1. Volume 89, Issue 3, Pages 598-612 (February 2016)
Visual Cue-Discriminative Dopaminergic Control of Visuomotor Transformation and Behavior Selection 
Yuanyuan Yao, Xiaoquan Li, Baibing Zhang, Chen Yin, Yafeng Liu, Weiyu Chen, Shaoqun Zeng, Jiulin Du 
Neuron 
Volume 89, Issue 3, Pages (February 2016)
DOI: /j.neuron
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1
Visual Stimulus-Specific Control of Visuomotor Transformation in the Zebrafish Escape Circuit
(A) Diagram of behavioral setup.
(B) Representative images showing expanding stimulus- and flash stimulus-evoked behavior of a zebrafish larva at 7 dpf. White dashed lines mark the front edge of an expanding stimulus shown in black, and white arrows indicate the movement direction. The coordinate axes were used to calculate the head direction of larvae.
(C) Distribution of larva head direction immediately before (dark) and after (blue) expanding stimulus-induced escape. The data were obtained from 20 escapes. The largest circle represents 40%.
(D) Probability of escape in response to different visual stimuli. The significance was computed relative to zero escape probability (expanding, p < 0.001; receding, p = 1; light approaching, p = 0.4; flash (15 ms), p = 0.2; flash (5 s), p = 0.2; Wilcoxon signed rank test).
(E) Effect of bilateral MC lesion on the probability of expanding stimulus-evoked escape. Top, images showing bilateral MCs (arrows) before and after MC-specific lesion; bottom, summary of escape probability.
(F) Samples of responses evoked by flash (triangle) and expanding stimuli (black bar) in the same MC of a 6 dpf larva. Visually evoked excitatory compound synaptic currents (CSCs) were recorded when the MC was held at the reversal potential of Cl− (ECl−, −60 mV).
(G) Summary of MC CSCs evoked by different visual stimuli. Expanding stimuli evoked larger responses in M cells.
(H and I) Spiking activities of tectal projection fibers in response to flash (15 ms in duration) (H) and expanding (I) stimuli at different intensities.
(J and K) MC CSCs evoked by flash (15 ms in duration) (J) and expanding (K) stimuli at different intensities.
Data obtained from the same larva are connected by a dashed line. The numbers in the brackets represent the numbers of larvae or MCs examined. Each open dot on the bars represents each data point. n.s., not significant; ∗∗p < 0.01, ∗∗∗p < 0.001; error bars, SEM.
See also Figures S1 and S2.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 2
Dopaminergic Signaling Is Important for Controlling Visuomotor Transformation
(A) Example (left) and summary (right) of data showing the effect of SCH bath application (50 μM) on MC eEPSCs evoked by electrical stimulation (filled arrowhead) of tectal projection fibers. The arrow in (A) indicates the peak under control condition (“Ctrl,” black).
(B) Example (left) and summary (right) of data showing the effect of local puffing (inset in the left panel) of SCH on MC CSCs evoked by 15 ms flash stimuli (open arrowhead in the left panel).
(C) Probability of escape evoked by 15 ms flash stimuli under the various treatments of dopaminergic signaling-relevant drugs. Apomorphine (“Apo”), non-selective dopamine receptor agonist; SCH-39166, D1R antagonist; quinpirole, D2R agonist; raclopride, D2R antagonist.
(D) Effect of bilateral MC lesion on the probability of escape evoked by 15 ms flash stimuli in the presence of SCH
(E) Example (left) and summary (right) of data showing the effect of local puffing of SCH on MC CSCs evoked by expanding stimuli.
(F) Summary of data showing the effect of SCH on the probability of expanding stimulus-evoked escape.
n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; error bars, SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 3
Hypothalamic Dopaminergic Neurons Control Visuomotor Transformation
(A) Projected confocal pseudocolor image of a 5 dpf ETvmat2:GFP larva, in which GFP was expressed in monoaminergic neurons. HC, caudal hypothalamus; HI, intermedial hypothalamus; OB, olfactory bulb; PreT, pretectum; PT, posterior tuberculum; Raphe, raphe nucleus; SP, subpallium. Green and red indicate relative dorsal and ventral distribution, respectively.
(B) Effects of two-photon laser-based lesion of dopaminergic neurons in the PT, HI, and HC on MC flash responses. Left top, schematic of experiment paradigm; left bottom, samples of MC responses under control or lesion conditions; right, summary.
(C) Example (top) and summary (bottom) of data showing the effect of MO-based th2 knockdown (“th2-MO”) on the number of dopamine-immunoreactive neurons in the HC.
(D) Example (top) and summary (bottom) of data showing the effect of MO-based th2 knockdown on MC responses evoked by 15 ms flash stimuli (arrowhead at top).
(E) Effects of HC lesion on the probability of flash-evoked escape behavior in the control (Ctrl) condition or in the presence of low doses of SCH (12 μM or 25 μM).
(F) Example (left) and summary (right) of data showing the effect of HC lesion on expanding stimulus-evoked responses of MCs.
(G) Effects of HC lesion on the probability of expanding stimulus-evoked escape behavior.
(H) Effect of the optogenetic activation of HC dopaminergic neurons on expanding stimulus-evoked MC responses. Left top, schematic of experimental paradigm: in vivo whole-cell recording was performed in one MC, and 440 nm laser was used to scan the middle layer of the ipsilateral HC in Tg(DAT:ChR2) larvae. Left bottom, sample traces showing MC responses evoked by expanding stimuli under the control condition (“Ctrl”) or optogenetic activation (“ChR2 activation”). Both sample traces are averaged of 5 trials. The italic blue lines represent 440 nm laser stimulation. Right, summary.
(I) Example (left) and summary (right) of data showing the effect of the optogenetic activation of HC dopaminergic neurons on flash-evoked MC responses.
(J and K) Effect of intracellular loading of the PKA inhibitor PKI (6-22) amide on flash- (J) or expanding stimulus-evoked (K) MC responses.
n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; error bars, SEM.
See also Figure S3.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 4
Hindbrain Inhibitory Interneurons Mediate Dopaminergic Control
(A) Confocal images from a 6 dpf Tg(GlyT2:GFP) larva showing the distribution of glycinergic interneurons around MCs in the hindbrain. The numbers “1,” “2,” and “3” indicate the location of glycinergic interneuron groups analyzed in (B)–(D). C, caudal; L, left; Ri, right, Ro, rostral. Each image shown is from one optical slice.
(B) 3D plot of the distribution of glycinergic interneurons, which exhibited spatial location-dependent functional properties. The frequencies of spontaneous and flash-evoked spiking activities are color- and symbol size-coded, respectively. Filled circles represent neurons irresponsive to flash stimuli. The three groups were roughly classified based on the spatial location and functional properties. The thick gray lines outline the MC morphology.
(C) Sample traces showing spontaneous firing activities of the three groups of glycinergic interneurons.
(D) Summary of 15 ms flash-evoked firing responses of the three groups of glycinergic interneurons.
(E) Schematic of two-photon lesion of glycinergic interneurons and in vivo whole-cell recording of bilateral MCs. +, intact side; −, lesion side.
(F–H) Effects of the lesion of the group 1, 2, 3, or both 2 and 3 on the total integrated charge (F), duration (G), and amplitude (H) of MC CSCs evoked by 15 ms flash stimuli.
(I) Effect of the lesion of both contralateral group 2 and ipsilateral group 3 on the total integrated charge of expanding stimulus-evoked MC CSCs.
(J–O) Retrograde tracing of glycinergic interneurons and optic tectal neurons by local injection of neurobiotin (NB) at the vicinity of MC ventral dendrites in a 6 dpf Tg(GlyT2:GFP) larva.
(J) Schematic of the locations of injection and imaging.
(K) Injection site (ellipse) at the right hindbrain. Arrowheads indicate non-specific signals on the skin.
(L) Schematic showing the location of images in (M)–(O). Single optic slices from different layers of the outlined areas are enlarged in (M)–(O).
(M) NB-positive neurons in the ipsilateral optic tectum.
(N) NB-positive glycinergic interneurons in the contralateral (“Left”), but not ipsilateral (“Right”), group 2. Asterisks indicate both NB- and GFP-positive cells. Based on z stack images, there were 6 NB-positive cells in the left brain, and 4 of them were also GFP-positive, whereas there were 3 NB-positive cells in the right, and none of them were co-labeled by GFP.
(O) NB-positive glycinergic interneurons in the ipsilateral (right side), but not contralateral (left side), group 3. Asterisks indicate both NB- and GFP-positive cells. Based on z stack images, there were 4 NB-positive cells in the left, and none of them were co-labeled by GFP, whereas there were 13 NB-positive cells in the right, and 10 of them were also GFP positive.
n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; error bars, SEM.
See also Figures S4 and S5.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 5
HC Dopaminergic Neurons Directly Modulate Hindbrain Glycinergic Interneurons via D1Rs
(A) Effect of the optogenetic activation of HC dopaminergic neurons on the membrane potential of hindbrain glycinergic interneurons. Left top, schematic of experimental paradigm: in vivo whole-cell recording was performed in glycinergic interneurons, and a 440 nm laser was used to scan the middle layer of the ipsilateral HC in double transgenic Tg (GlyT2:GFP,DAT:ChR2) larvae. Left bottom, sample traces showing that HC optogenetic activation-induced depolarization of glycinergic interneurons was blocked by SCH application. Right, summary.
(B and C) Effects of HC lesion on the spontaneous firing of the group 2 (B) and flash-evoked firing of the group 3 (C) glycinergic interneurons.
(D) Double immunostaining showing dopamine-immunoreactive fibers in the areas of the groups 2 (top) and 3 (bottom) glycinergic interneurons in a 6 dpf Tg(GlyT2:GFP) larva.
(E and F) Effects of apomorphine (“Apo”) application on the spontaneous firing of the group 2 (E) and flash-evoked firing of the group 3 (F) glycinergic interneurons.
(G) Flash-evoked CSCs in the group 3 glycinergic interneurons before and after application of apomorphine in the absence or presence of intracellular PKA inhibitor. Left, sample traces with the top two obtained from one interneuron and the bottom two from another interneuron. Right, summary.
(H) Effect of local puffing of SCH on flash-evoked CSCs in the group 3 glycinergic interneurons. Left, sample traces. Right, summary.
∗p < 0.05, ∗∗p < 0.01; error bars, SEM.
See also Figure S6.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 6 Properties of HC Dopaminergic Neurons’ Visual Responses
(A) In vivo confocal image of a 6-dpf Tg (DAT:Gal4,UAS:GCaMP5) larva showing GCaMP5 expression in HC dopaminergic neurons. Single optic slice at the middle layer of the HC was imaged. The dashed ellipse outlines the HC. C, caudal; L, left; Ri, right; Ro, rostral.
(B–E) In vivo calcium imaging data showing flash-evoked (B), receding-evoked (C), light approaching-evoked (D), and expanding stimulus-evoked (E) responses of HC dopaminergic neurons. Left panels, sample traces from the same larva. Each trace is the average of all responsive trials from all responsive neurons in the fish, except the traces in (E), which are the average of all ON-response trials (top) and all OFF-response trials (bottom), respectively. Right, pie charts showing the distribution of response types. ON response, OFF response, and no response are presented by pink, blue, and gray colors, respectively. Data were obtained from 405 HC dopaminergic neurons in 15 larvae.
(F–H) In vivo cell-attached recording data showing flash-evoked (F), receding-evoked (G), and expanding stimulus-evoked (H) responses of HC neurons. Left, sample traces showing the response pattern of responsive neurons. Each trace is the average of 4 (F) or 5 (G and H) trials. Right, pie charts showing the distribution of response types. Data were obtained from 49 (F), 38 (G), and 52 (H) HC neurons.
(I) Effect of bilateral lesion of the optic tectum on expanding stimulus- and flash stimulus-evoked responses of HC dopaminergic neurons. Data were obtained from 155 HC neurons in 5 larvae. Traces on pie charts represent typical responses from 42 (left) and 35 (right) cells.
(J and K) Effect of local puffing of PTX into the HC on expanding stimulus-evoked (J) and flash stimulus-evoked (K) responses of HC dopaminergic neurons. Data were obtained from 155 neurons in 6 larvae.
(J) Inhibition blockade effect on expanding stimulus-evoked responses of HC dopaminergic neurons. Top, sample traces from the same 7 cells showing expanding stimulus-evoked calcium responses before (left) and after (right) local puffing of PTX into the HC. Bottom, pie charts showing response type distribution of HC dopaminergic neuron before (left) and after (right) PTX application.
(K) Effect of inhibition blockade on flash-evoked responses of HC dopaminergic neurons. Top, sample traces from the same 2 cells showing flash-evoked calcium responses before (left) and after (right) local puffing of PTX into the HC. Bottom, pie charts showing response type distribution of HC dopaminergic neuron before (left) and after (right) PTX application.
See also Figure S7 and Tables S1–S5.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Figure 7 Working Model of Visuomotor Transformation Control
(A and B) Working model for differential control of visuomotor transformation at the escape circuit for non-threatening (A) and threatening (B) visual stimuli through a dopaminergic-inhibitory neural module.
(C) Sample traces of expanding stimulus- and flash stimulus-evoked postsynaptic potentials (PSPs) of MCs under different conditions.
(D) Latency of flash-evoked escape behaviors (top) or responses of dopaminergic neurons, group 3 glycinergic interneurons, and MCs (bottom). Top, flash-evoked escape behavior in control condition was observed in 5 out of 390 trials tested. The mean latency is 328 ± 63 ms for the control group (n = 5), 79 ± 3 ms for the PTX-treated group (n = 50), and 82 ± 4 ms for the SCH treated group (n = 35). Bottom, the mean latencies for the onset of HC dopaminergic neurons’ and glycinergic interneurons’ firing are 201 ± 18 ms (n = 28) and 177 ± 9 ms (n = 74), respectively. The mean latency is 79 ± 5 ms for the onset of MC EPSP1, 190 ± 14 ms for the onset of MC IPSP, 255 ± 16 ms for the peak of MC IPSP, and 931 ± 63 ms for the peak of MC EPSP2 (n = 21).
Error bars, SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions