1. Artificial Synaptic Rewiring Demonstrates that Distinct Neural Circuit Configurations Underlie Homologous Behaviors 
Akira Sakurai, Paul S. Katz 
Current Biology 
Volume 27, Issue 12, Pages e3 (June 2017)
DOI: /j.cub
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2. Current Biology 2017 27, 1721-1734.e3DOI: (10.1016/j.cub.2017.05.016)
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3. Figure 1
Abbreviated Phylogenetic Tree of Nudibranchia and the Swim CPGs in Melibe and Dendronotus
(A) Melibe and Dendronotus belong to a clade (indicated by bracket) that contains only genera that swim with left-right (LR) body flexions. This suggests that their most recent common ancestor (black dot) shared this behavior, and thus the behavior is homologous. Tree is based on [7–10]. NS indicates a non-swimmer.
(B) A schematic diagram of the Melibe swim CPG, which consists of Swim interneurons (Si1, Si2, and Si3) in left (L) and right (R) sides of the brain as indicated by numbered circles. Si4 is omitted for simplicity. Each neuron forms reciprocal inhibitory synapses with its contralateral counterpart. Si2 makes a slow excitatory synapse onto the contralateral Si3, which returns inhibitory synapses onto the contralateral Si1 and Si2. Based on Sakurai et al. [11].
(C) The swim motor pattern recorded intracellularly from six swim interneurons. The gray shading indicates the duration of a right Si3 burst. All neurons burst in LR alternation. The Si3 bursts are phase-delayed from the contralateral Si1 and Si2 bursts. The maximal membrane potentials during the swim motor pattern for Si1, Si2, and Si3 were −58.3 ± 4.7 (n = 22), −59.5 ± 4.9 (n = 16), and −53.3 ± 4.4 (n = 20) mV, respectively (mean ± SD).
(D) The Dendronotus swim interneurons form a circuit that is distinct from that of Melibe. The Si1 pair does not have reciprocally inhibitory synapses but is electrically connected and with the Si2 pair [6]. Si2 and Si3 form reciprocal inhibition with their contralateral counterparts. Si3 makes an excitatory synapse onto the contralateral Si2; they are also electrically coupled. Based on Sakurai et al. [6] and Sakurai and Katz [12].
(E) The Dendronotus Si1 fires irregularly, not in bursts; only Si2 and Si3 exhibit alternating bursts. The gray shading indicates the duration of a left Si2 burst. The Si3 bursts lead Si2 bursts by a few spikes. The gray shading indicates the duration of a right Si3 burst. The maximal membrane potentials during the swim motor pattern for Si1, Si2, and Si3 were −39.8 ± 6.3 (n = 17), −50.4 ± 5.3 (n = 26), and −44.4 ± 3.7 (n = 10) mV, respectively (mean ± SD).
In (B) and (D), lines terminating in filled circles indicate inhibitory synapses. Triangles indicate excitatory synapses; the long triangles indicate slow excitatory synapses (Si2-to-Si3 in Melibe), whereas the short triangles fast excitatory synapses (Si3-to-Si2 in Dendronotus). Resistor symbols indicate electrical connections. The thickness of the resistor line indicates the strength of connection [6, 11, 12]. Open circuits in (B) and (D) and boxes in (C) and (E) indicate neurons located in the left half of the brain; filled circuits and boxes indicate neurons in the right half of the brain. See also Table S1 and Movies S1 and S2.
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4. Figure 2 Curare Blocks Si3 Synapses in Both Species
The schematics show the Si3 synaptic connections onto the neurons in (A) and (B). See also Figure S1 and Data S1.
(A) Action potentials evoked in Si3 produced discrete, one-for-one IPSPs with a constant latency in the contralateral Si1, Si2, and Si3 (i). The latencies to the IPSPs in Si1, Si2, and Si3 were 15, 21, and 11 ms. The IPSPs decreased in amplitude with increasing doses of curare (ii–iv). Simultaneous recordings from four neurons were made in the presence of Hi-Di saline. A small amount of constant current (<0.5 nA) was injected into L-Si3 via bridge-balanced electrode to induce action potentials. Five to six traces were overlaid. In this example, the Si3-evoked IPSPs appeared as monophasic hyperpolarizing responses. However, in 56% preparations (n = 10 of 18), the Si3-evoked IPSPs in Si2 were biphasic with initial depolarization and subsequent hyperpolarization [11]. In such cases, 10−4 M curare blocked both synaptic components in all preparations examined. Traces were triggered at the peak of action potentials. The dotted lines indicate −54 mV (R-Si1), −50 mV (R-Si2), and −50 mV (R-Si3).
(B) In Dendronotus, the Si3 spikes produced one-to-one, constant latency EPSPs in the contralateral Si2 and IPSPs in the contralateral Si3 (i). The latency from the peak of Si3 spike to the IPSPs in Si3 was 19 ms and to the EPSP in Si2 11 ms. Both EPSPs and IPSPs decreased in amplitude with increasing doses of curare (ii–iv). Dotted lines indicate −40 mV. L-Si3 was depolarized by injecting 0.5–1 nA current through the recording electrode.
(C) The relationship between the dose (M) of curare and the strength of Si3 synapse. Normalized amplitude of the Si3-evoked IPSPs in the contralateral Si1–3 in Melibe (filled circles; n = 8 synapses in four animals) or the Si3-evoked EPSPs in the contralateral Si2 in Dendronotus (open squares; n = 6 synapses in six animals) were plotted against the concentration of curare in Hi-Di saline and fitted to a standard Hill model. The Hill slope and logEC50 for Melibe Si3-evoked IPSP are −1.1 and −5.5, whereas those for Dendronotus Si3-evoked EPSP are −1.0 and −6.3, respectively. Data are represented as mean ± SD.
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5. Figure 3 In Melibe, Curare Slows the Swim Motor Pattern
(A) Simultaneous intracellular recordings from R-Si1, L-Si2, and R-Si3 show the motor pattern in normal saline (left). Bath application of 0.1 mM curare (gray bar, right) slowed down the rhythm by extending the duration of bursts and causing them to become irregular. Schematics above the traces indicate the neural circuit. The blocked Si3 synapses are shown in gray (right).
(B) Curare (0.1 mM) significantly decreased the burst frequency (inverse burst period). In normal saline, the burst frequency was 0.26 ± 0.09 (n = 16 animals) Hz, which decreased to 0.06 ± 0.02 Hz (n = 16 animals) in curare. The burst frequency recovered to 0.19 ± 0.08 Hz (n = 8 animals) after more than 30 min washout of curare. There was a statistically significant effect of curare on the burst frequency (F(2,22) = 47.9, p < by one-way repeated-measures [RM] ANOVA).
(C) Curare (0.1 mM) significantly increased the coefficient of variation (CoV) in the burst period. CoV of the burst period during the swim motor pattern was measured from the right Si1 or Si2 in each individual in the duration of at least 40 s. In normal saline (white), the CoV was 0.04 ± 0.02 (n = 16 animals), which increased to 0.21 ± 0.12 (n = 16 animals) in curare (dark gray). The CoV decreased to 0.08 ± 0.03 (n = 8 animals) after washout of curare (light gray). There was a statistically significant effect of curare on the CoV (F(2,22) = 20.8, p < by one-way RM ANOVA).
(D) Curare had no effect on the intraburst spike frequencies of the swim interneurons. The bar graph shows averaged intraburst spike frequencies (Hz) measured from Si1, Si2, and Si3, in the normal saline (white), in curare (dark gray), and after washout (light gray). Curare had no significant effect on intraburst firing (Si1, F(2,14) = 1.44, p = 0.27 by one-way RM ANOVA, n = 14 cells; Si2, F(2,14) = 0.92, p = 0.42 by one-way RM ANOVA, n = 12 cells; Si3, F(2,16) = 2.27, p = 0.14 by one-way ANOVA, n = 13 cells).
In (B)–(D), data are represented as mean ± SD. Bracket with asterisks indicating significant differences by all pairwise multiple comparison procedures (Holm-Sidak method, two asterisks for p < and one asterisk for p = 0.011). Individual data points are shown on each bar as open circles.
(E) Suppression of firing in Si3s by hyperpolarizing current injection (−4 nA) had similar effect as curare application by extending the burst duration of Si1 and Si2. Schematic above the traces indicate both Si3s were simultaneously hyperpolarized by injecting a negative current step.
(F) The Si3 pair do not act as a half-center oscillator when not receiving periodic synaptic input from Si2. Suppression of firing in both Si1 and Si2 by hyperpolarizing current injection (−4 nA) made Si3 fire tonically with no LR alternation.
In (A), (E), and (F), dotted lines indicate 0 mV. See also Data S2.
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6. Figure 4
Dynamic-Clamp Replacement of Curare-Blocked Synapses Restores Periodicity in Melibe
(A) Simultaneous intracellular microelectrode recordings of the left and right Si1s and Si3s. In normal saline (left), the motor pattern progressed for a period of less than 5 s. In the presence of curare (0.1 mM), the period increased and became irregular (right, gray bar). The burst period shortened and became regular when the Si3-to-Si1 synapses that were blocked by curare were replaced with artificial synapses (300 nS) generated by the dynamic clamp (indicated in red). The Isyn traces indicate the artificial synaptic current injected when the dynamic clamp was on (red bar). When the clamp was turned off, the burst period lengthened and became irregular again. Dotted lines indicate 0 mV.
(B) The burst frequency (Hz) increased with increased strength of the Si3 synapse. The increase in the conductance of Si3-to-Si1/2 synapse in dynamic clamping caused significant increase in the burst frequency (F(5,18) = 8.3, p < by one-way RM ANOVA, n = 6 animals). All pairwise multiple comparisons (Holm-Sidak method) revealed significant differences between 300 and 0 nS (p < 0.001), 200 and 0 nS (p = 0.003), and 400 and 0 nS (p = 0.018).
(C) The CoV of the burst periods decreased significantly when Si3-to-Si1 synapse was introduced by the dynamic clamp (F(5,18) = 4.3, p = by one-way RM ANOVA, n = 6 animals). All pairwise multiple comparisons (Holm-Sidak method) revealed significant differences between 200 and 0 nS (p = 0.025), 300 and 0 nS (p = 0.034), and 100 and 0 nS (p = 0.033).
(D) The intraburst spike frequency of Si1 or Si2 showed no change after replacement with artificial dynamic-clamp synapses (F(5,18) = 0.35, p = 0.88 by one-way RM ANOVA).
In (B)–(D), experiments were obtained by using either Si1 (n = 3 preparations) or Si2 (n = 3 preparations). In each graph, the white bar shows the control value in normal saline (N.S.) before curare, a gray bar shows the value in 0.1 mM curare, and red bars show the values under the dynamic clamp with different conductance (from 50 to 400 nS) of the artificial synapses. Symbols represent individual preparations. Due to technical difficulty, bilateral dynamic clamping as shown in (A) was performed in three preparations (two with the Si3-to-Si1 synapses and one with the Si3-to-Si2 synapses). In other preparations, only one Si3-to-Si1 synapse (n = 1 preparation) or one Si3-to-Si2 synapse (n = 2 preparations) was replaced by the dynamic clamp. Data are represented as mean ± SD. See also Figures S2A and S3A and Data S3.
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7. Figure 5
In Dendronotus, Curare Eliminates Bursting Activity in Both Si2 and Si3
(A) Simultaneous intracellular microelectrode recordings from left and right Si2s and Si3s show a typical bursting pattern in normal saline (left). In the presence of 0.1 mM curare (gray bar, right), both Si2 and Si3 ceased bursting and showed irregular spiking.
(B) Curare (0.1 mM) halted bursting activity in most of the preparations (n = 30 of 35). In the normal saline, the burst frequency was 0.21 ± 0.05 Hz (n = 35 animals), which decreased to 0.02 ± 0.05 Hz in curare (n = 35 animals). The burst frequency recovered to 0.25 ± 0.06 Hz after washout of curare (n = 8 animals). There was a statistically significant effect of curare on the burst frequency (F(2,41) = 122.4, p < by one-way RM ANOVA). The burst frequency (Hz) was measured from the right Si2.
(C) Curare significantly reduced the intraburst spike frequencies of the swim interneurons. The bar graph shows averaged intraburst spike frequencies (Hz) measured from Si2, and Si3, in the normal saline (white), in curare (dark gray), and after washout (light gray). In both neurons, the spike frequency dropped significantly when curare was applied (Si2, F(2,41) = 62.9, p < by one-way RM ANOVA, n = 35 cells; Si3, F(2,21) = 16.3, p < by one-way RM ANOVA, n = 27 cells). When bursting in LR alternation was not detected in the presence of curare, then average spike frequency over a time period of >30 s was used as the intraburst spike frequency.
In (B) and (C), brackets with an asterisk indicate significant difference by all pairwise multiple comparison procedures (Holm-Sidak method, two asterisks for p < 0.001 and one asterisk for p = 0.02). Data are represented as mean ± SD. Individual data points are shown on each bar as open circles.
(D) Injection of hyperpolarizing current into both Si3s (at down arrows) halted bursting in the Si2s, which went silent or spiked sporadically.
(E) Injection of hyperpolarizing current into both Si2s (down arrows) caused the both Si3s to fire tonically. Large Si3-evoked EPSPs can be seen in the two Si2s. In (A)–(E), dotted lines indicate 0 mV.
See also Data S4.
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8. Figure 6
In Dendronotus, Bursting Activity Is Recovered by Replacing the Blocked Synapses with Artificial Dynamic-Clamp Synapses
(A) In normal saline (left), both Si2s and Si3s exhibited normal bursting activity. In the presence of 0.1 mM curare (right, gray bar), Si3 synapses were blocked and regular bursting ceased. When just the Si3-to-Si2 synapses were replaced by artificial dynamic synapses (80 nS, red bar), all four neurons immediately resumed alternating bursts. When the dynamic clamp was turned off, the bursting immediately ceased again. Isyn shows the current injected by the dynamic clamp. Dotted lines indicate 0 mV.
(B) In preparations that exhibited rhythmic bursting (n = 8 of 12 preparations), the burst frequency increased with increased artificial synaptic conductance from Si3 to Si2 (F(5,21) = 7.95, p < by one-way RM ANOVA, n = 8 animals). All pairwise multiple comparison (Holm-Sidak method) revealed significant differences between 120 and 0 nS (p < 0.001), 80 and 0 nS (p = 0.01), 120 and 40 nS (p = 0.02), and 160 and 0 nS (p = 0.02).
(C) The intraburst spike frequency of Si2 significantly increased with increasing artificial synaptic conductance (F(5,21) = 6.4, p < by one-way RM ANOVA, n = 8 animals). All pairwise multiple comparison (Holm-Sidak method) showed that adding a synaptic conductance from 80 to 120 nS caused a significant increases in the spike frequency (p = 0.008). When bursting in LR alternation was not detected in the presence of curare, then average spike frequency of the time period of >30 s was used as the intraburst spike frequency.
In (B) and (C), a white bar shows the control value in normal saline (N.S.) before curare, a gray bar shows the value in 0.1 mM curare, and red bars show the values under the dynamic clamp with different conductance (from 40 to 200 nS) of the artificial Si3-to-Si2 synapses. Symbols represent individual preparations that exhibited rhythmic bursting. The graphs do not contain data from four preparations, in which dynamic clamp failed to induce rhythmic bursting. Data are represented as mean ± SD. See also Figures S2B and S3B and Data S5.
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9. Figure 7
Bursting Activity in Dendronotus Is Recovered through Cross-Species Rewiring
(A) In normal saline (left), the two SI1s fire irregularly while the two Si2s burst in alternation. In the presence of 0.1 mM curare (right, gray bar), all bursting ceased. The Melibe circuit configuration was mimicked by addition of electrical coupling (80 nS) between Si1 and Si2 ipsilaterally and creation of inhibitory synapses (100 nS) between Si1s and from Si2 to Si1 as shown by red synapses in circuit diagram. When the dynamic clamp was turned on (red bar), both Si1s and Si2s immediately burst in LR alternation. They ceased bursting immediately after turning off of the dynamic clamp. Isyn shows the current injected by the dynamic clamp. Dotted lines indicate 0 mV.
(B) The addition of electrical coupling together with the inhibitory synapses caused a significant increase in the Si2 burst frequency (F(5,23) = 12.73, p < by one-way RM ANOVA, n = 12 animals). In all 12 preparations rhythmic bursting was observed during dynamic clamping. All pairwise multiple comparison (Holm-Sidak method) revealed significant differences between 40 and 0 nS (p < 0.001), 60 and 0 nS (p < 0.001), 80 and 0 nS (p < 0.001), 100 and 0 nS (p = 0.002), and 20 and 0 nS (p = 0.003). While changing the conductance of electrical coupling, the strength of the IPSPs was fixed at 50 or 100 nS.
(C) With increased electrical coupling, the intraburst spike frequency of Si2 increased significantly (F(5,23) = 24.02, p < by one-way RM ANOVA, n = 12 animals). All pairwise multiple comparison (Holm-Sidak method) revealed significant differences between 40 and 0 nS (p < 0.001), 60 and 0 nS (p < 0.001), 80 and 0 nS (p < 0.001), 100 and 0 nS (0 < 0.001), 100 and 20 nS (p = 0.002), 80 and 20 nS (p = 0.002), and 60 and 20 nS (p = 0.016).
(D) Creating redundant reciprocal inhibition was not sufficient to cause stable rhythmic bursting. Adding only inhibitory synapses (100 nS, purple bar) between Si1s and from Si2 to Si1 failed to produce bursting in any of the preparations examined (n = 5).
(E) Adding only the electrical connection between ipsilateral Si1 and Si2 induced rhythmic bursting in Dendronotus. In the presence of curare (0.1 mM), electrical coupling (100 nS) was added between Si1 and Si2 ipsilaterally by the dynamic clamp (orange bar). Although the bursting is not as clear as that shown in Figure 6, both Si1 and Si2 exhibited bursting in LR alternation in nine of 12 preparations. Dotted lines indicate 0 mV.
(F) The addition of electrical connections alone significantly increased the burst frequency (F(5,20) = 11.2, p < by one-way RM ANOVA, n = 9 animals). All pairwise multiple comparison (Holm-Sidak method) revealed significant differences as follows: 80 versus 0 nS, p < 0.001; 100 versus 0 nS, p < 0.001; 80 versus 20 nS, p = 0.025; 100 versus 20 nS, p = 0.025; 80 versus 40 nS, p = 0.032; 100 versus 40 nS, p = 0.033; 60 versus 0 nS, p < Rhythmic bursting was observed in nine of 12 preparations by adding artificial electrical connection of up to 100 nS. The graphs do not contain data from three preparations, in which the dynamic clamp failed to induce rhythmic bursting.
(G) Artificial electrical connections increased the intraburst spike frequency in Si2 (F(5,20) = 5.22, p = by one-way RM ANOVA, n = 9 animals). All pairwise multiple comparison (Holm-Sidak method) revealed significant differences in 80 versus 0 nS (p = 0.036) and 60 versus 0 nS (p = 0.049). The graphs do not contain data from three preparations, in which the dynamic clamp failed to induce rhythmic bursting. In each graph, data are represented as mean ± SD. Symbols represent individual preparations.
See also Figure S4 and Data S6.
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