gsc2 neurons localize to the nucleus incertus.

(A, A’) WISH for gsc2 and (B-C’) double-label WISH for (B, B’) gsc2 and sst1.1 or (C, C’) gsc2 and rln3a was performed on (A-B’) 4 days post-fertilization (dpf) or (C, C’) 6 dpf larvae. (A, C, C’) Dorsal views. (A’, B, B’) Lateral views. (B’, C’) Enlarged views of boxed regions in B and C, respectively. Scale bars, 100 µm. (D-F) Fluorescent double-label WISH for (D) rln3a and gsc2, (E) rln3a and nmbb, and (F) rln3a and cckb. Dorsal views of 6 dpf larvae, Z-projections. Scale bar, 10 µm. (G) Schematic depicting distribution of neuronal subtypes in the nucelus incertus (NI) of larval zebrafish. Green dots, gsc2 expression; purple dots, rln3a expression; blue dots, nmbb expression; pink dots and shading, cckb expression. IPN, interpeduncular nucleus; PAG, periaqueductal grey.

Subset of neuropeptides expressed in larval zebrafish NI.

WISH for (A-A’) ccka, (B-B’) cckb, (C-C’) nmba, (D-D’) nmbb and (E-E’) nts expression in 6 dpf larvae. Dorsal views of the same larvae were imaged at (A, B, C, D, E) dorsal and (A’, B’, C’, D’, E’) ventral planes. White arrowheads indicate the NI. Scale bar, 100 µm.

Overlapping expression of rln3a and nmbb in the zebrafish NI.

Fluorescent double-label WISH for rln3a and nmbb transcripts. Dorsal views of 6 dpf larvae. (A) Z-projection and (A’-A’’’) higher magnification image of NI from larva in A. (B-C’’) NI in two additional larvae. (A’-C’’) Optical sections showing neurons expressing (A’, B, C) rln3a, (A’’, B’, C’) nmbb and (A’’’, B’’, C’’) composite images. White arrowheads indicate neurons that co-express both genes. (A) Scale bar, 100 µm. (A’-C’’) Scale bar, 10 µm.

Transgenic driver lines recapitulate gsc2 and rln3a expression patterns.

(A, D) CRISPR/Cas9 genome editing strategies used to generate (A) Tg(gsc2:QF2)c721 and (D) Tg(rln3a:QF2; he1.1:YFP)c836 driver lines. (B, C, E, F) Dorsal views of 6 dpf larvae. (B, E) WISH for (B) gsc2 and (E) rln3a. (C, F) Confocal Z-projections of (C) Tg(gsc2:QF2)c721; Tg(QUAS:GFP)c578 and (F) Tg(rln3a:QF2; he1.1:YFP)c836; Tg(QUAS:GFP)c578 larvae. Scale bars, 100 µm. sgRNA: single guide RNA, hsp70: heat shock cognate 70-kd protein, tandem duplicate 1 promoter, 5’ UTR: 5’ untranslated region, HA: homology arm, he1.1: promoter of hatching enzyme gene.

QF2 driver lines recapitulate gsc2 and rln3a expression patterns in the adult brain.

(A) Schematic lateral view of adult zebrafish brain (after Wullimann et al., 1996), indicating positions of coronal sections (70 µm) shown in (B-G). (B, D, F) WISH for (B) gsc2 and (D, F) rln3a. (C, E, G) Confocal Z-projections of labeled neurons in (C) Tg(gsc2:QF2)c721; Tg(QUAS:GFP)c578 and (E, G) Tg(rln3a:QF2; he1.1:YFP)c836; Tg(QUAS:GFP)c578 brains. Scale bars, 100 µm.

gsc2 neurons reside outside the IPN in the adult brain.

(A-C’) Confocal optical sections of 70 µm vibratome slices from a representative TgBAC(gng8:Eco.NfsB-2A-CAAX-GFP)c375; Tg(gsc2:QF2)c721; Tg(QUAS:NLS-mApple)c718 adult brain. Images are ordered from (A, B, C) anterior to (A’, B’, C’) posterior. Arrowheads indicate gsc2 cell bodies. Scale bar 100 µm.

rln3a and gsc2 NI neurons are largely GABAergic.

(A-F’’) Confocal images of 6 dpf larvae. (A-C) Lateral views. (D-F’’) Dorsal views. (A) Z-projection of Tg(gsc2:QF2)c721; Tg(QUAS:GFP)c578; Tg(slc17a6b:DsRed)nns9Tg larva. (B-F’’) Optical sections. (B) PAG and (C) NI of a Tg(rln3a:QF2; he1.1:YFP)c836; Tg(QUAS:mApple; he1.1:CFP)c788; Tg(slc17a6b:GFP)zf139Tg larva. (D) Tg(gsc2:QF2)c721; Tg(QUAS:mApple-CAAX; he1.1:mCherry)c636; Tg(gad1b:GFP)nn25Tg larva. (D’) Magnified view of boxed region in D. White arrowhead indicates a gad1b-positive gsc2-positive neuron. (E-F’’) Tg(rln3a:QF2; he1.1:YFP)c836; Tg(QUAS:mApple; he1.1:CFP)c788; Tg(gad1b:GFP)nn25Tg larva. (E-E’’) View of PAG. (F-F’’) View of NI. (E’, F’) Magnified views of boxed regions in E and F. (E’’, F’’) Individual neurons indicated by arrowheads in E’ and F’ respectively. Top panels: GABAergic, middle panels: rln3a, bottom panels: composite. (G) Boxplot showing the percentage of gsc2 and rln3a NI neurons, and rln3a PAG neurons that express Tg(gad1b:GFP)nn25Tg, n = 3 larvae. Scale bars, 100 µm.

gsc2 and rln3a neurons distinct projection patterns.

(A-J) Confocal optical sections of (A-E) Tg(gng8:Eco.NfsB-2A-CAAX-GFP)c375; Tg(gsc2:QF2)c721; Tg(QUAS:mApple-CAAX; he1.1:mCherry)c636 and (F-J) Tg(gng8:Eco.NfsB-2A-CAAX-GFP)c375; Tg(rln3a:QF2; he1.1:YFP)c836; Tg(QUAS:mApple-CAAX; he1.1:mCherry)c636 6 dpf larvae ordered from dorsal to ventral. (K-M) 3D reconstructions of confocal Z-stacks generated using Zen software (Zeiss), Tg(rln3a:QF2; he1.1:YFP)c836; Tg(QUAS:GFP-CAAX)c591; Tg(QUAS:NLS-GFP; he1.1:CFP)c682 larvae at 7 dpf showing efferents from (K) intact rln3a PAG (asterisks) and NI (arrows) neurons or following two-photon laser-mediated ablation of (L) PAG or (M) NI rln3a cell bodies at 6 dpf. Scale bars, 100 µm.

gsc2 and rln3a NI neurons innervate different dorsoventral IPN regions.

(A-H’) Confocal images of 6 dpf larvae. (A-B, E-F’) TgBAC(gng8:Eco.NfsB-2A-CAAX-GFP)c375 and Tg(gsc2:QF2)c721 driving (A, A’) Tg(QUAS:NLS-mApple; he1.1:CFP)c718 or (B, E-F’) Tg(QUAS:mApple-CAAX; he1.1:mCherry)c636. (C-D, G-H’) TgBAC(gng8:GAL4FF)c426; Tg(UAS-E1B:NTR-mCherry)c264 and Tg(rln3a:QF2; he1.1:YFP)c836 driving (C, C’) Tg(QUAS:NLS-GFP; he1.1:CFP)c682 or (D, G-H’) Tg(QUAS:NLS-GFP; he1.1:CFP)c682 and Tg(QUAS:GFP-CAAX)c591. (A’, C’) Higher magnification images of larvae in A and C, respectively. (A, A’, C, C’) Z-projections. (B, D) optical sections. (A-D) Lateral views. (E-H’) Dorsal views. Optical sections at the level of the (E, E’, G, G’) dorsal IPN or (F, F’, H, H’) ventral IPN of the same larvae. (E’, F’, G’, H’) Labeled efferent projections only. (I, J) Confocal Z-projections of coronal sections (70 μm) through adult brains of (I) Tg(gsc2:QF2)c721; Tg(QUAS:GFP-CAAX; he1.1:YFP)c631 or (J) Tg(rln3a:QF2; he1.1:YFP)c836; Tg(QUAS:GFP-CAAX)c591 fish. (K) Schematic of the IPN showing distinct dorsoventral regions innervated by rln3a and gsc2 neurons. Scale bars, 100 µm.

Increased calcium signaling in gsc2 neurons upon optogenetic activation of the dHb.

Calcium transients were imaged at 2.6 Hz before, during, and after illumination with 561 nm light in 7 dpf larvae. (A) Drawings depicting imaging of calcium transients and optogenetic activation using confocal microscopy. (B-C’) Representative maximum intensity projections of GCaMP7a fluorescence in (B) dHb and (B’) gsc2 neurons of the same larva, or (C) dHb and (C’) rln3a NI neurons of the same larva. Scale bar, 100 μm. (D-E’’) Tg(gsc2:QF2)c721 or (F-H’’) Tg(rln3a:QF2; he1.1:YFP)c836 driver lines in (D-H) TgBAC(gng8:GAL4FF)c426; Tg(UAS:GCaMP7a)zf415; Tg(QUAS:GCaMP7a)c594 larvae (D, E, F, G, H) with or (D’, E’, F’, G’, H’) without Tg(UAS:ReaChR-RFP)jf50. The average change in GCaMP7a signaling (%ΔF/F) is shown for (D, D’, F, F’) the dorsal habenulae, (E, E’) gsc2 neurons, (G, G’) rln3a NI neurons, and (H, H’) rln3a PAG neurons. Shading indicates standard deviation. Gaps at light onset and offset are due to latency in switching the laser configuration. (D’’, E’’, F’’, G’’, H’’) Average Fpost/Fpre is shown for (D’’, F’’) the dHb, (E’’) gsc2 neurons, (G’’) rln3a NI neurons, and (H’’) rln3a PAG neurons of ReaChR+ and ReaChR- larvae. Fpost is the area under the curve for 15 frames during 561 nm illumination and Fpre is the area under the curve for 15 frames preceding 561 nm illumination. Black bars indicate means. (D’’) n = 6 ReaChR- larvae, mean ratio = 0.7499333 ± 0.1489034, n = 5 ReaChR+ larvae, mean ratio = 2.94612 ± 0.4078562, ***p = 0.00040797. (E’’) n = 6 ReaChR- larvae, mean ratio = 1.074067 ± 0.1540597, n = 5 ReaChR+ larvae, mean ratio = 1.86018 ± 0.1681724, **p = 0.0073. (F’’) n = 5 ReaChR- larvae, mean ratio = 1.22092 ± 0.292814, n = 5 ReaChR+ larvae, mean ratio = 11.08328 ± 6.544268, *p = 0.0317. (G’’) n = 5 ReaChR- larvae, mean ratio = 1.82404 ± 0.3183693, n = 5 ReaChR+ larvae, mean ratio = 1.97298 ± 0.5857324, p = 0.8288. (H’’) n = 5 ReaChR- larvae, mean ratio = 2.13364 ± 0.2748605, n = 5 ReaChR+ larvae, mean ratio = 1.8276 ± 0.267812, p = 0.4482. Extended y-axis in F’’ to display higher values.

gsc2 and rln3a NI neurons differ in their spontaneous activity and response to an aversive cue.

Calcium transients were imaged at 5.2 Hz in 7dpf larvae during a mild electric shock (25 V, 200 ms duration). (A) Drawing depicting delivery of shock to an immobilized larva during imaging. (B, C) Examples of maximum intensity projections for NI neurons in (B) Tg(gsc2:QF2)c721; Tg(QUAS:GCaMP7a)c594 or (C) Tg(rln3a:QF2; he1.1:YFP)c836; Tg(QUAS:GCaMP7a)c594 larvae. Scale bars, 10 μm. (D, E) GCaMP7a signaling (%ΔF/F) for representative individual (D) gsc2 or (E) rln3a neurons. Arrows indicate local maxima identified as peaks by the MATLAB findpeaks function (MinPeakProminence: 0.3, MinPeakWidth: 10). (D’, E’) Average %ΔF/F for all recorded (D’) gsc2 neurons (93 from 11 larvae) or (E’) rln3a neurons (76 from 10 larvae). Shading indicates standard deviation. (F, G) Average (F) number of peaks during the recording period (as depicted by arrows in examples D and E) and (G) Fpost/Fpre for gsc2 neurons and rln3a neurons. In G, Fpost and Fpre are the area under the curve for 300 frames post-shock and for 300 frames prior to shock. Black bars in F indicate mean peaks for gsc2 neurons (5.560527 ± 0.6295351, n = 11 larvae) and rln3a neurons (9.90797 ± 1.180382, n = 10 larvae), **p = 0.0035. In G, Fpost and Fpre correspond to the area under the curve for 300 frames post-shock and for 300 frames prior to shock, respectively, and black bars indicate mean Fpost/Fpre for gsc2 neurons (1.578618 ± 0.1185055, n= 11 larvae) and rln3a neurons (1.1005 ± 0.1266781, n= 10 larvae) ***p = 0.00030144.

Loss of rln3a NI neurons increases spontaneous locomotor activity.

(A-C’’’) Single optical sections from two-photon imaging of 6 dpf (A, A’) Tg(gsc2:QF2)c721; Tg(QUAS:GFP)c578 or (B-C’’’) Tg(rln3a:QF2; he1.1:YFP)c836; Tg(QUAS:GFP)c578 larvae (A, B, C, C’’) before and (A’, B’, C’, C’’’) after laser-mediated ablation of (A, A’) gsc2 neurons, (B, B’) rln3a NI neurons, or (C, C’) left and (C’’, C’’’) right rln3a PAG neurons. Scale bars, 10 μm. (D-D’’’) Average locomotor activity during 5 seconds prior to and after shock. Shock delivery is denoted by the gray line. (E) Total locomotor activity during 5 seconds pre-and post-shock. Unablated: n = 27 larvae, pre mean = 0.8563379 ± 0.2340588 cm, post mean = 5.888059 ± 0.6411924 cm, gsc2 neurons ablated: n = 17 larvae, pre mean = 1.185243 ± 0.3124427 cm, post mean = 7.226035 ± 1.202651 cm, rln3a NI neurons ablated: n = 15 larvae, pre mean = 1.304913 ± 0.2634792 cm, post mean = 8.08791 ± 1.445184 cm, rln3a PAG neurons ablated: n = 17 larvae, pre mean = 0.7242808 ± 0.2307174 cm, post mean = 6.916558 ± 1.065436 cm. Kruskal-Wallis rank sum test: ***p = 2.2 x 10-16. Dunn’s post-hoc tests with adjustment for multiple comparisons: no statistically significant differences within pre-and post-shock epochs, p < 0.001*** for each pre-shock vs. post-shock comparison. Unablated control group includes Tg(gsc2:QF2)c721; Tg(QUAS:GFP)c578 and Tg(rln3a:QF2; he1.1:YFP)c836; Tg(QUAS:GFP)c578 siblings of ablated larvae. (F-F’’’) Representative trajectories of 7 dpf (F’) larvae with ablated gsc2 neurons, (F’’) ablated rln3a NI neurons or (F’’’) ablated rln3a PAG neurons, and (F) sibling controls during the first 115 seconds of the recording (baseline activity). (G) Total locomotor activity during the first 115 seconds of the recording. Unablated: mean = 19.86593 ± 3.186432 cm, ablated gsc2 neurons: mean = 17.86737 ± 3.837193 cm, ablated rln3a NI neurons: mean = 42.8003 ± 5.273192 cm, ablated rln3a PAG neurons: mean = 15.72976 ± 3.553839 cm. Kruskal-Wallis rank sum test: ***p = 0.0009853. Dunn’s post-hoc tests with adjustment for multiple comparisons: ablated rln3a NI neurons vs. unablated **p = 0.0019, ablated rln3a vs. gsc2 NI neurons **p = 0.0019, or ablated rln3a NI vs. rln3a PAG neurons **p = 0.0019. (H) Average bout length during the pre-shock period, with bouts defined as continuous periods of movement and no more than one second of prolonged immobility. Unablated: mean = 7.353653 ± 1.338161 seconds, ablated gsc2 neurons: mean = 6.177544 ± 1.14287 seconds, ablated rln3a NI neurons: mean = 10.37861 ± 1.166383 seconds, ablated rln3a PAG neurons: mean = 4.215133 ± 0.7386169 seconds. Kruskal-Wallis rank sum test: **p = 0.001344. Dunn’s post-hoc tests with adjustment for multiple comparisons: ablated rln3a NI neurons vs. unablated *p = 0.03884, ablated rln3a vs. gsc2 NI neurons ablated *p = 0.03884, or ablated rln3a NI vs. rln3a PAG neurons ***p = 0.00055. (I) Bout number during the pre-shock period. Unablated: mean = 7.740741 ± 1.031681 bouts, ablatex gsc2 neurons: mean = 6.882353 ± 1.38501 bouts, ablated rln3a NI neurons: mean = 8.133333 ± 1.045929 bouts, ablated rln3a PAG neurons: mean = 7.411765 ± 1.331349 bouts. Kruskal-Wallis rank sum test: p = 0.8895.

Confirmation of selective ablation of NI neuronal clusters.

(A-D’) WISH for (A, A’, C, C’) gsc2 or (B, B’, D, D’) rln3a was performed on 7 dpf larvae. (A’, B’) Tg(gsc2:QF2)c721; Tg(QUAS:GFP)c578 larvae whose gsc2 neurons were ablated at 6 dpf. (C’, D’) Tg(rln3a:QF2; he1.1:YFP)c836; Tg(QUAS:GFP)c578 larvae whose rln3a NI neurons were ablated at 6 dpf. (A, B, C, D) Unablated sibling controls for larvae in A’, B’, C’ and D’ respectively. (D,D’) Higher background due to the longer incubation time required to detect rln3a transcripts, which are reduced in Tg(rln3a:QF2; he1.1:YFP)c836 heterozygotes relative to wild type.

Properties of gsc2 and rln3a NI neurons.

Zebrafish lines used in this study.

Plasmids used in this study.

Oligonucleotides used in this study.

Summary of Statistical Tests Used

Deposited data and code.