Article Figures & Data
- Figure 1.
Establishment of Tg fish that express botulinum toxin in GCs or PCs. Sagittal sections of adult gSA2AzGFF152B;Tg(UAS:BoTxBLC-GFP) (A–G), Tg(cbln12:Gal4FF);Tg(UAS:BoTxBLC-GFP) (H–N), and Tg(aldoca:BoTxBLC-GFP) (O–U) brains were stained with anti-GFP (green), and anti-Neurod1, or anti-parvalbumin 7 (Pvalb7, magenta) antibodies. A–C, H–J, O–Q, Cerebellum region. D–F, K–M, R–T, High-magnification views of the boxes in A, H, O. G, N, U, Low-magnification views. Cb, cerebellum; CCe, corpus cerebelli; Di, diencephalon; GL, granular layer; Hb, hindbrain; Hyp, hypothalamus; LCa, lobus caudalis cerebelli; ML, molecular layer; PCL; PC layer; SM, stratum marginale; Tel, telencephalon; TeO, tectum opticum; TL, torus longitudinalis; Vam, medial division of valvula cerebelli. Scale bars: 400 μm (A; applies to A–C, H–J, O–Q), 200 μm (D; applies to D–F, K–M, R–T), 1 mm (G, N, U).
- Figure 2.
Active avoidance conditioning of wild-type fish. A, Tank used for active avoidance conditioning. A white opaque tank (L41 cm × W17 cm × H12 cm) with transparent walls at both ends, and a trapezoidal wedge (L10–20 cm × W17 cm × H5 cm) in the center, were used. Green LEDs and a pair of electrodes were placed on each side. Top view (left panel) and side view (right panel). B, C, Protocol for active avoidance. In the habituation session, a fish was allowed to swim freely for 20 min in the tank. In the training session, when a fish was located in a side compartment, the LED was turned on for 15 s (CS). If the fish did not escape to the other side after 10 s, an electric shock was administered for 5 s (US) in each trial. When the fish moved before the electric shocks, the trial was successful and was followed by a 30-s interval and the next trial. When fish had eight successful trials among 10 consecutive trials, they were considered to have established active avoidance in the training session, and were subjected to the next trial session. When fish did not establish active avoidance within 60 trials, the training session was terminated. When fish established active avoidance in three consecutive training sessions, they were subjected to the test session. In the test session, only light stimuli with LEDs were administered. When the fish had eight successful trials among 10 consecutive trials in the training session, they were considered to be learners. When fish did not establish active avoidance in the training session or did not succeed in the test session, they were considered to be non-learners. D, Acquisition of active avoidance conditioning in wild-type adult fish. Percentages of learners and non-learners are indicated (n = 43). E, Number of trials when learner fish established active avoidance in the training and test sessions (n = 22). The graph shows averages and SEs of the data. F, Time from CS to escape in each session of learner fish (n = 7). The graph shows averages and SEs of the data. G, H, Swimming behaviors. Turning frequency (turns/min) and swimming speed (mm/s) of learners and non-learners during free swimming (learner; n = 22, non-learner; n = 21). The graph shows averages and SEs of the data (ns indicates non-significance, Welch’s t test). I, Freezing response of non-learners. Average swimming speed (mm/s) of seven non-learners before and after the onset of CS in the 44th–53rd trials of training session 1 was calculated. J, Test for responsiveness to electric shocks in wild-type adult fish (n = 7). Swimming speed for 2 s before and after electric shocks was calculated (**p < 0.01, Welch’s t test). ns, not significant.
- Figure 3.
Expression of botulinum toxin in GCs suppresses active avoidance conditioning. A, B, Turning frequency (turns/min) and swimming speed (mm/s) of gSA2AzGFF152B;Tg(UAS:BoTxBLC-GFP) (152B::BoTx) and control sibling fish during free swimming (152B::BoTx; n = 47, control; n = 43). The graph shows the averages and SEs of the data (ns indicates non-significance, Welch’s t test. C, D, Turning frequency and swimming speed of Tg(cbln12:Gal4FF);Tg(UAS:BoTxBLC-GFP) (cbln12::BoTx) fish during free swimming (cbln12::BoTx; n = 39, control; n = 38). The graph shows the averages and SEs of the data (ns indicates non-significance, *p < 0.05, Welch’s t test). E, F, Response to electric shocks in 152B::BoTx (n = 7) and cbln12::BoTx (n = 7) fish. Swimming speed was calculated for 2 s before and after the electric shocks (***p < 0.001, *p < 0.05, Welch’s t test). G, H, Percentages of active avoidance learners for 152B::BoTx (n = 47) and control sibling fish (n = 43; G), and for cbln12::BoTx (n = 39), and control sibling fish (n = 38; H; ***p < 0.001, *p < 0.05, Fisher’s exact test). ns, not significant.
- Figure 4.
Expression of botulinum toxin in PCs suppresses active avoidance conditioning. A, B, Turning frequency (turns/min) and swimming speed (mm/s) of Tg(aldoca:BoTxBCL-GFP) (aldoca:BoTx) and control sibling fish during free swimming (aldoca:BoTx; n = 45, control; n = 35). The graph shows the averages and SEs of the data (ns indicates non-significance, Welch’s t test). C, Response to electric shocks in aldoca:BoTx fish (n = 7). Swimming speed was calculated for 2 s before and after the electric shocks (***p < 0.001, Welch’s t test). D, Percentages of active avoidance learners of aldoca:BoTx (n = 45) and control (n = 35) fish (**p < 0.01, Fisher’s exact test). E, Number of trials required to establish active avoidance conditioning (aldoca:BoTx: n = 6; control: n = 16). The graph shows the averages and SEs of the data (ns indicates non-significance, Welch’s t test).
- Figure 5.
NTR-mediated ablation of PCs in adult fish suppresses active avoidance conditioning. A–L, Ablation of PCs. Adult Tg(aldoca:NTR-TagRFPT) fish were treated with MTZ for 18 h (A–F) or left untreated (G–L). The fish were subjected to behavior assays and subsequent histologic analysis 11 d after MTZ treatment. Sagittal sections were stained with anti-Pvalb7 antibody (green). Expression of NTR-TagRFPT (TagRFP, magenta) is also shown. D–F, J–L, High-magnification views of the boxes in A, G. Arrows and arrow heads indicate Pvalb7-positive dendrites of PCs (in the cerebellum) and Type I neurons (in the optic tectum), respectively. The dotted line in G indicates the limit of the cerebellum. Note that the Pvalb7 signal in PCs but not in Type I neurons disappeared and no ML was observed in the MTZ-treated fish. M, N, Turning frequency (turns/min) and swimming speed (mm/s) of Tg(aldoca:NTR-TagRFPT) (aldoca:NTR) and control fish during free swimming (aldoca:NTR; n = 13, control; n = 43). The graph shows the averages and SEs of the data (ns indicates non-significance, *p < 0.05, Welch’s t test). O, Response to electric shocks in adult aldoca:NTR fish treated with MTZ (n = 7). Swimming speed was calculated for 2 s before and after the electric shocks (***p < 0.001, Welch’s t test). P, Swimming speed for 2 s after US in each strain. The graph shows the averages and SEs of the data (ns indicates non-significance, one-way repeated measures ANOVA with Tukey’s post hoc test). Q, Percentage of active avoidance learners of aldoca:NTR (n = 13) and control wild-type (n = 43) fish (**p < 0.01, Fisher’s exact test). Va, valvula cerebelli. The other abbreviations are described in Figure 1. Scale bars: 400 μm (A; applies to A–C, G–I) and 200 μm (D; applies to D–F, J–L). ns, not significant.
- Figure 6.
Expression of botulinum toxin in GCs or PCs also perturbs classical conditioning responses. A, Protocol for classical fear conditioning. A compartment on one side of the tank in Figure 2A was used. In the habituation session, a light stimulus (CS) was provided for 2 s per trial (10 trials). In the training session, a paired CS and US (0.2-s electric shock given 1.8 s after the onset of CS) was administered in each trial (10 trials). In the test session, CS alone was administered (10 trials). The interval between trials was 30 s, and the interval between sessions was 20 min. B, Changes in swimming speed before and after the CS of wild-type (WT, n = 25), 152B::BoTx (n = 23), and aldoca:BoTx (n = 24) fish. Swimming speed was measured for 1.5 s before and after the CS in each trial, and average changes in swimming speed in the training and test sessions were calculated. The graph shows the averages and SEs of the data (Training session; lines factor: p = 1.483e-07, trials factor: p = 1.189e-07, lines × trials interaction: p = 1.398e-06, two-way repeated measures ANOVA; ***p < 0.001, **p< 0.01, *p < 0.05, two-way repeated measures ANOVA with Tukey’s post hoc test. Test session; lines factor: p = 1.398e-06, trials factor: p = 0.171, lines × trials interaction: p = 0.3649, two-way repeated measures ANOVA; WT vs 152B::BoTx in test session: p < 1e-22, WT vs aldoca:BoTx in test session: p < 1e-22, 152B::BoTx vs aldoca:BoTx in test session: p = 0.9425, one-way ANOVA with Tukey’s post hoc test). C, Percentages of Pavlovian conditioning learners of WT (n = 14), 152B::BoTx (n = 12), and aldoca:BoTx (n = 13) fish (*p < 0.05, Fisher’s exact test with BH post hoc test). D, Pavlovian panic responses during active avoidance conditioning. Data from 10 wild-type fish that were subjected to active avoidance conditioning were used. Since wild-type fish established active avoidance in the 13th trial at the earliest, swimming speed for 1.5 s before and after the CS was measured in each trial from the fourth to the 13th trial. Average speed is plotted in the graph.
Tg lines Expression Sample number Lateral sections (%) Medial sections (%) LCa (%) gSA2AzGFF152B;
Tg(UAS:BoTxBLC-GFP)GCs (CCe>> LCa, EG) 1 14.7 69.5 0.2 2 13.7 63.9 0.9 3 6.4 48.6 0.2 Tg(cbln12:Gal4FF);
Tg(UAS:BoTxBLC-GFP)GCs (CCe, LCa, EG, TL) 1 38.0 35.6 44.2 2 37.7 42.1 55.0 3 55.6 50.9 50.5 Tg(aldoca:BoTxBLC-GFP) PCs 1 96.4 97.7 NA 2 95.9 96.8 3 100 99.4 Sagittal sections from three gSA2AzGFF152B;Tg(UAS:BoTxBLC-GFP) or Tg(cbln12:Gal4FF);Tg(UAS:BoTxBLC-GFP) adult fish were stained with anti-GFP and anti-Neurod1 antibodies. Sagittal sections from three Tg(aldcoa:BoTxBLC-GFP) adult fish were stained with anti-GFP and anti-Pvalb7 antibodies. Two typical lateral and medial sections from each fish were used. For the GCs, the percentage of the GFP+ area in the GL area was determined by using ImageJ software. For the PCs, the number of GFP+ and Pvalb7+ cells was counted manually. The percentage of GFP+ cells to Pvalb7+ cells was determined. CCe, corpus cerebelli; EG, eminentia granularis; GC, granule cells; LCa, lobus caudalis cerebelli; NA, not applicable; TL, torus longitudinalis.
Figure Measurement Type of test Comparison Statistical value 2G Number of turns during free swimming Welch’s t test Learner WT vs non-learner WT p = 0.5924
t(41) = 0.53972H Swimming speed during free swimming Welch’s t test Learner WT vs non-learner WT p = 0.4405
t(41) = 0.77912J Swimming speed before and after
electric shockWelch’s t test Before US vs after US in WT p = 0.002975
t(12) = −4.5393A Number of turns during free swimming Welch’s t test Control vs 152B::BoTx p = 0.6224
t(86) = −0.49423B Swimming speed during free swimming Welch’s t test Control vs 152B::BoTx p = 0.7603
t(86) = −0.30623C Number of turns during free swimming Welch’s t test Control vs cbln12::BoTx p = 0.03522
t(75) = −2.1453D Swimming speed during free swimming Welch’s t test Control vs cbln12::BoTx p = 0.3511
t(75) = −0.93973E Swimming speed before and after
electric shockWelch’s t test Before US vs after US in
152B::BoTxp = 4.043e-05
t(12) = −6.3143F Swimming speed before and after
electric shockWelch’s t test Before US vs after US in
cbln12::BoTxp = 0.01427
t(12) = −3.1303G Learning rate of active avoidance Fisher’s exact test Control vs 152B::BoTx p = 5.574e-08 3H Learning rate of active avoidance Fisher’s exact test Control vs cbln12::BoTx p = 0.01080 4A Number of turns during free swimming Welch’s t test Control vs aldoca:BoTx p = 0.2164
t(78) = 1.2464B Swimming speed during free swimming Welch’s t test Control vs aldoca:BoTx p = 0.6001
t(78) = −0.52644C Swimming speed before and after
electric shockWelch’s t test before US vs after US in
aldoca:BoTxp = 0.0001143
t(12) = −6.2484D Learning rate of active avoidance Fisher’s exact test Control vs aldoca:BoTx p = 0.002116 4E Number of trials in training session 1 Welch’s t test Control vs aldoca:BoTx p = 0.3255
t(20) = −1.0314E Number of trials in training session 2 Welch’s t test Control vs aldoca:BoTx p = 0.7545
t(20) = 0.31824E Number of trials in training session 3 Welch’s t test Control vs aldoca:BoTx p = 0.3865
t(20) = 0.88564E Number of trials in test session Welch’s t test Control vs aldoca:BoTx p = 0.2301
t(20) = −1.3605M Number of turns during free swimming Welch’s t test Control vs aldoca:NTR p = 0.2728
t(54) = −1.1435N Swimming speed during free swimming Welch’s t test Control vs aldoca:NTR p = 0.02462
t(54) = 2.5095O Swimming speed before and after
electric shockWelch’s t test Before US vs after US in
aldoca:NTRp = 0.0003567
t(12) = −6.3715P Swimming speed before and after
electric shockOne-way ANOVA F(18,621) = 2.88
p = 0.039435P Swimming speed before and after
electric shockOne-way ANOVA with Tukey’s
post hoc testWT vs 152B::BoTx p = 0.6201 5P Swimming speed before and after
electric shockOne-way ANOVA with Tukey’s
post hoc testWT vs cbln12::BoTx p = 0.08734 5P Swimming speed before and after
electric shockOne-way ANOVA with Tukey’s
post hoc testWT vs aldoca:BoTx p = 0.7949 5P Swimming speed before and after
electric shockOne-way ANOVA with Tukey’s
post hoc testWT vs aldoca:BoTx p = 0.9959 5Q Learning rate of active avoidance Fisher’s exact test Control vs aldoca:NTR p = 0.008326 6B Change of speed in training session Two-way repeated measures
ANOVA lines × trials interactionF(18,621) = 3.515
p = 1.398e-066B Change of speed in training session Two-way repeated measures
ANOVA lines factorF(2,69) = 19.92
p = 1.483e-076B Change of speed in training session Two-way repeated measures
ANOVA trials factorF(9,621) = 5.725
p = 1.189e-076B Change of speed in training session Two-way repeated measures
ANOVA with Tukey’s
post hoc testWT vs 152B::BoTx in trial 6 p = 0.03243 6B Change of speed in training session Two-way repeated measures
ANOVA with Tukey’s
post hoc testWT vs 152B::BoT in trial 7 p = 0.04779 6B Change of speed in training session Two-way repeated measures
ANOVA with Tukey’s
post hoc testWT vs 152B::BoT in trial 8 p = 4.1e-09 6B Change of speed in training session Two-way repeated measures
ANOVA with Tukey’s
post hoc testWT vs aldoca:BoTx in trial 8 p = 2.6e-05 6B Change of speed in training session Two-way repeated measures
ANOVA with Tukey’s
post hoc testWT vs 152B::BoTx in trial 9 p = 0.005936 6B Change of speed in test session Two-way repeated measures
ANOVA lines × trials
interactionF(18,621) = 1.083
p = 0.36496B Change of speed in test session Two-way repeated measures
ANOVA lines factorF(2,69) = 28.35
p = 1.398e-066B Change of speed in test session Two-way repeated measures
ANOVA trials factorF(9,621) = 1.431
p = 0.1716B Change of speed in training session Two-way repeated measures
ANOVA with Tukey’s
post hoc testWT vs 152B::BoTx p < 1e-22 6B Change of speed in training session One-way ANOVA with Tukey’s
post hoc testWT vs aldoca:BoTx p < 1e-22 6B Change of speed in training session One-way ANOVA with Tukey’s
post hoc testaldoca:BoTx vs
152B::BoTxp = 0.9425 6C Learning rate of Pavlovian fear
conditioningFisher’s exact test with BH
post hoc testWT vs 152B::BoTx p = 0.02892 6C Learning rate of Pavlovian fear
onditioningFisher’s exact test with BH
post hoc testWT vs aldoca:BoTx p = 0.02892 6C Learning rate of Pavlovian fear
conditioningFisher’s exact test with
BH post hoc test152B::BoTx vs
aldoca:BoTxp = 1.000 In all figures, the data distribution was normal. WT, wild-type.
- Movie 1.
Active avoidance of a wild-type fish, successful trial. A successful trial of active avoidance of a wild-type learner fish in the 25th trial of training session 1 is shown. The timing of the CS (light exposure with a green LED) and US (electric shock) are indicated. Note that the fish responded to the CS and escaped into the compartment on the other side of the tank.
- Movie 2.
Active avoidance of a wild-type fish, failed trial. A failed trial of active avoidance of a wild-type non-learner fish in the 25th trial of training session 1 is shown. The timing of the CS and US are indicated. Note also that the fish did not escape after the presentation of CS but responded to US.
- Movie 3.
Pavlovian fear conditioning of a wild-type fish. Behavior of a wild-type learner fish in trial 1 of the test session is shown. The timing of the CS is indicated. Note that the fish moved quickly after the presentation of CS.
- Movie 4.
Pavlovian fear conditioning of a 152B::BoTx fish. Behavior of a 152B::BoTx non-learner fish in trial 1 of the test session is shown. The timing of the CS is indicated. Note that the fish did not move after the presentation of CS.
- Movie 5.
Pavlovian fear conditioning of an aldoca:BoTx fish. Behavior of an aldoca:BoTx non-learner fish in trial 1 of the test session is shown. The timing of the CS is indicated. Note that the fish did not move after the presentation of CS.
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