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Rewiring neural circuits by the insertion of ectopic electrical synapses in transgenic C. elegans.

Rabinowitch I, Chatzigeorgiou M, Zhao B, Treinin M, Schafer WR - Nat Commun (2014)

Bottom Line: We added electrical synapses composed of the vertebrate gap junction protein Cx36 between Caenorhabditis elegans chemosensory neurons with opposite intrinsic responses to salt.In a second example, introducing Cx36 into an inhibitory chemical synapse between an olfactory receptor neuron and an interneuron changed the sign of the connection from negative to positive, and abolished the animal's behavioural response to benzaldehyde.These data demonstrate a synthetic strategy to rewire behavioural circuits by engineering synaptic connectivity in C. elegans.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Cell Biology, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK [2] Department of Medical Neurobiology, Hadassah Medical School, Hebrew University of Jerusalem, Jerusalem 9112102, Israel [3].

ABSTRACT
Neural circuits are functional ensembles of neurons that are selectively interconnected by chemical or electrical synapses. Here we describe a synthetic biology approach to the study of neural circuits, whereby new electrical synapses can be introduced in novel sites in the neuronal circuitry to reprogram behaviour. We added electrical synapses composed of the vertebrate gap junction protein Cx36 between Caenorhabditis elegans chemosensory neurons with opposite intrinsic responses to salt. Connecting these neurons by an ectopic electrical synapse led to a loss of lateral asymmetry and altered chemotaxis behaviour. In a second example, introducing Cx36 into an inhibitory chemical synapse between an olfactory receptor neuron and an interneuron changed the sign of the connection from negative to positive, and abolished the animal's behavioural response to benzaldehyde. These data demonstrate a synthetic strategy to rewire behavioural circuits by engineering synaptic connectivity in C. elegans.

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An engineered electrical synaptic connection between AWC and AIY flips the AIY benzaldehyde response profile.(a) AWC and AIY participate in a circuit for chemosensation and are naturally connected by an inhibitory chemical synapse. (b) Confocal projection image of strain AQ2660, in which AWC (green) expresses the calcium indicator YC3.60, and mCherry-tagged Cx36 puncta (red) are seen in the cell body (yellow arrow), dendrite (blue arrow) and axon (white arrows). Scale bar, 5 μm. (c) Confocal image of strain transgenic line AQ2646, in which mCherry-tagged Cx36 is expressed in AIY and AWC (red punctate fluorescence), and YC3.60 is expressed in AIY (green fluorescence). Yellow and white arrows indicate the cell body and axon respectively. Scale bar, 10 μm. An expanded version of this image and version showing the red channel alone are in Supplementary Fig. 3. (d,e) Calcium imaging of AWC and AIY responses to an upstep (d) or downstep (e) in benzaldehyde (Bz) concentration in wild-type worms and in worms expressing Cx36 in both AWC and AIY. (f–i) Calcium imaging of AWC (f,h) or AIY (g,i) responses to an upstep (f,g) or downstep (h,i) in Bz concentration in wild-type worms and in worms expressing Cx36 in AWC (f,h) or AIY (g,i) alone. At the top of each panel are averaged traces with shaded regions indicating s.e.m. At the bottom of each panel are percent mean fluorescent ratios, 10 s after compared with 10 s before stimulus onset. Two-tailed unpaired t-tests, *P<0.05, ***P<0.001, NS, not significant. Error bars represent s.e.m. Numbers on bars indicate sample sizes.
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f2: An engineered electrical synaptic connection between AWC and AIY flips the AIY benzaldehyde response profile.(a) AWC and AIY participate in a circuit for chemosensation and are naturally connected by an inhibitory chemical synapse. (b) Confocal projection image of strain AQ2660, in which AWC (green) expresses the calcium indicator YC3.60, and mCherry-tagged Cx36 puncta (red) are seen in the cell body (yellow arrow), dendrite (blue arrow) and axon (white arrows). Scale bar, 5 μm. (c) Confocal image of strain transgenic line AQ2646, in which mCherry-tagged Cx36 is expressed in AIY and AWC (red punctate fluorescence), and YC3.60 is expressed in AIY (green fluorescence). Yellow and white arrows indicate the cell body and axon respectively. Scale bar, 10 μm. An expanded version of this image and version showing the red channel alone are in Supplementary Fig. 3. (d,e) Calcium imaging of AWC and AIY responses to an upstep (d) or downstep (e) in benzaldehyde (Bz) concentration in wild-type worms and in worms expressing Cx36 in both AWC and AIY. (f–i) Calcium imaging of AWC (f,h) or AIY (g,i) responses to an upstep (f,g) or downstep (h,i) in Bz concentration in wild-type worms and in worms expressing Cx36 in AWC (f,h) or AIY (g,i) alone. At the top of each panel are averaged traces with shaded regions indicating s.e.m. At the bottom of each panel are percent mean fluorescent ratios, 10 s after compared with 10 s before stimulus onset. Two-tailed unpaired t-tests, *P<0.05, ***P<0.001, NS, not significant. Error bars represent s.e.m. Numbers on bars indicate sample sizes.

Mentions: We next tested whether existing neuronal connections could be reconfigured through ectopic Cx36 expression. To do this we focused on the AWC and AIY neurons, which mediate chemotaxis towards attractive odourants, such as benzaldehyde2526. The AWC sensory neurons respond to increases in odour concentration with reduced activity and to decreases in odour concentration with elevated activity. The AWCs make inhibitory chemical synapses with the AIY interneurons (Fig. 2a); although AIY ablation does not impair chemotaxis to odourants27, optogenetic AIY activation has been shown to promote forward travel and inhibit reorientations28. To determine whether heterologous connexin expression could modify the properties of these synapses and thereby impact behaviour, we expressed Cx36 in AWC and AIY using promoters derived from odr-1 (for AWCCx36) and the ttx-3 second intron (for AIYCx36) and imaged calcium responses to olfactory stimuli using genetically encoded calcium indicators (Fig. 2b,c; Supplementary Fig. 3). In wild-type animals, benzaldehyde presentation led to a calcium decrease in AWC and a reciprocal calcium increase in AIY, as expected from their known inhibitory connection (Fig. 2d). In animals expressing either the AWCCx36 or the AIYCx36 transgenes alone, these responses were not significantly altered (Fig. 2f,g). In contrast, animals expressing Cx36 in both neurons (AWCCx36AIYCx36) responded to benzaldehyde presentation with a calcium decrease in both AWC and AIY, suggesting that ectopic gap junctions between AWC and AIY reversed the sign of the AWC–AIY connection from negative to positive (Fig. 2d, red). A similar effect was observed when the AWCCx36AIYCx36 transgenes were carried on a chromosomally integrated array (Supplementary Fig. 4). We also observed an attenuation in the degree of AWC hyperpolarization in the AWCCx36AIYCx36 worms, consistent with a shunting of the hyperpolarizing current to AIY (Fig. 2d, red). Together these results indicate that ectopic gap junctions were formed between AWC and AIY, switching an inhibitory chemical synapse to a predomininantly excitatory electrical/chemical synapse.


Rewiring neural circuits by the insertion of ectopic electrical synapses in transgenic C. elegans.

Rabinowitch I, Chatzigeorgiou M, Zhao B, Treinin M, Schafer WR - Nat Commun (2014)

An engineered electrical synaptic connection between AWC and AIY flips the AIY benzaldehyde response profile.(a) AWC and AIY participate in a circuit for chemosensation and are naturally connected by an inhibitory chemical synapse. (b) Confocal projection image of strain AQ2660, in which AWC (green) expresses the calcium indicator YC3.60, and mCherry-tagged Cx36 puncta (red) are seen in the cell body (yellow arrow), dendrite (blue arrow) and axon (white arrows). Scale bar, 5 μm. (c) Confocal image of strain transgenic line AQ2646, in which mCherry-tagged Cx36 is expressed in AIY and AWC (red punctate fluorescence), and YC3.60 is expressed in AIY (green fluorescence). Yellow and white arrows indicate the cell body and axon respectively. Scale bar, 10 μm. An expanded version of this image and version showing the red channel alone are in Supplementary Fig. 3. (d,e) Calcium imaging of AWC and AIY responses to an upstep (d) or downstep (e) in benzaldehyde (Bz) concentration in wild-type worms and in worms expressing Cx36 in both AWC and AIY. (f–i) Calcium imaging of AWC (f,h) or AIY (g,i) responses to an upstep (f,g) or downstep (h,i) in Bz concentration in wild-type worms and in worms expressing Cx36 in AWC (f,h) or AIY (g,i) alone. At the top of each panel are averaged traces with shaded regions indicating s.e.m. At the bottom of each panel are percent mean fluorescent ratios, 10 s after compared with 10 s before stimulus onset. Two-tailed unpaired t-tests, *P<0.05, ***P<0.001, NS, not significant. Error bars represent s.e.m. Numbers on bars indicate sample sizes.
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f2: An engineered electrical synaptic connection between AWC and AIY flips the AIY benzaldehyde response profile.(a) AWC and AIY participate in a circuit for chemosensation and are naturally connected by an inhibitory chemical synapse. (b) Confocal projection image of strain AQ2660, in which AWC (green) expresses the calcium indicator YC3.60, and mCherry-tagged Cx36 puncta (red) are seen in the cell body (yellow arrow), dendrite (blue arrow) and axon (white arrows). Scale bar, 5 μm. (c) Confocal image of strain transgenic line AQ2646, in which mCherry-tagged Cx36 is expressed in AIY and AWC (red punctate fluorescence), and YC3.60 is expressed in AIY (green fluorescence). Yellow and white arrows indicate the cell body and axon respectively. Scale bar, 10 μm. An expanded version of this image and version showing the red channel alone are in Supplementary Fig. 3. (d,e) Calcium imaging of AWC and AIY responses to an upstep (d) or downstep (e) in benzaldehyde (Bz) concentration in wild-type worms and in worms expressing Cx36 in both AWC and AIY. (f–i) Calcium imaging of AWC (f,h) or AIY (g,i) responses to an upstep (f,g) or downstep (h,i) in Bz concentration in wild-type worms and in worms expressing Cx36 in AWC (f,h) or AIY (g,i) alone. At the top of each panel are averaged traces with shaded regions indicating s.e.m. At the bottom of each panel are percent mean fluorescent ratios, 10 s after compared with 10 s before stimulus onset. Two-tailed unpaired t-tests, *P<0.05, ***P<0.001, NS, not significant. Error bars represent s.e.m. Numbers on bars indicate sample sizes.
Mentions: We next tested whether existing neuronal connections could be reconfigured through ectopic Cx36 expression. To do this we focused on the AWC and AIY neurons, which mediate chemotaxis towards attractive odourants, such as benzaldehyde2526. The AWC sensory neurons respond to increases in odour concentration with reduced activity and to decreases in odour concentration with elevated activity. The AWCs make inhibitory chemical synapses with the AIY interneurons (Fig. 2a); although AIY ablation does not impair chemotaxis to odourants27, optogenetic AIY activation has been shown to promote forward travel and inhibit reorientations28. To determine whether heterologous connexin expression could modify the properties of these synapses and thereby impact behaviour, we expressed Cx36 in AWC and AIY using promoters derived from odr-1 (for AWCCx36) and the ttx-3 second intron (for AIYCx36) and imaged calcium responses to olfactory stimuli using genetically encoded calcium indicators (Fig. 2b,c; Supplementary Fig. 3). In wild-type animals, benzaldehyde presentation led to a calcium decrease in AWC and a reciprocal calcium increase in AIY, as expected from their known inhibitory connection (Fig. 2d). In animals expressing either the AWCCx36 or the AIYCx36 transgenes alone, these responses were not significantly altered (Fig. 2f,g). In contrast, animals expressing Cx36 in both neurons (AWCCx36AIYCx36) responded to benzaldehyde presentation with a calcium decrease in both AWC and AIY, suggesting that ectopic gap junctions between AWC and AIY reversed the sign of the AWC–AIY connection from negative to positive (Fig. 2d, red). A similar effect was observed when the AWCCx36AIYCx36 transgenes were carried on a chromosomally integrated array (Supplementary Fig. 4). We also observed an attenuation in the degree of AWC hyperpolarization in the AWCCx36AIYCx36 worms, consistent with a shunting of the hyperpolarizing current to AIY (Fig. 2d, red). Together these results indicate that ectopic gap junctions were formed between AWC and AIY, switching an inhibitory chemical synapse to a predomininantly excitatory electrical/chemical synapse.

Bottom Line: We added electrical synapses composed of the vertebrate gap junction protein Cx36 between Caenorhabditis elegans chemosensory neurons with opposite intrinsic responses to salt.In a second example, introducing Cx36 into an inhibitory chemical synapse between an olfactory receptor neuron and an interneuron changed the sign of the connection from negative to positive, and abolished the animal's behavioural response to benzaldehyde.These data demonstrate a synthetic strategy to rewire behavioural circuits by engineering synaptic connectivity in C. elegans.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Cell Biology, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK [2] Department of Medical Neurobiology, Hadassah Medical School, Hebrew University of Jerusalem, Jerusalem 9112102, Israel [3].

ABSTRACT
Neural circuits are functional ensembles of neurons that are selectively interconnected by chemical or electrical synapses. Here we describe a synthetic biology approach to the study of neural circuits, whereby new electrical synapses can be introduced in novel sites in the neuronal circuitry to reprogram behaviour. We added electrical synapses composed of the vertebrate gap junction protein Cx36 between Caenorhabditis elegans chemosensory neurons with opposite intrinsic responses to salt. Connecting these neurons by an ectopic electrical synapse led to a loss of lateral asymmetry and altered chemotaxis behaviour. In a second example, introducing Cx36 into an inhibitory chemical synapse between an olfactory receptor neuron and an interneuron changed the sign of the connection from negative to positive, and abolished the animal's behavioural response to benzaldehyde. These data demonstrate a synthetic strategy to rewire behavioural circuits by engineering synaptic connectivity in C. elegans.

Show MeSH
Related in: MedlinePlus