointing arrows), and all connected by gap junctions to a single interneuron, RIH (oval), whose output contributes to the navigation circuit.
channel TRP-4 for mechanosensory transduction (Li et al., 2006; Kindt et al., 2007a; Kang et al., 2010). These neurons are dopaminergic and are involved in modifying locomotion on physical contact with food (Sawin et al., 2000). Four OLQ neurons have similar morphology to the CEPs. However, they use different mechanoreceptors, the transient receptor potential V channel OSM-9 (Colbert et al., 1997; Chatzigeorgiou and Schafer, 2011), and the transient receptor potential A (TRPA) channel TRPA-1 (Kindt et al., 2007b). These neurons are involved in controlling foraging and head withdrawal. Two FLP neurons have multidendritic processes. This is a rare morphology for C. elegans neurons, most of which have a simple bipolar structure. They use the degenerin/epithelial-like sodium channel (DEG/ENaC) channel MEC-10 to detect both gentle and harsh mechanical contact with the nose (Huang and Chalfie, 1994; Chatzigeorgiou and Schafer, 2011). The FLP neurons form part of an escape mechanism responsive to noxious physical stimulation of the nose. What drew our attention to the nose touch circuit was the observation that FLP responses to gentle nose touch, as measured by calcium imaging, depended only partially on the cell-autonomous activity of the DEG/ENaC channel MEC-10, which is completely necessary for responses in FLP evoked by harsh mechanical contact with the nose, but on the other hand, nonecell autonomously, on functional OSM-9 in the OLQs (Chatzigeorgiou and Schafer, 2011). This suggested that the activity of other sensory neurons in the circuit facilitated FLP activation. The CEPs, OLQs, and FLPs are
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all connected via gap junctions to a single neuron RIH, an interneuron with a role in navigation (Fig. 1.1). We found that this facilitation is conveyed by the gap junctions connecting the OLQ neurons to RIH, and the FLP neurons to RIH. Loss of OSM-9 abolished the input from OLQ that contributed to part of the FLP response to gentle touch (Chatzigeorgiou and Schafer, 2011).
3. SIMPLIFIED MATHEMATICAL MODEL OF THE NOSE TOUCH CIRCUIT To better understand how the nose touch hub-and-spoke gap junction circuit works, we formulated a simplified mathematical model describing current flow between two input neurons and a hub interneuron (Fig. 1.2) (Rabinowitch et al., 2013). First we took into account the flow of current through the membrane of each individual neuron in the circuit. The neuron’s membrane is typically modeled as a resistor and capacitor operating in parallel (eg, hub neuron in Fig. 1.2). The resistor, which represents membrane conductance Gm, corresponds to the passive ion channels embedded in the cell’s membrane, through which current can leak into or out of the cell. Electrical and chemical gradients input 1
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Figure 1.2 Model of a simplified hub-and-spoke circuit. Each neuron is modeled by a capacitor in parallel to a driving force and resistor in series. In the sensory input neurons an additional driving force and resistor in series, representing the sensory receptor, are connected in parallel. Gap junctions between the input neurons and the hub are represented by resistors.
Caenorhabditis elegans Mechanosensory Circuits
formed across the membrane create a driving force, represented as a voltage source, EL, that drives current through these membrane channels. Accumulation of electrical charge on the membrane produces membrane capacitance, Cm, represented in the equivalent circuit as a capacitor. The activity of C. elegans neurons is characterized mostly by graded potentials rather than by spiking temporal patterns, which allowed us to ignore discrete brief electrical events at faster timescales. In addition to these passive components, the input neurons include in parallel a sensory receptor driving force, ER, and conductance, gR. We express this conductance in terms of membrane conductance as b ¼ gR/Gm (see input neurons in Fig. 1.2). The gap junctions connecting between the input and hub neurons were modeled as additional resistors with conductance gGJ. We define the gap junction coupling strength as a ¼ gGJ/Gm. A system of three differential equations fully captures the flow of current in or out of each neuron. We focused our analysis on the steady-state membrane potential of the model neurons. Further details about the analysis and the assumptions underlying the model can be found in Rabinowitch et al., 2013. The model serves as an effective tool for addressing questions and deriving experimental predictions about coincidence detection and gain control in gap junction circuits. In particular, we have investigated both theoretically and experimentally two possible mechanisms for nose touch detection: lateral facilitation and shunting inhibition.
4. LATERAL FACILITATION The activity of one input neuron might be enhanced by the activity of a second input neuron through lateral facilitation (Fig. 1.3A). Such lateral facilitation can be mediated by gap junctions by allowing current to flow from one input neuron to the other, via the hub. However, because current flows away from one neuron to facilitate the other, the activity in that first neuron should diminish, implying that lateral facilitation is asymmetric. To investigate what determines the direction and extent of lateral facilitation in the model, we compared the steady-state membrane potentials of input 1, either when both inputs 1 and 2 are activated (Fig. 1.3A), or when input 1 is activated but completely isolated from the circuit (Fig. 1.3B). A positive difference between the two conditions corresponds to a facilitation of input 1 by input 2. As an example, we have plotted the percent facilitation of input neuron 1 as a function of receptor strength for the case whereby the coupling strengths of both gap junctions are equal, a1 ¼ a2 ¼ 0.5 (Fig. 1.3C). Notably, neuron 1 can be facilitated by neuron 2 only when the strength of receptor 1 is considerably weaker than that of receptor 2. This stands to reason because in order for neuron 2 to facilitate neuron 1 the membrane potential of neuron 2 must be higher than that of neuron 1. However, it is not just the relative level of activity between the two neurons that matters, the gap junction coupling strengths also play an important role. In Fig. 1.3D, we have plotted the percent
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Figure 1.3 Lateral facilitation. (A) Hub-and-spoke circuit configuration, in which both input neurons are active. (B) Only input 1 is active and is isolated from the circuit. (C, D) Percentage difference between the steady-state voltage of input neuron 1 when connected to the circuit compared with when in isolation, as a function of transduction strength (C) or gap junction coupling strength (D). (E) Input neuron 1 (FLP) calcium response to gentle nose touch decreases (downward arrow in schematic diagram) when input neuron 1 is isolated from the hub-and-spoke circuit. (F) Input neuron 1 (FLP) calcium response to gentle nose touch increases (upward arrow in schematic diagram) when the electrical coupling between input neuron 2 (CEP) and the hub output neuron (RIH) is enhanced by synaptic engineering.
facilitation of input neuron 1 as a function of gap junction coupling in a circuit in which the receptor strength of neuron 1 is much weaker than that of neuron 2 (b1 ¼ 0.5 < b2 ¼ 5). The extent to which neuron 2 influenced the activity of neuron 1 directly depended on the coupling strength of input neuron 1 and the hub (Fig. 1.3D). Very weak coupling entailed almost no change in V1, because very little current could make its way to neuron 1. In contrast, stronger coupling resulted in either considerable facilitation or weakening of V1. The coupling strength between neuron 2 and the hub determined whether neuron 1 activity would be facilitated or weakened. Strong coupling of neuron 2 and the hub allows for current to flow from neuron 2 to the hub and then to neuron 1. Weak coupling between neuron 2 and the hub isolates neuron 2 and draws current away from neuron 1 into the hub, reducing the activity of neuron 1. As described earlier, we have found that the response of the FLP neurons to gentle touch is facilitated by the OLQ neurons (Fig. 1.3E) (Chatzigeorgiou and Schafer, 2011). Consistent with our model, this might be due to relatively weakened responsiveness of FLP to gentle touch compared with the OLQ response. Our model also predicts that strong coupling of both FLP and OLQ to the hub interneuron RIH is important for enabling the facilitation of FLP. Thus, according
Caenorhabditis elegans Mechanosensory Circuits
to our model, FLP responses might be even further facilitated if, for example, the gap junction coupling between another input neuron and the hub RIH were stronger (Fig. 1.3D). To test this prediction, we artificially increased the gap junction coupling strength between the CEP input neurons and RIH and measured the effect on FLP calcium responses. We did this by using a synaptic engineering approach (Rabinowitch et al., 2014; Rabinowitch and Schafer, 2015). We expressed the mouse gap junction protein Connexin36 in the CEP and RIH neurons, to form additional gap junctions between these neurons. Because vertebrate gap junction proteins, such as Connexin36, belong to a different family than invertebrate gap junction proteins, which are called innexins, we expected no nonspecific heterotypic gap junctions to form between the engineered Connexin36 and endogenous C. elegans innexins. We found that, indeed, as the model predicted, increased gap junction coupling between CEP neurons and RIH enhanced the FLP response to gentle touch to the nose (Fig. 1.3F) (Rabinowitch et al., 2013).
5. INHIBITION BY SHUNTING Lateral facilitation consists of one active neuron contributing to the activity of another active neuron. Another form of interaction in gap junction circuits may occur when one input neuron is active and another input neuron is not. This might happen following certain stimulation patterns that evoke a specific response only in neurons that are tuned to the particular features of the stimulus, whereas other neurons remain silent. We wished to examine using our model, the effect that the inactivity of input neuron 2 might have on input neuron 1 (Fig. 1.4A). To this end we considered the percent difference in the membrane potential of neuron 1, V1, when neuron 2 is inactive compared with when neuron 2 is removed from the circuit, ie, is ablated (Fig. 1.4A and B). We found that for all values of gap junction coupling strength, a1, a2, and receptor strength, b1, b2, the membrane potential of input neuron 1, V1, was always inhibited by the inactivity of input neuron 2; ie, the difference in V1 steady-state values between silencing input 2 and ablating it was always negative. This kind of electrical suppression of membrane potential is called shunting inhibition. We demonstrated shunting inhibition experimentally, taking advantage of the fact that each neuron class in the nose touch circuit relies on different receptors for transduction (Fig. 1.1), and is therefore selectively silent in mutants lacking the relevant functional receptor. Thus, in worms with dysfunctional OSM-9 receptors, and thus silent OLQ neurons, FLP and RIH calcium responses to nose touch were diminished, unless the OLQ neurons were ablated. Similarly, in trp-4 mutants with silent CEPs, FLP and RIH responses were reduced unless the CEPs were ablated, and in worms with defective MEC-10, and thus silent FLPs, CEP and RIH responses were attenuated, but normal if the FLPs were ablated (Rabinowitch et al., 2013).
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Figure 1.4 Inhibition by shunting. (A) Hub-and-spoke circuit configuration, in which input neuron 1 is active and input neuron 2 is silent. (B) Circuit configuration, in which input neuron 1 is active and input neuron 2 is ablated. (C, D) Percentage difference between the steady-state voltage of input neuron 1 (C), or the hub output neuron (D) when one input neuron is active and the other is silent compared with when one input neuron is active and the other is ablated, as a function of gap junction coupling strength. (E) Input neuron 1 (FLP) calcium response to gentle nose touch decreases (downward arrow in schematic diagram) when the coupling between input neuron 2 (CEP) and the hub output neuron (RIH) is enhanced by synaptic engineering. (F) Input neuron 1 (CEP) calcium response to gentle nose touch decreases (downward arrow in schematic diagram) when the electrical coupling between input neuron 1 and the hub output neuron (RIH) is enhanced by synaptic engineering.
We further found from the model that the degree of shunting inhibition depended strongly on gap junction coupling strength. As can be seen in the example in Fig. 1.4C and D, stronger coupling to the hub of input neuron 2 produced more shunting inhibition of V1 as well as the hub membrane potential, V0, because more current could get drawn away from neuron 1 through the hub into neuron 2. In contrast, stronger gap junction coupling between input neuron 1 and the hub also increased the shunting inhibition of neuron 1, whereas it reduced the inhibition of the hub. The reason is that more current drawn out of input neuron 1 could remain in the hub rather than flow into input neuron 2 (Fig. 1.4D). We used synaptic engineering once more to test these predictions. As earlier, we examined worms with a gap junction inserted between CEP and the hub, RIH. First, we considered CEP as input neuron 2, and used trp-4 mutants to simulate silencing of the CEPs. In this case it was a2, the coupling strength between neuron 2 and the hub that was increased by the engineered gap junction (Fig. 1.4E). As predicted by the model, strengthening a2 resulted in decreased calcium responses to nose touch in both FLP (input neuron 1 in this case) and RIH (the hub). Second, we switched roles between CEP and FLP. Now we used mec-10 mutants to silence
Caenorhabditis elegans Mechanosensory Circuits
FLP (input neuron 2). Consistent with the model, strengthening a1 this time, led to reduced calcium responses in CEP (input neuron 1), but to increased responses in RIH (the hub) (Fig. 1.4F).
6. CONCLUSIONS AND FUTURE PERSPECTIVES These studies illustrate how the function of a simple microcircuit can rely on the unique properties of electrical synapses. Electrical coupling between sensory neurons, either directly or indirectly through a hub interneuron, can enhance coincident responses through lateral facilitation. Conversely, in the absence of coincident activity, current can be shunted through hub neurons to inactive inputs, which act as current sinks and inhibit overall network activity. Together, lateral facilitation and inhibition by shunting allow the hub-and-spoke gap junction circuit to function as an analog coincidence detector, with inputs summing nonlinearly in the network output. Synthetic increases in gap junction coupling to the hub can increase both facilitation and inhibition, making network output depend more strongly on coincidence. Although this work concerns a specific hub and spoke circuit in the worm, the computational properties of the circuit follow straightforwardly from its architecture; thus, it seems reasonable to imagine hub-and-spoke circuits might perform similar functions in more complex brains. The use of ectopic connexin expression to create new electrical synapses has been useful in the analysis of electrically coupled circuits in C. elegans, and it may have broader applications as well (Rabinowitch et al., 2014, 2016). Other invertebrates, such as Drosophila, also lack endogenous connexin genes, so targeted Cx36 expression should allow the creation of synthetic electrical synapses in other genetically tractable invertebrate model organisms. Moreover, the vertebrate counterparts of innexins, the pannexins, are thought to form only hemichannels and are therefore unlikely to form gap junctions with a nematode innexin partner. Thus, the converse approach of ectopically expressing worm innexins in vertebrate neurons may allow the insertion of ectopic electrical synapses in animals such as the mouse. Connexin and innexin transgenesis may not only prove to be useful tools for the analysis of neural circuit mechanisms, but also allow for neural repair or even the engineering of new functions in transformed animals. This could represent a first step toward synthetic neurobiology.
OUTSTANDING QUESTIONS/FUTURE DIRECTIONS • • • •
Do other hub-and-spoke circuits in C. elegans perform coincidence detection? Do hub-and-spoke circuits in other animals perform coincidence detection, and how does the existence of action potentials affect their function? What kind of functions do other gap junction circuit motifs in C. elegans perform? Is synaptic engineering effective in model organisms other than C. elegans?
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REFERENCES Altun, Z.F., Chen, B., Wang, Z.-W., Hall, D.H., 2009. High resolution map of Caenorhabditis elegans gap junction proteins. Dev. Dyn. 238, 1936e1950. Chalfie, M., Sulston, J.E., White, J.G., Southgate, E., Thomson, J.N., Brenner, S., 1985. The neural circuit for touch sensitivity in Caenorhabditis elegans. J. Neurosci. 5, 956e964. Chatzigeorgiou, M., Schafer, W.R., 2011. Lateral facilitation between primary mechanosensory neurons controls nose touch perception in C. elegans. Neuron 70, 299e309. Colbert, H.A., Smith, T.L., Bargmann, C.I., 1997. OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J. Neurosci. 17, 8259e8269. Hart, A.C., Sims, S., Kaplan, J.M., 1995. Synaptic code for sensory modalities revealed by C. elegans GLR-1 glutamate receptor. Nature 378, 82e85. Huang, M., Chalfie, M., 1994. Gene interactions affecting mechanosensory transduction in Caenorhabditis elegans. Nature 367, 467e470. Jang, H., Kim, K., Neal, S.J., Macosko, E., Kim, D., Butcher, R.A., Zeiger, D.M., Bargmann, C.I., Sengupta, P., 2012. Neuromodulatory state and sex specify alternative behaviors through antagonistic synaptic pathways in C. elegans. Neuron 75, 585e592. Kang, L., Gao, J., Schafer, W.R., Xie, Z., Xu, X.Z.S., 2010. C. elegans trp family protein TRP-4 is a poreforming subunit of a native mechanotransduction channel. Neuron 67, 381e391. Kaplan, J.M., Horvitz, H.R., 1993. A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 90, 2227e2231. Kawano, T., Po, M.D., Gao, S., Leung, G., Ryu, W.S., Zhen, M., 2011. An imbalancing act: gap junctions reduce the backward motor circuit activity to bias C. elegans for forward locomotion. Neuron 72, 572e586. Kindt, K.S., Quast, K.B., Giles, A.C., De, S., Hendrey, D., Nicastro, I., Rankin, C.H., Schafer, W.R., 2007a. Dopamine mediates context-dependent modulation of sensory plasticity in C. elegans. Neuron 55, 662e676. Kindt, K.S., Viswanath, V., Macpherson, L., Quast, K., Hu, H., Patapoutian, A., Schafer, W.R., 2007b. Caenorhabditis elegans TRPA-1 functions in mechanosensation. Nat. Neurosci. 10, 568e577. Li, W., Feng, Z., Sternberg, P.W., Shawn Xu, X.Z., 2006. A C. elegans stretch receptor neuron revealed by a mechanosensitive TRP channel homologue. Nature 440, 684e687. Macosko, E.Z., Pokala, N., Feinberg, E.H., Chalasani, S.H., Butcher, R.A., Clardy, J., Bargmann, C.I., 2009. A hub-and-spoke circuit drives pheromone attraction and social behavior in C. elegans. Nature 458, 1171e1175. Rabinowitch, I., Schafer, W.R., 2015. Engineering new synaptic connections in the C. elegans connectome. Worm 4, e992668. Rabinowitch, I., Chatzigeorgiou, M., Schafer, W.R., 2013. A gap junction circuit enhances processing of coincident mechanosensory inputs. Curr. Biol. 23, 963e967. Rabinowitch, I., Chatzigeorgiou, M., Zhao, B., Treinin, M., Schafer, W.R., 2014. Rewiring neural circuits by the insertion of ectopic electrical synapses in transgenic C. elegans. Nat. Commun. 5, 4442. Rabinowitch, I., Laurent, P., Zhao, B., Walker, D., Beets, I., Schoofs, L., Bai, J., Schafer, W.R., Treinin, M., 2016. Neuropeptide-driven cross-modal plasticity following sensory loss in Caenorhabditis elegans. PLoS Biol. 14, e1002348. Sawin, E.R., Ranganathan, R., Horvitz, H.R., 2000. C. elegans locomotory rate is modulated by the environment through a dopaminergic pathway and by experience through a serotonergic pathway. Neuron 26, 619e631. Simonsen, K., Moerman, D., Naus, C.C., 2014. Gap junctions in C. elegans. Membr. Physiol. Membr. Biophys. 5, 40. Varshney, L.R., Chen, B.L., Paniagua, E., Hall, D.H., Chklovskii, D.B., 2011. Structural properties of the Caenorhabditis elegans neuronal network. PLoS Comput. Biol. 7, e1001066.
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Way, J.C., Chalfie, M., 1989. The mec-3 gene of Caenorhabditis elegans requires its own product for maintained expression and is expressed in three neuronal cell types. Genes Dev. 3, 1823e1833. White, J.G., Southgate, E., Thomson, J.N., Brenner, S., 1986. The structure of the nervous system of the nematode Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 314, 1e340. Xu, M., Jarrell, T.A., Wang, Y., Cook, S.J., Hall, D.H., Emmons, S.W., 2013. Computer assisted assembly of connectomes from electron micrographs: application to Caenorhabditis elegans. PLoS One 8, e54050.
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Electrical Coupling in Caenorhabditis elegans Mechanosensory Circuits I. Rabinowitch1, W.R. Schafer2 1
Fred Hutchinson Cancer Research Center, Seattle, WA, United States; 2MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
1. INTRODUCTION The nematode Caenorhabditis elegans is in many ways an ideal organism to investigate microcircuits and their roles in behavior. It is currently the only organism with a complete physical connectome; each of its 302 neurons has been individually identified and its synaptic and gap junctional connections mapped at the level of electron microscopy. It is also highly accessible to genetic manipulation, with a sequenced genome, a short generation time, and amenability to transgenesis and gene replacement. Moreover, its transparency and compactness have made it well suited for optogenetic manipulation and recording of neural activity in behaving animals. Together, these tools make it possible to dissect how the interactions between defined neurons generate the functional properties of microcircuits, and how those properties relate to whole animal behavior. Gap junctions form an important component of the C. elegans connectome. The published C. elegans “wiring diagram” includes approximately 900 gap junctions along with 8000 chemical synapses (White et al., 1986). Analyses using modern machine vision methods (Xu et al., 2013) suggest this is an underestimate, with over 4000 gap junctions reported in data published online (wormwiring.org). Like other invertebrates, C. elegans gap junctions are formed from innexins rather than connexins (Altun et al., 2009; Simonsen et al., 2014). The C. elegans genome contains 25 innexin genes, 20 of which are neuronally expressed (Altun et al., 2009). These show varying patterns of expression, some expressed widely and others in only a few neurons. A few have been shown to have behavioral phenotypes; for example, loss-of-function mutations in unc-7 and unc-9, which are expressed in motorneurons and premotor interneurons, result in strongly uncoordinated movement (Kawano et al., 2011). The pattern of gap junction connections in the worm nervous system has been analyzed with the goal of identifying motifs of potential functional importance. In particular, the frequencies of all possible three-neuron and four-neuron connectivity patterns have been determined and compared with their expected frequencies in a random network (Varshney et al., 2011). Overrepresented patterns, such as a triangular connection of three neurons, might represent microcircuit elements with a conserved function in computation. One Network Functions and Plasticity ISBN 978-0-12-803471-2, http://dx.doi.org/10.1016/B978-0-12-803471-2.00001-1
© 2017 Elsevier Inc. All rights reserved.
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overrepresented motif observed in the four-neuron analysis is the hub and spoke, in which a single hub neuron is connected to each of the other three neurons (“spokes”). Another overrepresented four-neuron motif is the “diamond” motif whereby all neuron pairs except for one are connected by gap junctions (Varshney et al., 2011). The hub-and-spoke architecture, whereby multiple neurons are connected to one central neuron, is a recurring circuit motif in the C. elegans connectome (Varshney et al., 2011); indeed, larger hub-andspoke circuits, with a single hub receiving gap junctions from a large number of “spoke” inputs, are not uncommon. For example, the hub-and-spoke circuit in which many sensory neurons of varying modalities are connected by gap junctions to interneurons called RMG has been shown to control aggregation behavior, and to modulate responses to nematode pheromones (Macosko et al., 2009; Jang et al., 2012). We have undertaken an analysis of a simpler hub-and-spoke network involved in nose touch (Chatzigeorgiou and Schafer, 2011; Rabinowitch et al., 2013). This circuit involves a small number of input neurons of a single (mechanosensory) modality, and the behavioral output, an escape response called a reversal, is robust and easily correlated with the activity of the hub neuron. From these studies, we have aimed to uncover general principles of how hub-and-spoke circuits process information and control behavior.
2. THE NOSE TOUCH CIRCUIT The natural habitat of C. elegans consists of soil and rotting fruit. With no sense of vision, it relies heavily on a range of mechanosensory cues to navigate, locate food, interact with conspecifics, and avoid threats. Such complex interaction with the environment presents several challenges to the worm’s mechanosensory system. It must be able to discriminate between different textures and patterns, distinguishing, for example, between food (bacteria), soil particles, a mating partner, and a predator. In addition, its dynamic range must be extensive enough to detect both the gentlest and harshest mechanical inputs. Gap junctions might be useful building blocks for neural circuits that implement these features. We demonstrate this in the nose touch circuit, one of several neural circuits involved in mechanosensation in C. elegans. Other circuits include the polymodal nociceptive circuit involving the ASH neurons (Kaplan and Horvitz, 1993; Hart et al., 1995), the gentle body touch circuit (Chalfie et al., 1985), and the harsh body touch circuit (Way and Chalfie, 1989). The nose touch circuit is important for the transduction and processing of mechanosensory information sensed by the nose, often the first body part to come into contact with the changing texture that the worm encounters as it navigates through its surroundings. The circuit comprises several classes of mechanosensory neurons. The neurons in each class share a distinct morphology, are equipped with specific mechanoreceptors, and are linked to separate downstream circuits (Fig. 1.1): Four CEP neurons extend their dendrites to the tip of the nose and require the transient receptor potential N
Caenorhabditis elegans Mechanosensory Circuits
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Figure 1.1 The C. elegans nose touch circuit. The C. elegans nose touch circuit consists of the CEP, OLQ, and FLP mechanosensory neurons (rectangles), each expressing specific mechanoreceptors (indicated by different colors), each contributing to a specific circuit (indicated by downward pointing arrows), and all connected by gap junctions to a single interneuron, RIH (oval), whose output contributes to the navigation circuit.
channel TRP-4 for mechanosensory transduction (Li et al., 2006; Kindt et al., 2007a; Kang et al., 2010). These neurons are dopaminergic and are involved in modifying locomotion on physical contact with food (Sawin et al., 2000). Four OLQ neurons have similar morphology to the CEPs. However, they use different mechanoreceptors, the transient receptor potential V channel OSM-9 (Colbert et al., 1997; Chatzigeorgiou and Schafer, 2011), and the transient receptor potential A (TRPA) channel TRPA-1 (Kindt et al., 2007b). These neurons are involved in controlling foraging and head withdrawal. Two FLP neurons have multidendritic processes. This is a rare morphology for C. elegans neurons, most of which have a simple bipolar structure. They use the degenerin/epithelial-like sodium channel (DEG/ENaC) channel MEC-10 to detect both gentle and harsh mechanical contact with the nose (Huang and Chalfie, 1994; Chatzigeorgiou and Schafer, 2011). The FLP neurons form part of an escape mechanism responsive to noxious physical stimulation of the nose. What drew our attention to the nose touch circuit was the observation that FLP responses to gentle nose touch, as measured by calcium imaging, depended only partially on the cell-autonomous activity of the DEG/ENaC channel MEC-10, which is completely necessary for responses in FLP evoked by harsh mechanical contact with the nose, but on the other hand, nonecell autonomously, on functional OSM-9 in the OLQs (Chatzigeorgiou and Schafer, 2011). This suggested that the activity of other sensory neurons in the circuit facilitated FLP activation. The CEPs, OLQs, and FLPs are
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all connected via gap junctions to a single neuron RIH, an interneuron with a role in navigation (Fig. 1.1). We found that this facilitation is conveyed by the gap junctions connecting the OLQ neurons to RIH, and the FLP neurons to RIH. Loss of OSM-9 abolished the input from OLQ that contributed to part of the FLP response to gentle touch (Chatzigeorgiou and Schafer, 2011).
3. SIMPLIFIED MATHEMATICAL MODEL OF THE NOSE TOUCH CIRCUIT To better understand how the nose touch hub-and-spoke gap junction circuit works, we formulated a simplified mathematical model describing current flow between two input neurons and a hub interneuron (Fig. 1.2) (Rabinowitch et al., 2013). First we took into account the flow of current through the membrane of each individual neuron in the circuit. The neuron’s membrane is typically modeled as a resistor and capacitor operating in parallel (eg, hub neuron in Fig. 1.2). The resistor, which represents membrane conductance Gm, corresponds to the passive ion channels embedded in the cell’s membrane, through which current can leak into or out of the cell. Electrical and chemical gradients input 1
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Figure 1.2 Model of a simplified hub-and-spoke circuit. Each neuron is modeled by a capacitor in parallel to a driving force and resistor in series. In the sensory input neurons an additional driving force and resistor in series, representing the sensory receptor, are connected in parallel. Gap junctions between the input neurons and the hub are represented by resistors.
Caenorhabditis elegans Mechanosensory Circuits
formed across the membrane create a driving force, represented as a voltage source, EL, that drives current through these membrane channels. Accumulation of electrical charge on the membrane produces membrane capacitance, Cm, represented in the equivalent circuit as a capacitor. The activity of C. elegans neurons is characterized mostly by graded potentials rather than by spiking temporal patterns, which allowed us to ignore discrete brief electrical events at faster timescales. In addition to these passive components, the input neurons include in parallel a sensory receptor driving force, ER, and conductance, gR. We express this conductance in terms of membrane conductance as b ¼ gR/Gm (see input neurons in Fig. 1.2). The gap junctions connecting between the input and hub neurons were modeled as additional resistors with conductance gGJ. We define the gap junction coupling strength as a ¼ gGJ/Gm. A system of three differential equations fully captures the flow of current in or out of each neuron. We focused our analysis on the steady-state membrane potential of the model neurons. Further details about the analysis and the assumptions underlying the model can be found in Rabinowitch et al., 2013. The model serves as an effective tool for addressing questions and deriving experimental predictions about coincidence detection and gain control in gap junction circuits. In particular, we have investigated both theoretically and experimentally two possible mechanisms for nose touch detection: lateral facilitation and shunting inhibition.
4. LATERAL FACILITATION The activity of one input neuron might be enhanced by the activity of a second input neuron through lateral facilitation (Fig. 1.3A). Such lateral facilitation can be mediated by gap junctions by allowing current to flow from one input neuron to the other, via the hub. However, because current flows away from one neuron to facilitate the other, the activity in that first neuron should diminish, implying that lateral facilitation is asymmetric. To investigate what determines the direction and extent of lateral facilitation in the model, we compared the steady-state membrane potentials of input 1, either when both inputs 1 and 2 are activated (Fig. 1.3A), or when input 1 is activated but completely isolated from the circuit (Fig. 1.3B). A positive difference between the two conditions corresponds to a facilitation of input 1 by input 2. As an example, we have plotted the percent facilitation of input neuron 1 as a function of receptor strength for the case whereby the coupling strengths of both gap junctions are equal, a1 ¼ a2 ¼ 0.5 (Fig. 1.3C). Notably, neuron 1 can be facilitated by neuron 2 only when the strength of receptor 1 is considerably weaker than that of receptor 2. This stands to reason because in order for neuron 2 to facilitate neuron 1 the membrane potential of neuron 2 must be higher than that of neuron 1. However, it is not just the relative level of activity between the two neurons that matters, the gap junction coupling strengths also play an important role. In Fig. 1.3D, we have plotted the percent
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Figure 1.3 Lateral facilitation. (A) Hub-and-spoke circuit configuration, in which both input neurons are active. (B) Only input 1 is active and is isolated from the circuit. (C, D) Percentage difference between the steady-state voltage of input neuron 1 when connected to the circuit compared with when in isolation, as a function of transduction strength (C) or gap junction coupling strength (D). (E) Input neuron 1 (FLP) calcium response to gentle nose touch decreases (downward arrow in schematic diagram) when input neuron 1 is isolated from the hub-and-spoke circuit. (F) Input neuron 1 (FLP) calcium response to gentle nose touch increases (upward arrow in schematic diagram) when the electrical coupling between input neuron 2 (CEP) and the hub output neuron (RIH) is enhanced by synaptic engineering.
facilitation of input neuron 1 as a function of gap junction coupling in a circuit in which the receptor strength of neuron 1 is much weaker than that of neuron 2 (b1 ¼ 0.5 < b2 ¼ 5). The extent to which neuron 2 influenced the activity of neuron 1 directly depended on the coupling strength of input neuron 1 and the hub (Fig. 1.3D). Very weak coupling entailed almost no change in V1, because very little current could make its way to neuron 1. In contrast, stronger coupling resulted in either considerable facilitation or weakening of V1. The coupling strength between neuron 2 and the hub determined whether neuron 1 activity would be facilitated or weakened. Strong coupling of neuron 2 and the hub allows for current to flow from neuron 2 to the hub and then to neuron 1. Weak coupling between neuron 2 and the hub isolates neuron 2 and draws current away from neuron 1 into the hub, reducing the activity of neuron 1. As described earlier, we have found that the response of the FLP neurons to gentle touch is facilitated by the OLQ neurons (Fig. 1.3E) (Chatzigeorgiou and Schafer, 2011). Consistent with our model, this might be due to relatively weakened responsiveness of FLP to gentle touch compared with the OLQ response. Our model also predicts that strong coupling of both FLP and OLQ to the hub interneuron RIH is important for enabling the facilitation of FLP. Thus, according
Caenorhabditis elegans Mechanosensory Circuits
to our model, FLP responses might be even further facilitated if, for example, the gap junction coupling between another input neuron and the hub RIH were stronger (Fig. 1.3D). To test this prediction, we artificially increased the gap junction coupling strength between the CEP input neurons and RIH and measured the effect on FLP calcium responses. We did this by using a synaptic engineering approach (Rabinowitch et al., 2014; Rabinowitch and Schafer, 2015). We expressed the mouse gap junction protein Connexin36 in the CEP and RIH neurons, to form additional gap junctions between these neurons. Because vertebrate gap junction proteins, such as Connexin36, belong to a different family than invertebrate gap junction proteins, which are called innexins, we expected no nonspecific heterotypic gap junctions to form between the engineered Connexin36 and endogenous C. elegans innexins. We found that, indeed, as the model predicted, increased gap junction coupling between CEP neurons and RIH enhanced the FLP response to gentle touch to the nose (Fig. 1.3F) (Rabinowitch et al., 2013).
5. INHIBITION BY SHUNTING Lateral facilitation consists of one active neuron contributing to the activity of another active neuron. Another form of interaction in gap junction circuits may occur when one input neuron is active and another input neuron is not. This might happen following certain stimulation patterns that evoke a specific response only in neurons that are tuned to the particular features of the stimulus, whereas other neurons remain silent. We wished to examine using our model, the effect that the inactivity of input neuron 2 might have on input neuron 1 (Fig. 1.4A). To this end we considered the percent difference in the membrane potential of neuron 1, V1, when neuron 2 is inactive compared with when neuron 2 is removed from the circuit, ie, is ablated (Fig. 1.4A and B). We found that for all values of gap junction coupling strength, a1, a2, and receptor strength, b1, b2, the membrane potential of input neuron 1, V1, was always inhibited by the inactivity of input neuron 2; ie, the difference in V1 steady-state values between silencing input 2 and ablating it was always negative. This kind of electrical suppression of membrane potential is called shunting inhibition. We demonstrated shunting inhibition experimentally, taking advantage of the fact that each neuron class in the nose touch circuit relies on different receptors for transduction (Fig. 1.1), and is therefore selectively silent in mutants lacking the relevant functional receptor. Thus, in worms with dysfunctional OSM-9 receptors, and thus silent OLQ neurons, FLP and RIH calcium responses to nose touch were diminished, unless the OLQ neurons were ablated. Similarly, in trp-4 mutants with silent CEPs, FLP and RIH responses were reduced unless the CEPs were ablated, and in worms with defective MEC-10, and thus silent FLPs, CEP and RIH responses were attenuated, but normal if the FLPs were ablated (Rabinowitch et al., 2013).
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Figure 1.4 Inhibition by shunting. (A) Hub-and-spoke circuit configuration, in which input neuron 1 is active and input neuron 2 is silent. (B) Circuit configuration, in which input neuron 1 is active and input neuron 2 is ablated. (C, D) Percentage difference between the steady-state voltage of input neuron 1 (C), or the hub output neuron (D) when one input neuron is active and the other is silent compared with when one input neuron is active and the other is ablated, as a function of gap junction coupling strength. (E) Input neuron 1 (FLP) calcium response to gentle nose touch decreases (downward arrow in schematic diagram) when the coupling between input neuron 2 (CEP) and the hub output neuron (RIH) is enhanced by synaptic engineering. (F) Input neuron 1 (CEP) calcium response to gentle nose touch decreases (downward arrow in schematic diagram) when the electrical coupling between input neuron 1 and the hub output neuron (RIH) is enhanced by synaptic engineering.
We further found from the model that the degree of shunting inhibition depended strongly on gap junction coupling strength. As can be seen in the example in Fig. 1.4C and D, stronger coupling to the hub of input neuron 2 produced more shunting inhibition of V1 as well as the hub membrane potential, V0, because more current could get drawn away from neuron 1 through the hub into neuron 2. In contrast, stronger gap junction coupling between input neuron 1 and the hub also increased the shunting inhibition of neuron 1, whereas it reduced the inhibition of the hub. The reason is that more current drawn out of input neuron 1 could remain in the hub rather than flow into input neuron 2 (Fig. 1.4D). We used synaptic engineering once more to test these predictions. As earlier, we examined worms with a gap junction inserted between CEP and the hub, RIH. First, we considered CEP as input neuron 2, and used trp-4 mutants to simulate silencing of the CEPs. In this case it was a2, the coupling strength between neuron 2 and the hub that was increased by the engineered gap junction (Fig. 1.4E). As predicted by the model, strengthening a2 resulted in decreased calcium responses to nose touch in both FLP (input neuron 1 in this case) and RIH (the hub). Second, we switched roles between CEP and FLP. Now we used mec-10 mutants to silence
Caenorhabditis elegans Mechanosensory Circuits
FLP (input neuron 2). Consistent with the model, strengthening a1 this time, led to reduced calcium responses in CEP (input neuron 1), but to increased responses in RIH (the hub) (Fig. 1.4F).
6. CONCLUSIONS AND FUTURE PERSPECTIVES These studies illustrate how the function of a simple microcircuit can rely on the unique properties of electrical synapses. Electrical coupling between sensory neurons, either directly or indirectly through a hub interneuron, can enhance coincident responses through lateral facilitation. Conversely, in the absence of coincident activity, current can be shunted through hub neurons to inactive inputs, which act as current sinks and inhibit overall network activity. Together, lateral facilitation and inhibition by shunting allow the hub-and-spoke gap junction circuit to function as an analog coincidence detector, with inputs summing nonlinearly in the network output. Synthetic increases in gap junction coupling to the hub can increase both facilitation and inhibition, making network output depend more strongly on coincidence. Although this work concerns a specific hub and spoke circuit in the worm, the computational properties of the circuit follow straightforwardly from its architecture; thus, it seems reasonable to imagine hub-and-spoke circuits might perform similar functions in more complex brains. The use of ectopic connexin expression to create new electrical synapses has been useful in the analysis of electrically coupled circuits in C. elegans, and it may have broader applications as well (Rabinowitch et al., 2014, 2016). Other invertebrates, such as Drosophila, also lack endogenous connexin genes, so targeted Cx36 expression should allow the creation of synthetic electrical synapses in other genetically tractable invertebrate model organisms. Moreover, the vertebrate counterparts of innexins, the pannexins, are thought to form only hemichannels and are therefore unlikely to form gap junctions with a nematode innexin partner. Thus, the converse approach of ectopically expressing worm innexins in vertebrate neurons may allow the insertion of ectopic electrical synapses in animals such as the mouse. Connexin and innexin transgenesis may not only prove to be useful tools for the analysis of neural circuit mechanisms, but also allow for neural repair or even the engineering of new functions in transformed animals. This could represent a first step toward synthetic neurobiology.
OUTSTANDING QUESTIONS/FUTURE DIRECTIONS • • • •
Do other hub-and-spoke circuits in C. elegans perform coincidence detection? Do hub-and-spoke circuits in other animals perform coincidence detection, and how does the existence of action potentials affect their function? What kind of functions do other gap junction circuit motifs in C. elegans perform? Is synaptic engineering effective in model organisms other than C. elegans?
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Way, J.C., Chalfie, M., 1989. The mec-3 gene of Caenorhabditis elegans requires its own product for maintained expression and is expressed in three neuronal cell types. Genes Dev. 3, 1823e1833. White, J.G., Southgate, E., Thomson, J.N., Brenner, S., 1986. The structure of the nervous system of the nematode Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 314, 1e340. Xu, M., Jarrell, T.A., Wang, Y., Cook, S.J., Hall, D.H., Emmons, S.W., 2013. Computer assisted assembly of connectomes from electron micrographs: application to Caenorhabditis elegans. PLoS One 8, e54050.
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