Specialized neurons in the right habenula mediate response to aversive olfactory cues

  1. Jung-Hwa Choi
  2. Erik R Duboue
  3. Michelle Macurak
  4. Jean-Michel Chanchu
  5. Marnie E Halpern  Is a corresponding author
  1. Carnegie Institution for Science, Department of Embryology, United States
  2. Jupiter Life Science Initiative, Florida Atlantic University, United States
  3. Wilkes Honors College, Florida Atlantic University, United States

Abstract

Hemispheric specializations are well studied at the functional level but less is known about the underlying neural mechanisms. We identified a small cluster of cholinergic neurons in the dorsal habenula (dHb) of zebrafish, defined by their expression of the lecithin retinol acyltransferase domain containing 2 a (lratd2a) gene and their efferent connections with a subregion of the ventral interpeduncular nucleus (vIPN). The lratd2a-expressing neurons in the right dHb are innervated by a subset of mitral cells from both the left and right olfactory bulb and are activated upon exposure to the odorant cadaverine that is repellent to adult zebrafish. Using an intersectional strategy to drive expression of the botulinum neurotoxin specifically in these neurons, we find that adults no longer show aversion to cadaverine. Mutants with left-isomerized dHb that lack these neurons are also less repelled by cadaverine and their behavioral response to alarm substance, a potent aversive cue, is diminished. However, mutants in which both dHb have right identity appear more reactive to alarm substance. The results implicate an asymmetric dHb-vIPN neural circuit in the processing of repulsive olfactory cues and in modulating the resultant behavioral response.

Editor's evaluation

This work presents a conceptual advance on our understanding of the habenula in vertebrate species, by revealing interesting functions to specific cell types within this region of the brain.

https://doi.org/10.7554/eLife.72345.sa0

Introduction

Fish use the sense of smell to search for food, detect danger, navigate, and communicate social information by detecting chemical cues in their aquatic environment (Yoshihara, 2014). As with birds and mammals, perception of olfactory cues is lateralized and influences behavior (Siniscalchi, 2017). In zebrafish, nine glomerular clusters in the olfactory bulb (OB) receive olfactory information from sensory neurons in the olfactory epithelium and, in turn, transmit signals to four forebrain regions: the posterior zone of the dorsal telencephalon (Dp), the ventral nucleus of the ventral telencephalon (Vv), the posterior tuberculum (PT), and the dorsal habenular region (dHb) (Miyasaka et al., 2014; Yoshihara, 2014). In contrast to all other target regions, which are located on both sides of the forebrain, only the right nucleus of the dHb is innervated by mitral cells that emanate from medio-dorsal (mdG) and ventro-medial (vmG) glomerular clusters in both OBs (Miyasaka et al., 2014; Yoshihara, 2014). Moreover, calcium imaging experiments suggest that the right dHb shows a preferential response to odorants compared to the left dHb (Chen et al., 2019; Dreosti et al., 2014; Jetti et al., 2014; Krishnan et al., 2014). The identity of the post-synaptic neurons within the right dHb that receive olfactory input and the purpose of this asymmetric connection are unknown.

The habenulae are highly conserved structures in the vertebrate brain and, in teleosts such as zebrafish, consist of dorsal and ventral (vHb) nuclei, which are equivalent to the medial and lateral habenulae of mammals, respectively (Amo et al., 2010). The neurons of the dHb are largely glutamatergic and contain specialized subpopulations that also produce acetylcholine, substance P, or somatostatin (deCarvalho et al., 2014; Hsu et al., 2016; Lee et al., 2019). In zebrafish, the number of neurons within each subtype differs between the left and right dHb (deCarvalho et al., 2014). The dHb have been implicated in diverse states such as reward, fear, anxiety, sleep, and addiction (Duboué et al., 2017; Hikosaka, 2010; Lee et al., 2019; Okamoto et al., 2012). Accordingly, the right dHb was shown to respond to bile acid and involved in food-seeking behaviors (Chen et al., 2019; Krishnan et al., 2014), whereas the left dHb was found to be activated by light and attenuate fear responses (Dreosti et al., 2014; Duboué et al., 2017; Zhang et al., 2017). However, the properties of the dHb neurons implicated in these behaviors, such as their neurotransmitter identity and precise connectivity with their unpaired target, the midbrain interpeduncular nucleus (IPN), have yet to be determined.

Here, we describe a group of cholinergic neurons defined by their expression of the lecithin retinol acyltransferase domain containing 2 a (lratd2a) gene [formerly known as family with sequence similarity 84 member B (fam84b)], that are predominantly located in the right dHb where they are selectively innervated by the olfactory mitral cells that originate from both sides of the brain (Miyasaka et al., 2009), and form efferent connections with a restricted subregion of the ventral IPN (vIPN). Activity of the lratd2a-expressing neurons is increased following exposure to the aversive odorant cadaverine and, when these neurons are inactivated, adult zebrafish show a diminished repulsive response. Our findings provide further evidence for functional specialization of the left and right habenular nuclei and reveal the neuronal pathway that mediates a lateralized olfactory response.

Results

lratd2a-expressing neurons in the right dHb receive bilateral olfactory input

A subset of medio-dorsal mitral cells that are labeled by Tg(lhx2a:gap-YFP) were previously shown to project their axons bilaterally through the telencephalon and terminate in the right dHb (Miyasaka et al., 2009), in the vicinity of a small population of lratd2a-expressing neurons [(deCarvalho et al., 2013) and Figure 1A]. To characterize this subset of dHb neurons, we used CRISPR/Cas9-mediated targeted integration (Kimura et al., 2014) to introduce the QF2 transcription factor (Ghosh and Halpern, 2016; Subedi et al., 2014) under the control of lratd2a transcriptional regulation (Figure 1B). QF2 does not disrupt transcription at the lratd2a locus (Figure 1—figure supplement 1) and drives expression of QUAS-regulated genes encoding fluorescent protein reporters in a similar pattern to endogenous lratd2a gene expression in the nervous system (deCarvalho et al., 2013). In 5 days post fertilization (dpf) larval zebrafish, this includes a subset of neurons in the OB, the bilateral vHb, and asymmetrically distributed neurons in the dHb. There are 2.5 times more cells in the right dHb than the left (Figure 1C–E’’ and Figure 1—figure supplement 2) and those on the left consistently show weaker lratd2a expression and less GFP labeling than those on the right (Figure 1A and Figure 1—figure supplements 12). Double labeling confirmed that axons of the bilateral lhx2a positive olfactory mitral cells terminate precisely at the lratd2a-expressing neurons in the right dHb (Figure 1C–D’’, Figure 1—figure supplement 3B), co-localized with the pre-synaptic protein synaptophysin (Figure 1—figure supplement 3A). The lratd2a-expressing dHb neurons, in turn, project to a restricted region of the ventral IPN (Figure 1F–G’’).

Figure 1 with 3 supplements see all
lratd2a-expressing neurons in the right dHb connect asymmetric pathway from the olfactory bulb to ventral IPN.

(A) Pattern of lratd2a expression at 5 days post fertilization (dpf), open arrowhead indicates right dHb and black arrowheads the bilateral vHb. (B) Sequences of WT (top) and transgenic fish (bottom) with QF2 integrated within the first exon of the lratd2a gene. PAM sequences are green, the sgRNA-binding site red and donor DNA blue. Confocal dorsal views of Tg(lratd2a:QF2), Tg(QUAS:mApple-CAAX) and Tg(lhx2a:gap-YFP) labeling in a (C-C’’) 5 dpf larva and in transverse sections of the adult brain at 3 months post-fertilization (mpf) at the level of the (D-D’’) dHb and (E-E’’) olfactory bulb. Axons of lhx2a olfactory mitral cells (open arrowheads, C and D) terminate at lratd2a dHb neurons. (F-F’’) Lateral view of Tg(lratd2a:QF2), Tg(QUAS:mApple-CAAX), Tg(gng8:GFP) larva at 6 dpf with mApple-labeled dHb terminals at the ventral interpeduncular nucleus (vIPN). Dorsal habenular nuclei (dHb), dorsal interpeduncular nucleus (dIPN). (G-G’’) Axonal endings of lratd2a dHb neurons are restricted to the ventralmost region of the vIPN in transverse section of 2.5 mpf adult brain. Scale bar, 50 μm. A-P, anterior to posterior; L-R, left-right; OB, olfactory bulb.

Aversive olfactory cues activate lratd2a neurons in the right dHb

Several studies using transgenic expression of the genetically encoded calcium indicator GCaMP have demonstrated that dHb neurons are activated by olfactory cues in larval zebrafish (Jetti et al., 2014; Krishnan et al., 2014). We had previously examined the habenular response of adult zebrafish to cadaverine, a known aversive olfactory cue that is released from decaying fish (Hussain et al., 2013), and to chondroitin sulfate, a component of alarm substance (also known as Schreckstoff), which is released from the skin of injured fish (Mathuru et al., 2012). However, we did not have the necessary transgenic tools to specifically record the activity of the lratd2a cell population (deCarvalho et al., 2014).

To determine whether olfactants activate the lratd2a-positive neuronal cluster in the dHb, we measured calcium signaling in Tg(lratd2a:QF2)c644, Tg(QUAS:GCaMP6f)c587 larvae at 7 dpf. We monitored the responses of individual cells in the right dHb, as GCaMP6f labeling was weakly or not detected in neurons on the left (data not shown). The observed changes in fluorescence intensity after the addition of two applications of an aversive cue were compared to those observed after two additions of vehicle alone (i.e. deionized water), all delivered 1 min apart (Figure 2A and B). Cadaverine elicited a significant increase in GCaMP6f labeling in the same neurons following the first and second applications (16-fold increase averaged over both doses; Figure 2C). On average, a greater than fourfold increase in calcium signaling was measured in individual lratd2a-expressing neurons following addition of chondroitin sulfate compared to their response to vehicle alone (Figure 2D).

Increased activity of lratd2a-expressing dHb neurons upon exposure to aversive olfactory cues.

(A–B) Average change in fluorescence (ΔF/F) in seconds (sec) for all lratd2a positive neurons in the larval right dHb over a 5 min interval. Yellow bars indicate consecutive 5 s intervals of vehicle or odor delivery. Solid lines represent mean responses to (A) cadaverine or (B) to chondroitin sulfate and shadings represent the standard error of the mean (SEM). (C–D) Change in the intensity of GCaMP6f fluorescence for lratd2a neurons in the right dHb in response to consecutive delivery of (C) water or cadaverine [n = 30 neurons in three larvae, 0.009 ± 0.039 ΔF/F for water, 0.148 ± 0.074 ΔF/F for cadaverine] and of (D) water or chondroitin sulfate [n = 43 neurons in four larvae, 0.039 ± 0.037 ΔF/F for water, 0.176 ± 0.069 ΔF/F for chondroitin sulfate] at seven dpf, respectively. Two-way ANOVA reveals a significant effect of time [F(1, 29) = 32.09, p < 0.0001], interaction [F(3, 87) = 3.797, p = 0.0131] but no effect of vehicle vs. odorants [F(3, 87) = 1.169, p = 0.3262] for cadaverine; and a significant effect of time [F(1, 42) = 4.754, p = 0.0349], vehicle vs. odorants [F(3, 126) = 2.825, p = 0.0414] and interaction [F(3, 126) = 4.256, p = 0.0067] for chondroitin sulfate. Post-hoc analysis by Bonferroni’s multiple comparisons. (E–G) Colocalization of fos and lratd2a transcripts in transverse sections of adult olfactory bulbs (left panels) and habenulae (middle panels) detected by double labeling RNA in situ hybridization 30 min after addition of (E) water, (F) cadaverine, or (G) alarm substance to the test tank. (E’-G’) Higher magnification images (corresponding to dashed boxes in E-G) show lratd2a (brown) coexpressed with fos (blue) in cells of the right dHb (arrowheads). Scale bars, 100 μm. (H) Quantification of fos-expressing cells in the adult dHb after addition of water [3.58 ± 0.811 cells in the left and 5.61 ± 0.85 in the right dHb, n = 31 sections from 16 adult brains], cadaverine [5.32 ± 1.36 cells in the left and 15.73 ± 1.25 in the right dHb, n = 22 sections from 11 adult brains], or alarm substance [20.72 ± 2.70 cells in the left and 20.31 ± 2.53 in the right dHb, n = 29 sections from 17 adult brains]. For the right dHb, significantly more cells were fos positive after addition of cadaverine (p = 0.0031) or alarm substance (p < 0.0001). For the left dHb, a significant difference was only observed after addition of alarm substance (p < 0.0001). Two-way mixed ANOVA reveals a significant effect of group [F(2, 60) = 30.18, p < 0.0001], left vs. right [F(1, 30) = 13.02, p = 0.0011] and interaction [F(2, 38) = 7.881, p = 0.0014]. Post-hoc analysis by Bonferroni’s multiple comparisons. All numbers represent the mean ± SEM.

To examine whether the response to aversive odorants persists in the olfactory-dHb pathway of adult zebrafish, we used expression of the fos gene as an indicator of neuronal activation (deCarvalho et al., 2013; Hong et al., 2013). Consistent with previous findings (Dieris et al., 2017), cadaverine broadly activated OB mitral neurons in the dorsal glomerulus (dG), dorso-lateral glomerulus (dlG), medio-anterior glomerulus (maG), medio-dorsal glomerulus (mdG), lateral glomerulus (lG). In addition, we observed a threefold increase in the number of fos-expressing neurons in the right dHb following exposure of adult zebrafish to cadaverine relative to delivery of water alone (15.73 ± 1.25 vs 5.61 ± 1.07 cells, Figure 2E and F). The position of the fos-expressing cells in the right dHb corresponded to that of the lratd2a-expressing neurons (11.00% ± 1.07 of lratd2a-expressing region; Figure 2F’), supporting that the lratd2a subpopulation responds to cadaverine in both larvae and adults.

Exposure to alarm substance prepared from adult zebrafish increased the number of fos-expressing cells in the lateral glomerulus (lG) and dlG of the OB as would be expected (Mathuru et al., 2012; Yoshihara, 2014), but also in the dorso-lateral region of the dHb (Figure 2G and G’), where transcripts co-localized to lratd2a-expressing neurons. In contrast to cadaverine, alarm substance activated neurons equally in both the left and right dHb (20.72 ± 2.70 cells on the left and 20.31 ± 2.53 on the right; Figure 2H).

Synaptic inhibition of right dHb lratd2a neurons reduces aversive response to cadaverine

To confirm that lratd2a expressing dHb neurons play a role in processing of aversive olfactory cues, we inhibited synaptic transmission in these cells and tested adults for their reaction to cadaverine. The odorant was introduced at one end of a test tank and the time individuals spent within or outside of this region of the tank was measured. We used a preference index that is based on the position of an individual fish at a given time relative to the application site of the odorant (refer to Materials and methods).

The Tg(lratd2a:QF2) driver line is expected to inhibit lratd2a-expressing neurons in both the vHb as well as in the right dHb. We therefore devised an intersectional strategy to block the activity of neurons selectively in the right dHb, which combines Cre/lox-mediated recombination (Förster et al., 2017; Satou et al., 2013; Tabor et al., 2019) and the QF2/QUAS system (Ghosh and Halpern, 2016; Subedi et al., 2014). We produced transgenic fish expressing Cre recombinase under the control of the endogenous solute carrier family 5 member 7 a (slc5a7a) gene using CRISPR/Cas9 targeted integration (Kimura et al., 2014; Figure 3A–B). slc5a7a encodes a choline transporter involved in acetylcholine biosynthesis and, in zebrafish larvae, is strongly expressed in the right dHb and not in the vHb (Hong et al., 2013). Accordingly, in larvae bearing the three transgenes Tg(lratd2a:QF2)c601, Tg(slc5a7a:Cre)c662 and Tg(QUAS:loxP-mCherry-loxP-GFP-CAAX)c679, Cre-mediated recombination resulted in a switch in reporter labeling from red to green in right dHb neurons (Figure 3C). We followed a similar approach to inhibit synaptic transmission from lratd2a right dHb neurons using Botulinum neurotoxin (Lal et al., 2018; Sternberg et al., 2016; Zhang et al., 2017). A BoTxBLC-GFP fusion protein was placed downstream of a floxed mCherry reporter to generate Tg(QUAS:loxP-mCherry-loxP-BoTxBLC-GFP)c674. To validate the effectiveness of this transgenic line, a neuron specific promoter from a Xenopus neural-specific beta tubulin (Xla.Tubb2) gene was used to drive QF2 expression. Larvae bearing Tg(Xla.Tubb2:QF2;he1.1:mCherry)c663; Tg(slc5a7a:Cre)c662 and Tg(QUAS:loxP-mCherry-loxP-BoTxBLC-GFP)c674 showed a significantly reduced response to a touch stimulus, indicating that the neurotoxin was produced in the presence of Cre recombinase (Figure 3—figure supplement 2, Video 1).

Figure 3 with 4 supplements see all
Synaptic inhibition of lratd2a right dHb neurons attenuates response to cadaverine.

(A) Sequences upstream of the slc5a7a transcriptional start site before (WT) and after integration of Cre (blue indicates donor DNA) at sgRNA target site (red nucleotides and PAM sequences in green). (B) Schematic diagram of intersectional strategy using Cre/lox mediated recombination and the QF2/QUAS binary system. QF2 is driven by lratd2a regulatory sequences and the slc5a7a promoter drives Cre leading to reporter/effector expression in lratd2a neurons in the right dHb. (C) Dorsal view of GFP labeling in only the right dHb after Cre-mediated recombination in a 5 dpf Tg(lratd2a:QF2), Tg(slc5a7a:Cre), Tg(QUAS:loxP-mCherry-loxP-GFP-CAAX) larva. Scale bar, 25 μm. (D) BoTxBLC-GFP-labeled cells (open arrowhead) in the right dHb in Tg(lratd2a:QF2), Tg(slc5a7a:Cre), Tg(QUAS:loxP-mCherry-loxP-BoTxBLC-GFP) 5 dpf, 37 dpf, and 4 mpf zebrafish. Upper images show mCherry-labeled lratd2a Hb neurons, middle images show the subset of right dHb neurons that switched to GFP expression, and the bottom row are merged images. Scale bar, 50 μm. (E) Transverse section of BoTxBLC-GFP labeled axonal endings of dHb neurons that express Cre and lratd2a in a subregion of the vIPN (bracket) in Tg(lratd2a:QF2), Tg(slc5a7a:Cre), Tg(QUAS:loxP-mCherry-loxP-BoTxBLC-GFP) 37 dpf juveniles. Scale bar, 50 μm. (F, G) Preferred tank location prior to and after cadaverine addition of adults genotyped for absence (Cre-) or presence (Cre+) of Tg(slc5a7a:Cre). (F) Representative 1 min traces for single Cre- (blue) and Cre+ (purple) adults recorded over 10 min prior to (min 0–5) and after (min 6–10) addition of cadaverine to one end of the test tank (open arrows). (G) Preference index for all adults for an average of 2 min before (white) and for each of 3 min after (gray) the addition of cadaverine. In Cre- fish, aversive behavior was significantly increased at 2 min (p = 0.0116) and 3 min (p = 0.0344), n = 15 fish for each group. In contrast, Cre+ fish, showed no significant difference in their preferred location over time. Dashed red lines in F and G denote midpoint of test tank. Two-way ANOVA reveals significant effects of time [F(3, 27) = 29, p < 0.0001], but no effect of group [F(1, 14) = 2.381] and interaction [F(3, 33) = 1.813]. Post-hoc analysis by Bonferroni’s multiple comparisons. (H) Swimming speed during 1 min period before and after addition of alarm substance was similar for Cre- [3.68 ± 0.47 and 7.43 ± 1.1 cm/s] and Cre+ [4.02 ± 0.42 s and 7.93 ± 0.92 cm/s] adults. Two-way ANOVA reveals significant effects of time [F(1, 16) = 39.61, p < 0.0001], but no effect of group [F(1,16) = 0.2236] and interaction [F(1,16) = 0.0141]. Post-hoc analysis by Bonferroni’s multiple comparisons. (I) Duration in the upper half of the test tank prior to and after addition of alarm substance for Cre+ adults was 96.6 ± 15.72 s and 13.23 ± 3.34 s and for Cre- adults was 125.53 ± 18.6 s and 8.26 ± 2.5 s. Two-way ANOVA reveals a significant effect of time [F(1, 16) = 63.79, p < 0.0001], but no effect of group [F(1,16) = 1.048] and interaction [F(1,16) = 0.0141]. Post-hoc analysis by Bonferroni’s multiple comparisons. (J) Onset of fast swimming after application of alarm substance was observed at 25 ± 4.05 and at 22.7 ± 3.72 sec for Cre- and Cre+ fish, respectively [p = 0.679, unpaired t-test]. (K) Time interval between increased swimming speed and freezing behavior for Cre- (68.88 ± 23.36 s) and Cre+ (20.88 ± 3.93 s) adults [p = 0.051, unpaired t-test]. For H-K, all numbers represent the mean ± SEM.

Video 1
Behavior of Tg(Xla.Tubb:QF2), Tg(QUAS:loxP-mCherry-loxP-BoTxBLC-GFP) 4 dpf larvae with or without the slc5a7a:Cre transgene in response to touch stimulus.

BoTxBLC-GFP was selectively expressed in lratd2a/slc5a7a neurons of the right dHb (Figure 3D) in individuals bearing the three transgenes Tg(lratd2a:QF2)c601, Tg(slc5a7a:Cre)c662, and Tg(QUAS:loxP-mCherry-loxP-BoTxBLC-GFP)c674. Axons labeled by BoTxBLC-GFP terminated at the vIPN (Figure 3E), in the same location as those observed in Tg(lratd2a:QF2), Tg(QUAS:mApple-CAAX) fish (Figure 1G) suggesting that botulinum neurotoxin inhibits synaptic transmission within this restricted region of the vIPN. We confirmed that BoTxBLC-GFP labeling persisted in the right dHb neurons throughout development, although some variability was observed in the number of BoTxBLC-GFP-positive cells between individuals (Figure 3—figure supplement 3).

To determine whether the lratd2a neurons in the right dHb contributed to the aversive response to cadaverine, we monitored the behavior within and between groups of adults that had or did not have the BoTxBLC-GFP transgene. Fish lacking the transgene showed a significantly reduced preference for the side of the tank where cadaverine had been applied (Figure 3G).

When the response of individual fish within each group was compared over time (Figure 3—figure supplement 1), adults with or without BoTxBLC-GFP initially avoided the side of the test tank where cadaverine had been introduced. However, aversion was sustained for 4 min in control fish, but not in those expressing BoTxBLC-GFP in lratd2a neurons. These findings, from statistical tests on individuals both within and between groups, suggest that lratd2a neurons in the right dHb are required for a prolonged aversive response to cadaverine.

Disruption of synaptic transmission in lratd2a-expressing Hb neurons alone did not alter the response of zebrafish to alarm substance, which typically triggers erratic, rapid swimming and bottom dwelling, followed by freezing behavior (Diaz-Verdugo et al., 2019; Jesuthasan and Mathuru, 2008). Similar to controls, both juveniles and adults expressing BoTxBLC-GFP under the control of Tg(lratd2a:QF2)c601 showed rapid swimming/darting behavior within 22–25 s after delivery of alarm substance, first doubling their speed of swimming (Figure 3H–K and Figure 3—figure supplement 4), and then freezing for the duration of the 5 min recording period. Blocking the activity of lratd2a neurons in the right dHb is therefore insufficient to diminish the robust behavioral changes elicited by alarm substance (Figure 3H–K and Figure 3—figure supplement 4).

Zebrafish mutants with habenular defects show altered responses to aversive cues

We examined the response to aversive odorants by tcf7l2zf55 mutants that develop with symmetric left-isomerized dHb and lack the vHb, but are viable to adulthood (Hüsken et al., 2014; Muncan et al., 2007). In agreement with the transformation of dHb identity, projections from OB mitral cells do not terminate in the right dHb of homozygous mutants nor are lratd2a-expressing neurons or their efferents to the vIPN detected (Figure 4A–D).

Figure 4 with 1 supplement see all
Attenuated response to aversive odorants by left-isomerized dHb mutants.

(A–B) (A) Absence of lratd2a-expressing right dHb neurons and (B) right-isomerized expression of kctd12.1 in tcf7l2 mutant larvae at five dpf. (C) Dorsal views of olfactory mitral neuronal projections of Tg(lhx2a:gap-YFP) larvae at 6 dpf. Open arrowhead indicates axon terminals of mitral cells in the WT right dHb that are absent in the mutant. (D) Dorsal views of dHb neuronal projections to the ventral IPN in Tg(lratd2a:QF2), Tg(QUAS:mApple-CAAX) larvae at 6 dpf. (E) Representative traces (1 min) for tcf7l2 mutant and WT sibling adults after application of cadaverine. (F) Preference index for mutants and WT siblings for an average of 2 min before (white) and for each of 3 min after (gray) the addition of cadaverine. Only WT fish showed a significant difference in their preferred location at 1 min (p = 0.0439) and at 2 min (p = 0.0184). For each group, n = 15 adults. Two-way ANOVA reveals a significant effect of time [F(3, 24) = 3.665, p = 0.046], group [F(1, 14) = 6.197, p = 0.026] and interaction [F(3, 30) = 7.953, p = 0.001]. Post-hoc analysis by Bonferroni’s multiple comparisons. Dashed red lines denote midpoint of test tank. (G) Swimming speed for 30 s before and after addition of alarm substance was1.13 ± 0.22 cm/s and 1.89 ± 0.56 cm/s for tcf7l2 homozygotes and 2.84 ± 0.48 cm/s and 4.88 ± 0.63 cm/s for their WT siblings, n = 10 fish for each group. Two-way ANOVA reveals significant effects of time [F(1, 9) = 19.31, p = 0.0021] and group [F(1, 9) = 13.91, p = 0.0047], but no effect of interaction [F(1, 9) = 3.933]. Post-hoc analysis by Bonferroni’s multiple comparisons. (H) Duration in the upper half of the test tank prior to and after addition of alarm substance for tcf7l2 adults was 143.58 ± 24.80 s and 38.77 ± 19.56 s and 43.68 ± 16.35 s and 8.19 ± 6.16 s for their WT siblings, n = 10 fish for each group. Two-way ANOVA reveals significant effects of time [F(1, 9) = 3755, p = 0.0002], group [F(1, 9) = 12.42, p = 0.0065] and interaction [F(1, 9) = 5.877, p = 0.0383]. Post-hoc analysis by Bonferroni’s multiple comparisons. (I) Onset of fast swimming after application of alarm substance occurred at 15 ± 2.65 s for WT and at 136 ± 44.86 s for tcf7l2 fish [p = 0.015, unpaired t-test]. (J) The time interval between increased swimming speed and freezing behavior was 124.2 ± 43.13 s for WT and 257.3 ± 27.43 s for tcf7l2 fish [p = 0.018, unpaired t-test]. For F-J, all numbers represent the mean ± SEM.

Following application of cadaverine, tcf7l2zf55 homozygous adults failed to exhibit the characteristic aversive behavior of their wild-type siblings (Figure 4E and F and Figure 4—figure supplement 1). Exposure to alarm substance also did not elicit a significant increase in swimming speed from baseline (1.13 ± 0.22 cm/s before and 1.89 ± 0.56 cm/s after) relative to WT siblings (2.84 ± 0.48 cm/s before and 4.88 ± 0.63 cm/s after, Figure 4G, I and J). Homozygous tcf7l2zf55 mutants tended to swim more slowly (Figure 4G) and spend more time in the top half of a novel test tank than wild-type adults, although the latter behavior was suppressed in the presence of alarm substance (Figure 4H). tcf7l2zf55 mutants are thus partly responsive to this fearful cue.

To further assess the role of lratd2a-expressing neurons in aversive olfactory processing, we looked at homozygous mutants of the brain-specific homeobox (bsx) gene, which develop right-isomerized dHb [(Schredelseker and Driever, 2018) and Figure 5A] and are viable to adulthood (Schredelseker and Driever, 2018). As might be expected when both dHb have right identity, equivalent populations of lratd2a-expressing neurons were found on both sides of the brain (Figure 5C). Instead of innervating only the right dHb as in controls, the axons of lhx2a:gap-YFP-labeled olfactory mitral cells terminated in the left and right dHb [(Dreosti et al., 2014) and Figure 5B], where the clusters of lratd2a neurons are situated (data not shown). Projections from the lratd2a dHb neurons coursed bilaterally through the left and right fasciculus retroflexus (FR) and innervated the same limited region of the ventral IPN (Figure 5C).

Figure 5 with 1 supplement see all
Enhanced reactivity to alarm substance in mutants with right-isomerized dHb.

(A) Asymmetric expression pattern of kctd12.1 is right-isomerized in bsx homozygotes at five dpf. (B) Projections of Tg(lhx2a:gap-YFP) labeled olfactory mitral cells terminate bilaterally (open arrowheads) in the dHb of bsxm1376 homozygous mutants at five dpf. (C) In bsx mutants, axons from both left (open arrowhead) and right dHb lratd2a neurons project to the same region of the vIPN. Scale bar, 50 μm. (D) Bilateral fos-expressing neurons in right-isomerized mutants. fos (blue) and lratd2a (brown) transcripts in the olfactory bulbs (upper panels) and dHb (bottom panels) of 10-month-old bsxm1376 heterozygotes and homozygous mutants detected by RNA in situ hybridization 30 min after addition of cadaverine to the test tank. Brackets indicate fos-expressing cells. Scale bar, 100 μm. (E) Quantification of fos-expressing cells in the dHb after application of cadaverine in bsxm1376/+ [9.47 ± 2.37 cells on the left and 15.41 ± 2.19 cells on the right, n = 17 sections from nine adults] and bsxm1376/m1376 adults [17.64 ± 1.59 cells on the left and 18.5 ± 1.76 cells on the right, n = 14 sections from eight adults]. Two-way mixed ANOVA reveals significant effect of group [F(1, 16) = 5.178, p = 0.037] and left vs. right [F(1, 16) = 6.885, p = 0.0184], but no effect of interaction [F(1, 10) = 3.85]. Post-hoc analysis by Bonferroni’s multiple comparisons. (F) Preference index for bsx adults for an average of 2 min before (white) and for each of 3 min after (gray) the addition of cadaverine. Both bsx homozygotes and heterozygotes showed reduced responsiveness to cadaverine. Two-way ANOVA reveals significant effect of interaction [F(3, 34) = 5.483, p = 0.005], but no effect of time [F(3, 25) = 0.987] and group [F(1, 14) = 2.728]. Post-hoc analysis by Bonferroni’s multiple comparisons. (G) Swimming speed for 30 s before and after addition of alarm substance. In heterozygous adults, swimming speed was 1.22 ± 0.31 cm/s before and 3.52 ± 0.44 cm/sec after and, in homozygotes, 0.46 ± 0.08 cm/s before and 2.80 ± 0.37 cm/s after, n = 15 adults for each group. Two-way ANOVA reveals significant effect of time [F(1, 14) = 113.4, p < 0.0001], but no effect of group [F(1, 14) = 4.459] and interaction [F(1, 14) = 0.0023]. Post-hoc analysis by Bonferroni’s multiple comparisons. (H) Duration in the upper half of the test tank prior to and after addition of alarm substance for bsxm1376/m1376 adults was 194.86 ± 25.66 s and 51.89 ± 14.84 s and was 63.55 ± 10.11 s and 7.95 ± 3.24 s for bsxm1376/+, n = 15 fish for each group. Two-way ANOVA reveals significant effect of time [F(1, 14) = 44.35, p < 0.0001], group [F(1, 14) = 22.45, p = 0.0003] and interaction [F(1, 14) = 20.89, p = 0.0004]. Post-hoc analysis by Bonferroni’s multiple comparisons. (I) Onset of fast swimming after application of alarm substance was observed at 33.13 ± 19.19 s in bsxm1376/+ and at 37.73 ± 4.27 s in bsxm1376/m1376 fish [p = 0.816, unpaired t-test]. (J) Time interval between increased swimming speed and freezing behavior for bsxm1376/+ (103.1 ± 30.75 s) and for bsxm1376/m1376 (186.7 ± 28.23 s) [p = 0.055, unpaired t-test]. For E-J, all numbers represent the mean ± SEM.

To measure the reaction to cadaverine in bsxm1376/m1376 adults with bilaterally symmetric lratd2a neurons, we counted the number of cells expressing fos in the dHb and found an increase in the left nucleus compared to heterozygous siblings (Figure 5D–E). Despite the symmetric activation of dHb neurons, bsx homozygotes and heterozygotes both showed reduced responsiveness to cadaverine (Figure 5F and Figure 5—figure supplement 1). Overall, homozygous mutants were slower swimmers than heterozygotes (Figure 5G–J); however, after exposure to alarm substance, their swimming speed relative to baseline was twofold faster than that of their heterozygous siblings (Figure 5G), indicative of an enhanced response to this aversive cue.

Discussion

From worms to humans, stimuli including odors are differently perceived by left and right sensory organs to elicit distinct responses (Güntürkün and Ocklenburg, 2017; Güntürkün et al., 2020). Honeybees, for example, show an enhanced performance in olfactory learning when their right antenna is trained to odors (Guo et al., 2016; Letzkus et al., 2006; Rogers and Vallortigara, 2008). In mice, over one third of mitral/tufted cells were found to be interconnected between the ipsilateral and contralateral olfactory bulbs for sharing of odor information received separately from each nostril, and for coordinated perception (Grobman et al., 2018). The zebrafish provides a notable example of a lateralized olfactory pathway, with the discovery of a subset of bilateral mitral cells that project to the dorsal habenulae but terminate only at the right nucleus (Miyasaka et al., 2014; Miyasaka et al., 2009). This finding prompted us to ask what is different about the post-synaptic dHb neurons that receive this olfactory input and what function does this asymmetric pathway serve.

Aversive olfactory cues activate identified neurons in the right dHb

We previously showed that the olfactory mitral cells that express lhx2a and are located in medio-dorsal and ventro-medial bilateral glomerular clusters (Miyasaka et al., 2014; Miyasaka et al., 2009) and project their axons to a subregion of the right dHb where the lratd2a gene is transcribed (deCarvalho et al., 2013). From transgenic labeling with membrane-tagged fluorescent proteins and synaptophysin, we now confirm that the lhx2a olfactory neurons precisely terminate at a cluster of lratd2a/slc5a7a expressing cholinergic neurons present in the right dHb.

Through calcium imaging using a genetically encoded calcium indicator, we validated that the right dHb is responsive when larval zebrafish are exposed to aversive odors such as cadaverine or chondroitin sulfate (Jetti et al., 2014; Krishnan et al., 2014), a component of alarm substance (Mathuru et al., 2012), more specifically, that the lratd2a-expressing neurons of the right dHb significantly respond to these aversive olfactory cues above their response to water alone. As has also been observed by others (Jesuthasan et al., 2021), application of vehicle alone, even when introduced slowly into a testing chamber, is sufficient to elicit a change in GCaMP fluorescence.

In adults, we used fos expression as a measure of neuronal activation and showed that transcripts colocalized to lratd2a-expressing cells. Interestingly, cadaverine predominantly activated neurons in the right dHb in larvae and adults, whereas neurons responsive to alarm substance were detected in both the left and right dHb nuclei of adult zebrafish. Different types of olfactory cues activate distinct glomeruli in the OB (Friedrich and Korsching, 1997; Yoshihara, 2014), and consistent with the prior studies, we observed that, in adults, cadaverine significantly increased fos expression in the mdG and dG regions, the location of lhx2a neurons that project to the right dHb. By contrast, alarm substance predominantly activated neurons in the lG and dlG regions of the OB that innervate the telencephalon and posterior tuberculum (Miyasaka et al., 2014; Miyasaka et al., 2009), suggesting that both dHb receive input via this route rather than through direct olfactory connections. Indeed, we found that more neurons reacted to alarm substance than cadaverine throughout the brain, including in the Dp, Vv and thalamic areas (data not shown).

In previous experiments (deCarvalho et al., 2013), we did not detect activated neurons in the right dHb of adult zebrafish following exposure to cadaverine or alarm substance. Several factors could account for the difference from the earlier study: we now have the transgenic tools to examine lratd2a neurons directly, we used higher concentrations of cadaverine and alarm substance and, in contrast to delivering odorants to groups of zebrafish, we tested the neuronal response in individual adults.

It has been suggested that lateralized olfactory and visual functions of the dHb are more prominent early in development and less so at later stages (Fore et al., 2020). However, the presence of lratd2a-expressing neurons in the right dHb and their preferential response to cadaverine from larval to adult stages supports the persistence of lateralized activity and illustrates the value of examining defined neuronal populations.

Right dHb neurons mediate aversive behavioral responses

As a group, zebrafish in which the synaptic activity of lratd2a neurons in the right dHb was inhibited by BoTxBLC-GFP did not exhibit repulsion to cadaverine, although some individuals showed a mild aversive response that was not sustained relative to sibling controls. This subtle behavioral effect suggests that the entire population of lratd2a neurons may not be effectively inactivated in all animals. The described intersectional approach to suppress neuronal activity may be incomplete, as evidenced by the observed variability in BoTxBLC-GFP labeling between individual larvae. Additionally, compensatory mechanisms occurring between the onset of Cre expression in slc5a7a neurons (at 3 dpf) and adulthood could allow for partial recovery of the response to repulsive cues. It is also possible that the lratd2a neurons are a heterogeneous population, with different subsets controlling the magnitude of the aversive response or the duration. More finely tuned techniques for temporal or spatial regulation of neuronal inactivation would help resolve these issues.

Despite both being aversive cues (Hussain et al., 2013; Mathuru et al., 2012), cadaverine and alarm substance elicit different behavioral responses by adult zebrafish. Control fish show active repulsion to cadaverine for the first to 2–4 min of a 5 min testing period, whereas alarm substance triggers immediate erratic behavior such as rapid swimming and darting that is typically followed by prolonged freezing (Hussain et al., 2013; Mathuru et al., 2012). Therefore, it is not necessarily expected that the same neuronal populations will mediate the response to both substances.

Perturbation of the lratd2a-expressing right dHb neurons either selectively by BoTxBLC-mediated synaptic inactivation, or in tcf7l2zf55 homozygous mutants that completely lack them, reduced aversion to cadaverine, either in the length or degree of the response. In contrast to juveniles or adults with BoTxBLC inactivated neurons that displayed a similar response to alarm substance as controls, tcf7l2zf55 mutants, showed no difference in their swimming behavior before and after its addition. One explanation is that many regions throughout the brain are likely involved in directing the complex repertoire of behaviors elicited by alarm substance and inactivation of lratd2a neurons in the habenular region alone is insufficient to weaken the overall response. Furthermore, the tcf7l2zf55 mutation could disrupt other brain regions that regulate behaviors elicited by alarm substance since the tfc7l2 gene is expressed in neurons throughout the brain, including the anterior tectum, dorsal thalamus and the hindbrain (Young et al., 2002).

Similar to tcf7l2zf55, the bsxm1376 mutation is pleiotropic resulting in right-isomerization of the dHb due to the absence of the parapineal (Schredelseker and Driever, 2018), and also the loss of the terminal tuberal hypothalamus, mammillary hypothalamic regions and secondary prosencephalon (Schredelseker et al., 2020). We did not observe enhanced or prolonged aversion to cadaverine in bsxm1376 homozygotes relative to controls. However, although homozygous mutants did show a hyperactive response to alarm substance, we cannot discount the involvement of other affected brain regions. Albeit technically challenging in adults, a more selective test such as optogenetic activation of only the lratd2a dHb neurons in wild-type and mutant zebrafish could help resolve their contribution to the alarm response.

The identification of a subset of neurons in the right dHb that receive olfactory input and terminate their axons at a defined subregion of the ventral IPN lays the groundwork for tracing an entire pathway from olfactory receptors to the neurons directing the appropriate behavioral response. The midline IPN has been morphologically defined into subregions (deCarvalho et al., 2014 ; Lima et al., 2017; Quina et al., 2017), but their connectivity and functional properties are not well studied. Recent work has begun to assign different functions to given subregions, such as the role of the rostral IPN in nicotine aversion (Morton et al., 2018; Quina et al., 2017). Neurons in the ventral IPN project to the raphe nucleus (Agetsuma et al., 2010; Lima et al., 2017), but the precise identity of raphe neurons that are innervated by the lratd2a-expressing dHb neurons remains to be determined. Transcriptional profiling of the IPN should yield useful information on its diverse neuronal populations and likely lead to the identification of the relevant post-synaptic targets in the ventral IPN and their efferent connections. Elaboration of this pathway may also help explain the advantage of lateralization in the processing of aversive information. It has been argued, for instance, that the antennal specialization to aversive odors in bees is correlated with directed turning away from the stimulus and escape (Rogers and Vallortigara, 2019). Directional turning has also been observed in larval zebrafish (Horstick et al., 2020), but whether it is correlated with laterality of the Hb-IPN pathway is unclear. Beyond olfaction, left-right asymmetry appears to be a more general feature of stress-inducing, aversive responses as demonstrated for the rat ventral hippocampus (Sakaguchi and Sakurai, 2017) and human pre-frontal cortex, where heightened anxiety also activates more neurons on the right than on the left (Avram et al., 2010; Ocklenburg et al., 2016).

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Danio rerio)Tg(lratd2a:QF2)c601This paperTransgenic,Halpern lab
Genetic reagent (Danio rerio)Tg(slc5a7a:Cre)c662This paperTransgenic,Halpern lab
Genetic reagent (Danio rerio)Tg(Xla.Tubb2:QF2;
he1.1:mCherry)c663
This paperTransgenic,Halpern lab
Genetic reagent (Danio rerio)Tg(QUAS:GCaMP6f)c587This paperTransgenic,Halpern lab
Genetic reagent (Danio rerio)Tg(QUAS:GFP)c403Subedi et al., 2014Transgenic,Halpern lab
Genetic reagent (Danio rerio)Tg(QUAS:mApple-CAAX;he1.1:mCherry)c636This paperTransgenic,Halpern lab
Genetic reagent (Danio rerio)Tg(QUAS:loxP-mCherry-loxP-GFP-CAAX)c679This paperTransgenic,Halpern lab
Genetic reagent (Danio rerio)Tg(QUAS:loxP-mCherry-loxP-BoTxBLC-GFP)c674This paperTransgenic,Halpern lab
Genetic reagent (Danio rerio)Tg(–10lhx2a:gap-EYFP)zf177Miyasaka et al., 2009RRID:ZFIN_ZDB-GENO-100504-13Transgenic
Genetic reagent (Danio rerio)Tg(lhx2a:syp-GFP)zf186Miyasaka et al., 2009RRID:ZFIN_ZDB-GENO-100504-13Transgenic
Genetic reagent (Danio rerio)tcf7l2zf55Muncan et al., 2007RRID:ZFIN_ZDB-GENO-071217-3Mutant
Genetic reagent (Danio rerio)bsxm1376Schredelseker and Driever, 2018RRID:ZFIN_ZDB-GENO-180802-1Mutant
Chemical compound, drugalpha-BungarotoxinInvitrogenCat# B-1601(1 mg/ml)
Chemical compound, drugCadaverineSigma-AldrichCat# 33,211(100 μM)
Chemical compound, drugChondroitin sulfate sodium salt from shark cartilageSigma-AldrichCat# C4384(100 μg/ml)
Chemical compound, drugT7 Endonuclease INEBCat# M0302L
Chemical compound, drugMAXIscript T7 Transcription KitInvitrogenCat# AM1312
Chemical compound, drugmMASSAGE mMACHINE T3 Transcription KitInvitrogenCat# AM1348
Chemical compound, drugGateway BP Clonase II Enzyme mixThermo Fisher ScientificCat# 11789020
Chemical compound, drugGateway LR Clonase II Enzyme mixThermo Fisher ScientificCat# 11791020
Chemical compound, drugDIG RNA Labeling MixRocheCat# 11277073910
Chemical compound, drug5-bromo-4-chloro-3-indolyl-phosphate, 4-toluidine salt (BCIP)RocheCat# 11383221001
Chemical compound, drug4-Nitro blue tetrazolium chloride, solution (NBT)RocheCat# 11383213001
Chemical compound, drug2-(4-Iodophenyl)–3-(4-nitrophenyl)–5-phenyltetrazolium Chloride (INT)FisherScientificCat# I00671G
AntibodyAnti-Digoxigenin-AP, Fab fragments antibody(Sheep polyclonal)RocheCat# 11093274910,(1:5000)
AntibodyAnti- Fluorescein -AP, Fab fragments antibody(Sheep polyclonal)RocheCat# 11426338910(1:5000)
Recombinant DNA reagentPlasmid: Gbait-hs-Gal4Kimura et al., 2014N/A
Recombinant DNA reagentPlasmid: NBeta-pEGFP-1Gift fromPaul KriegN/A
Recombinant DNA reagentPlasmid: Gbait-hsp70-QF2-SV40pAThis paperAddgene Plasmid#122,563Donor plasmid for integration,Halpern lab
Recombinant DNA reagentPlasmid: Gbait-hsp70-Cre-SV40pAThis paperAddgene Plasmid#122,562Donor plasmid for integration,Halpern lab
Recombinant DNA reagentPlasmid: pDR274Hwang et al., 2013Addgene Plasmid#42,250
Recombinant DNA reagentPlasmid: GFP sgRNAAuer et al., 2014N/A
Recombinant DNA reagentPlasmid: pT3TS-nCas9nJao et al., 2013#46,757
Recombinant DNA reagentpGEM-T Easy VectorPromegaCatalog# A1360
Software, AlgorithmFijiSchindelin et al., 2012https://imagej.net/Fiji
Software, AlgorithmMATLABThe MathWorkshttps://www.mathworks.com/
Software, AlgorithmExcelMicrosofthttp://products.office.com/en-us/excel
Software, AlgorithmGraphPad Prism9GraphPad Softwarehttps://www.graphpad.com/
Software, AlgorithmZebraLabViewpoint Life Scienceshttp://www.viewpoint.fr/en/home

Zebrafish

Zebrafish were maintained at 27 °C under a 14:10 hr light/dark cycle in a recirculating system with dechlorinated water (system water). The AB wild-type strain (Walker, 1998), transgenic lines Tg(lratd2a:QF2)c601, Tg(slc5a7a:Cre)c662, Tg(Xla.Tubb2:QF2;he1.1:mCherry)c663, Tg(QUAS:GCaMP6f)c587, Tg(QUAS:GFP)c403 (Subedi et al., 2014), Tg(QUAS:mApple-CAAX;he1.1:mCherry)c636, Tg(QUAS:loxP-mCherry-loxP-GFP-CAAX)c679, and Tg(QUAS:loxP-mCherry-loxP-BoTxBLC-GFP)c674, Tg(–10lhx2a:gap-EYFP)zf177 (formally known as Tg(lhx2a:gap-YFP)) (Miyasaka et al., 2009), Tg(lhx2a:syp-GFP)zf186 (Miyasaka et al., 2009), and mutant strains tcf7l2zf55 (Muncan et al., 2007) and bsxm1376 (Schredelseker and Driever, 2018) were used. For imaging, embryos and larvae were transferred to system water containing 0.003% phenylthiourea (PTU) to inhibit melanin pigmentation. All zebrafish protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Carnegie Institution for Science [Protocol #122] or Dartmouth College [Protocol #00002253(m3a)].

Generation of transgenic lines by Tol2 transgenesis

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The MultiSite Gateway-based construction kit (Kwan et al., 2007) was used to create transgenic constructs for Tol2 transposition. A 16 bp QUAS sequence (Potter et al., 2010), was cloned into the 5’ entry vector (pDONRP4-P1R, #219 of Tol2kit v1.2) via a BP reaction (11789020, Thermo Fisher Scientific). Middle entry vectors (pDONR221, #218 of Tol2kit v1.2 Kwan et al., 2007) were generated for QF2, mApple-CAAX, loxP-mCherry-stop-loxP and GCaMP6f. Sequences corresponding to the SV40 poly A tail, the SV40 poly A tail followed by a secondary marker consisting of the zebrafish hatching enzyme one promoter (Xie et al., 2012) driving mCherry, or to BoTxBLC-GFP (Lal et al., 2018; Sternberg et al., 2016; Zhang et al., 2017) were cloned into the 3’ entry vector (pDONRP2R-P3, #220 of Tol2kit v1.2 Kwan et al., 2007). Final constructs were created using an LR reaction (11791020, Thermo Fisher Scientific) into a Tol2 destination vector (pDestTol2pA2, #394 of the Tol2kit v1.2 Kwan et al., 2007; Supplementary file 1).

To produce Tol2 transposase mRNA, pCS-zT2TP was digested by NotI and RNA synthesized using the mMESSAGE mMACHINE SP6 Transcription Kit (AM1340, Thermo Fisher Scientific). RNA was purified by phenol/chloroform-isoamyl extraction, followed by chloroform extraction and isopropanol precipitation (Suster et al., 2011). A solution containing QF2/QUAS plasmid DNA (~25 ng/μl), transposase mRNA (~25 ng/μl) and phenol red (0.5%) was microinjected into one-cell stage zebrafish embryos, which were raised to adulthood. To identify transgenic founders, F0 adult fish were outcrossed to AB and embryos were assessed for the presence of the secondary marker by screening for mCherry labeling of hatching gland cells after 24 hr post fertilization (hpf) and raised to adulthood.

Generation of transgenic lines by genome editing

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For generating transgenic lines at targeted sites, we performed CRISPR/Cas9-mediated genome editing using the method of Kimura et al., 2014, which relies on homology-independent repair of double-strand breaks for integration of donor DNA. To construct the donor DNA, we combined GFP bait sequences (Gbait) and the hsp70 promoter fragment (Kimura et al., 2014), with a QF2 sequence, which contains the DNA binding and transcriptional activation domains of the QF transcription factor of Neurospora crassa (Ghosh and Halpern, 2016; Subedi et al., 2014). The Gbait-hsp70 sequence was amplified with forward 5’- GGCGAGGGCGATGCCACCTACGG-3’ and reverse 5’- CCGCGGCAAGAAACTGCAATAAAAAAAAC-3’ primers, using Gbait-hsp70:Gal4 donor DNA (Kimura et al., 2014). QF2 sequence was amplified with forward 5’- ACTAGTATGCCACCCAAGCGCAAAACGC-3’ and reverse 5’- CTGCAGCAACTATGTATAATAAAGTTGAAA-3’ primers, using pDEST:QF2 template DNA. Subsequently, the Gbait-hsp70 fragment and QF2 fragment were independently inserted into pGEM T-easy (A1360, Promega) and subsequently combined into one vector by SacII digestion and ligation (Addgene, plasmid #122563). The Cre sequence was amplified using pCR8GW-Cre-pA-FRT-kan-FRT as template DNA (Suster et al., 2011) (forward 5’-ACTAGTGCCACCATGGCCAATTTACTG-3’ and reverse 5’-CTGCAGGGACAAACCACAACTAGA-3’ primers), and inserted into pGEM T-easy. The Gbait-hsp70 fragment was subcloned into the Cre vector by SacII digestion and ligation (Addgene, plasmid #122562).

Production of sgRNAs and Cas9 RNA was performed as described previously (Hwang et al., 2013; Jao et al., 2013). Potential sgRNAs were designed using Zifit (Sander et al., 2010). Pairs of synthetic oligonucleotides (lratd2a sense, 5’-TAGGACTGGACACCGAAGAAGA-3’; lratd2a anti-sense, 5’-AAACTCTTCTTCGGTGTCCAGT-3’; slc5a7a sense, 5’-TAGGCTCTTTGTGCACTGTTGG-3’; slc5a7a anti-sense, 5’-AAACCCAACAGTGCACAAAGAG-3’), 5’-TAGG-N18-3’ and 5’-AAAC-N18-3’, were annealed and inserted at the BsaI site of the pDR274 vector (Addgene, plasmid #42250). To make sgRNA and Cas9 mRNA, template DNA for sgRNAs and pT3TS nCas9n (Addgene, plasmid #46757) were digested by DraI and XbaI, respectively. The MAXIscript T7 Transcription Kit (AM1312, Thermo Fisher Scientific) was used for synthesis of sgRNAs from linearized DNA template and the mMESSAGE mMACHINE T3 Transcription Kit (AM1348, Thermo Fisher Scientific) for synthesis of Cas9 RNA. RNA was purified by phenol/chloroform and precipitated by isopropanol.

A solution containing sgRNA for the targeted gene (~50 ng/μl), sgRNA (~50 ng/μl) to linearize donor plasmids at the Gbait site (Auer et al., 2014; Kimura et al., 2014), the Gbait-hsp70-QF2-pA and Gbait-hsp70-Cre-pA (~50 ng/μl) plasmids, Cas9 mRNA (~500 ng/μl), and phenol red (0.5%) was microinjected into one-cell stage embryos. To verify integration of donor DNA at the target locus, PCR was performed using primers that correspond to sequences flanking the integration site and within the donor plasmid (hsp70 reverse, 5’-TCAAGTCGCTTCTCTTCGGT-3’). (For lratd2a, the forward primer is 5’-CTGCTGAAGTGGCATTTATGGGC-3’ and the reverse primer is 5’-CCTGGAAGTCCCCGACATAC-3’; for slc5a7a the forward primer is 5’-CACATCTCTCTGACGTCCATC-3’ and the reverse is 5’-GTTGCTGCGCAGGACTTAAAA-3’). Sequence analysis of PCR products confirmed integration at the targeted sites.

RNA in situ hybridization

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Whole-mount in situ hybridization was performed as previously described (deCarvalho et al., 2014; Gamse et al., 2002). In brief, larvae and dissected brains were fixed in 4% paraformaldehyde (P6148, Sigma-Aldrich) in 1 X PBS (phosphate-buffered saline) at 4 °C overnight. To synthesize RNA probes, the following restriction enzymes and RNA polymerases were used: lratd2a (BamHI/T7), fos (NotI/SP6), slc5a7a (NotI/SP6), kctd12.1 (EcoRI/T7) (deCarvalho et al., 2013; Hong et al., 2013). Probes were labeled with UTP-digoxigenin (11093274910, Roche) and samples incubated at 70 °C in hybridization solution containing 50% formamide. Hybridized probes were detected using alkaline phosphatase-conjugated antibodies (Anti-Digoxigenin-AP, #11093274910, and Anti-Fluorescein-AP, #11426338910, Sigma-Aldrich) and visualized by staining with 4-nitro blue tetrazolium (NBT, #11383213001, Roche), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP, #11383221001, Roche) and 2-(4-Iodophenyl)–3-(4-nitrophenyl)–5-phenyltetrazolium Chloride (INT, #I00671G, Fisher Scientific).

Preparation of odorants

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Alarm substance was freshly prepared on the day of testing. Adult zebrafish (6 female and six male) were anesthetized in 0.02% tricaine (E10521, Ethyl 3-aminobenzoate methanesulfonate; Sigma-Aldrich). Shallow lesions were made on the skin (10 on each side) using a fresh razor blade and single fish were consecutively immersed in a beaker containing distilled water (25 ml for fos experiment and 50 ml for behavioral analyses) for 30 s at 4 °C. The solution was filtered using a 0.2 μm filter (565–0020, ThermoFisher Scientific) and stored at 4 °C until used (Mathuru et al., 2012). The cadaverine (#33211, final concentration 100 μM) and chondroitin sulfate (#c4384, 100 μg/ml) were purchased from Sigma-Aldrich, and stock solutions prepared in distilled water.

Calcium imaging in larval zebrafish

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To monitor activity within the same population of lratd2a neurons in response to odorants or to a vehicle control, calcium signaling was performed on seven dpf Tg(lratd2a:QF2)c644; Tg(QUAS:GCaMP6f)c587 larvae. Larvae were paralyzed by immersion in α-bungarotoxin (20 µl of 1 mg/ml solution in system water, B1601, ThermoFisher Scientific) Duboué et al., 2017; Severi et al., 2014 followed by washing in fresh system water. Individuals were embedded in 1.5% low melting agarose (SeaPlaque Agarose, Lonza) in a petri dish (60 mm) with a custom-designed mold. After solidification, the agarose around the nostrils was carefully removed with forceps for access to odorants, and the individual immersed in fresh system water. The dish was placed under a 20 X (NA = 0.5) water immersion objective on a Zeiss LSM 980 confocal microscope. Images were acquired in XYT acquisition mode with 512 × 200 pixel resolution at a rate of 2 Hz and digitized eight bit from single focal planes. The vehicle control (deionized water) followed by the odorant solution (0.2 ml) was slowly expelled through plastic tubing (Tygon R-3606; 0.8 mm ID, 2.4 mm OD) attached to a 1 ml syringe (BD 309659) and with the other end positioned in front of an individual’s face. Two sequential applications of each were spaced by 1 min intervals. To calculate fluorescence intensity, regions of interest (ROI) were manually drawn around each cell in the average focal plane with the polygon tool and ROI manage in Fiji (Schindelin et al., 2012). To normalize calcium activity for each neuron to baseline fluorescence, the fractional change in fluorescence (ΔF/F) was calculated before the application of odorants (average of 320 frames from each neuron), according to the formula F = (Fi-F0)/F0, where Fi is the fluorescence intensity at a single time point and F0 is the baseline fluorescence. All data and images were analyzed using custom programs in MATLAB (MathWorks, version 7.3), Excel software and GraphPad Prism9.

Assay of Fos expression in adult zebrafish

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Individual adult zebrafish (7–9 months old) were placed in a tank with 1 L system water and acclimated for at least 1 hr prior to odorant exposure. Each odorant solution (1 ml) was gently pipetted into the tank water and the fish was kept there as the odorant diffused. After 30 min, the fish was sacrificed in an ice water slurry, and the brain dissected out and fixed in 4% paraformaldehyde in 1 X PBS overnight at 4 °C. Fixed brains were embedded in 4% low melting agarose in 1 X PBS and sectioned at 50 μm (for juvenile brains) or 70 μm (for adult brains) using a vibratome (VT1000S, Leica Biosystems, Inc). For more precise counting of fos expressing cells in adult brains, habenular sections were 35 μm thick. Sections were covered in 50% glycerol in 1 X PBS under coverslips. Bright-field images were captured with a Zeiss AxioCam HRc camera mounted on a Zeiss Axioskop. A Leica SP5 confocal microscope was used for fluorescent images. Data from fos RNA in situ hybridization experiments were quantified using ImageJ/Fiji software (Schindelin et al., 2012).

Behavioral assays

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Behavioral assays were performed using 5–7 week-old juvenile zebrafish and adults that were of 4–8 months of age. Responses to odorants were measured between 10:00 a.m. and 4:00 p.m. and fish were starved for 1 day prior to testing (Koide et al., 2009). Individual adults were placed in a 1.5 L test tank (Aquatic Habitats) in 1 L of system water and allowed to acclimate for at least 1 hr. For experiments with juveniles, individuals were acclimated to the behavior room for 1 hr, gently netted into the test tank (20 × 9 x 8.3 cm, 1.5 L mating cage) containing 0.6 L system water and maintained there for 5 min prior to testing. Swimming activity was recorded for 5 min (4 min for juveniles) before and after the application of odorants. Odorants (2 ml for adults, 1 ml for juveniles) were slowly expelled through plastic tubing (Tygon R-3606; 0.8 mm ID, 2.4 mm OD) attached on one end to a 3 ml syringe (BD 309657) and on the other positioned at one end of the test tank. A preference index was calculated using the formula: Preference to odorant = [(Total time spent in the half of the tank where odorant was delivered) − (Total time spent in the other half of the tank)]/Total time (Koide et al., 2009; Wakisaka et al., 2017). A preference index of –1 indicates that the position of an individual fish is maximally distanced from the application site of the odorant.

Quantification and statistical analyses

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Data were collected using custom written scripts in MATLAB (MathWorks). Statistical analyses were performed using GraphPad Prism9 (GraphPad Software, Inc). The Wilcoxon signed-rank test was used to compare a set of matched samples. An unpaired t-test was used to compare two independent data sets and two-way ANOVA followed by Bonferroni’s post hoc test was used to compare datasets greater than two. All tests were two-tailed and results depicted as non-significant (ns, p> 0.05) or significant (*, p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting file. Source data files have been provided for Figures 1-5. Behavioral analyses were performed using custom written scripts in MATLAB and uploaded as Source code Files.

References

  1. Book
    1. Ghosh A
    2. Halpern ME
    (2016) Transcriptional regulation using the Q system in transgenic zebrafish
    In: Detrich M, Leonard IZ, editors. Methods in Cell Biology. Academic Press. pp. 205–218.
    https://doi.org/10.1016/bs.mcb.2016.05.001
  2. Book
    1. Siniscalchi M
    (2017) Olfactory Lateralization
    In: Rogers LJ, Vallortigara G, editors. Lateralized Brain Functions: Methods in Human and Non-Human Species. Springer. pp. 103–120.
    https://doi.org/10.1007/978-1-4939-6725-4_4
  3. Book
    1. Walker C
    (1998) Chapter 3 Haploid Screens and Gamma-Ray Mutagenesis
    In: Detrich HW, Westerfield M, Zon LI, editors. Methods in Cell Biology. Academic Press. pp. 43–70.
    https://doi.org/10.1016/S0091-679X(08)61893-2
  4. Book
    1. Yoshihara Y
    (2014) Zebrafish Olfactory System
    In: Mori K, editors. The Olfactory System: From Odor Molecules to Motivational Behaviors. Springer. pp. 71–96.
    https://doi.org/10.1007/978-4-431-54376-3_5

Decision letter

  1. Claire Wyart
    Reviewing Editor; Institut du Cerveau et la Moelle épinière, Hôpital Pitié-Salpêtrière, Sorbonne Universités, UPMC Univ Paris 06, Inserm, CNRS, France
  2. Marianne E Bronner
    Senior Editor; California Institute of Technology, United States
  3. Caroline Lei Wee
    Reviewer; Harvard University, United States
  4. Manuel Mameli
    Reviewer; The University of Lausanne, Switzerland

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Specialized neurons in the right habenula mediate response to aversive olfactory cues" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Marianne Bronner as the Senior Editor. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The three reviewers have appreciated the novelty and originality of the study, but note that improved visualization, quantifications and statistical analyses will be necessary to fully support the conclusions of the manuscript. Without performing these quantifications and statistical tests for all figures as detailed below, the magnitude and significance of reported effects are not clear, nor do they take into account the variability of the measures and the dependence of some of the measures.

1. Anatomy (Figure 1):

The authors should verify the specificity of the novel transgenic lines generated in the study to target lratd2a+ neurons in the right dHb (some left dHb expression is seen in Figure 1C-E). In addition, improving the quantification of c-fos and lratd2a overlap is necessary.

2. Calcium imaging and response to chemical stimuli (Figure 2):

The effects of the aversive chemical stimuli on the calcium activity of the lratd2a+ cells in the dorsal habenula are not clear in the traces presented nor quantified for all cells in all fish recorded. As a matter of fact, the traces of single examples show responses to water alone and not a very clear effect of the olfactory cues. The statistics are lacking to compare the response to water, cadaverine and chondroitin sulfate. The authors should determine whether the olfactory stimulus used induced a larger response than water in the same cells. They should assess the variability across fish, and the reliability of the effects. We advise to use linear mixed models which are suited to take into account the variability of cells within the same fish, as well as the effect of clutch or day of the experiment. In order to to take into account the effect of time and pairing within the same cell that receives the water stimuli before the olfactory ones, we recommend using a linear mixed model with a fixed effect of time (measuring the average or max DFF before, during and after the chemical stimulation) and treatment (olfactory cue added), pairing values within the same cell and same fish.

3. Behavior (Figures 3 and 4):

The effects of genetic manipulations on the response to cadaverine and chondroitin sulfate should be quantified and compared across genotypes using a proper statistical tests. In the submitted manuscript, the authors only quantified an effect of the drug within each group using t-tests using only the last point before the drug was applied, instead of comparing between the groups the effects of different genotypes on the preference index at all time points (before and after the application of the olfactory cue). This is an issue for both figures, note that in Figure 4 the preference index during baseline might differ between the wild type and mutant group. The authors should use a two-way repeated measures analysis of variance (ANOVA), or Kruskal-Wallis test for non-parametric data, to assess differences between treatment groups over time for validation studies, and test the effect of the drug over time within each group and between conditions before and after. By doing so, the authors will determine whether there is an effect of time, whether there is an effect of genotype and if there is an effect of time, measure the effect of an interaction. Note that if the authors opt for the ANOVA, they should perform post hoc comparison tests using either the Tukey method (commonly used to make pairwise comparisons) by correcting for multiple testing or t-tests with Bonferroni correction for multiple comparison. By doing so, the authors will determine whether there is an effect of time, whether there is an effect of genotype and measure the effect of a possible interaction.

Reviewer #1 (Recommendations for the authors):

Figure 1: Anatomy

To show that lratd2a+ neurons receive inputs from the olfactory bulb, the authors rely on gross low scale overlap of YFP+ axons on the soma of mApple-CAAX in the triple transgenic line lratd2a:QF2; QUAS:mApple-CAAX; lhx2a:YFP,

– A: First thing, add a quantification of (a) the number of neurons that are expressing lratd2a in the wild type fish at 7, 14, 21dpf and adult stage and (b) the number of neurons labeled in the lratd2a:QF2 transgenic line and the left and right side as it is central to the study – in the C panel, there seems to be expression of the transgene obtained by KI in the lratd2a locus (lratd2a:QF2)c601 in the left side.

– Next, add histological examinations or higher fluorescent images for panels D-D".

– Finally, can you specify if the overlap has been observed at the level of single planes or projections as it should be to suggest direct connectivity? If so, add a quantification to illustrate the overlap based on single planes from optical sections obtained on confocal with high mag and resolution and close up.

Figure 2: Calcium Imaging

– A: in order to understand whether the response of lratd2a+ neurons is specific to aversive cues: can you show the response to (a) water (as illustrated in B), (b) one other aversive cue as well as (c) one non aversive cues?

– A: for 14 dpf and response to chondroitin sulfate, please add the number of neurons missing.

– A: for 21 dpf and response to chondroitin sulfate, how come there are only 16 neurons measured instead of ~>35 ? Does it mean that the line is variegated and numbers can vary from 15 to 40 in the right habenula?

– B: since spontaneous activity is large and we cannot conclude for single examples, panels need to show the variability across cells and not only single trace : (a) use mean and ste to show the intrinsic variability of the response across cells and (b) quantify the response by calculating the peak and subtracting the baseline averaged over a similar time window before stimulation.

– C: the response to cadaverine measured with cfos is not clearly overlapping with expression of lratd2a: the authors should perform double fluorescent in situ labeling to demonstrate the overlap at the cellular level.

Figure 3: Behavior of adult zebrafish expressing botulinum toxin in a subset of neurons in the right dorsal habenula.

– A-E: the authors should validate that the intersectional genetics combining a KI line, the QF2/QUAS and Cre under the scl5a7a promoter is effective to target the lratd2a neurons only and which proportion of them. On these images, it appears that the KI line has expression on the left Habenula as well.

– G-J: The effect of the expression of the botulinum toxin is not clear at all (for the preference index: T-test a single time points on a subset of them can be misleading) statistics need to be improved : if the data is quantified and plotted every minute, we would expect to compare the conditions before baseline and establish that there is no difference in preference index before addition of cadaverine, and that a difference is observed after the addition and quantify for how long.

For the alarm substance, the data is not represented the same as for cadaverine: in the single measure of before/after, there is no difference across genotypes in speed, onset time of the fast swim, or time between fast swimming and freezing. But would there be a difference for cadaverine using the same single measure of before/after ? Probably not.

The statistics and choice of parameters needs to be sorted out and represented fully and fairly with consistency across compounds and figures.

Figure 4: Behavior of left-isomerized adult zebrafish.

– Expression of lratd2a is affected in the dorsal right and ventral right and left habenula so the mutant does not reveal only the role of lratd2a+ neurons in the dorsal right locus.

– Same issue here for the behavior: the pre-condition appears possibly different for the homozygous mutant and control sibling. The authors should test whether there are any difference of preference index in the two groups before drug application, and after drug application.

– Why are again different parameters plotted in H-G for the alarm substance compared to cadaverine? In addition instead of time onset and interval between fast and freeze, duration in the top of the tank is quantified. This choice looks arbitrary and all parameters should be chosen and kept the same for comparing the effects of cadaverine and alarm substance.

Reviewer #2 (Recommendations for the authors):

1. If calcium imaging experiments were to be repeated, water and odorant cues should be alternated so a direct comparison can be made for individual neurons. Also, both sides of the habenula could be simultaneously imaged, with lratd2a neurons labeled with another (e.g. RFP) marker, to allow for comparison between left and right habenular responses.

2. Can c-fos experiments be performed on the lratd2a transgenic background in adults to facilitate quantification?

3. While not necessary for this paper, chemogenetic approaches (e.g. TRPV1 from Prober lab) could be useful to activate the population

Reviewer #3 (Recommendations for the authors):

The authors of this work describe how cholinergic neurons expressing the lratd2a gene of the right dHb increase their activity to aversive odorant guiding aversive behaviors. The design of the study is very elegant, especially exciting is the combination of genetic tools that allow to label, as well as manipulate synaptic function. The authors present elegantly their data, and my impression is that this work deserves publication. The author may want to consider the following points:

1. The concepts of aversion and avoidance are confusing. Avoidance implies a form of learning (see reviews from J LeDoux) after an individual learns to anticipate an upcoming aversive stimulus. If I correctly interpreted the authors use a on-line reading of escape/aversive behaviour after inclusion of cadaverine. This should be probably better defined throughout the text.

2. At one point in the results the authors make use of a genetic approach allowing to control synaptic function with Botulinun neurotoxin. They state that "Axons labeled by BoTxBLC-GFP terminated at the vIPN.…. suggesting that botulinum neurotoxin inhibits synaptic transmission within this restricted region of the vIPN". I understand the technology is used in published data, yet it would be elegant to show along with the behavioural results an assessment of collapsed synaptic function.

3. The authors use a labeling strategy that allows claiming that the cholinergic neurons innervate a precise area of the IPN, supporting previous data in literature. In their data set the authors however study the functional responses to cadaverine only in somata of these neurons. It would be extremely relevat in my opinion to show that calcium transients are also detectable in the Cholinergic axons in the IPN. This would corroborate the functional integration of aversive signal within this neuronal circuit, and not only within the right habenula.

4. The authors may want to consider referring to the following reviews when citing habenula work in rats, mice, and humans in the context of physiology and disease: Hailan Hu et al., 2020; Lecca et al., 2014; Proulx et al., 2014.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Specialized neurons in the right habenula mediate response to aversive olfactory cues" for further consideration by eLife. Your revised article has been reviewed by 2 peer reviewers and the evaluation has been overseen by Marianne Bronner as the Senior Editor, and a Reviewing Editor.

The authors show an interesting role of a subpopulation of habenular neurons in the avoidance response to aversive olfactory cues. We thank the authors for improving the manuscript, particularly for repeating the calcium imaging experiments and for improving the data presentation. There are however some remaining issues that need to be addressed, as outlined below.

Essential revisions:

1) Overall the behavioral differences to cadaverine shown in Figure 3G (Cre- vs Cre+) are relatively mild, especially as the aversion indices are significantly different from baseline in both Cre- and Cre+ condition (Figure 3—figure supplement 2B). Given that this is a key experiment in the paper, a discussion regarding whether this is a limitation of the existing tools (e.g. insufficient neurons silenced) or a reflection of underlying biology (e.g. redundancy in circuits for avoidance, different circuits controlling duration vs magnitude of aversion) would be beneficial.

2) Presentation of Figure 2c-d can be improved further – the same neurons presumably are being imaged "before" and "after", however the way the data is currently plotted makes it look like they are independent neurons.

3) It is counterintuitive that a negative aversion index means stronger aversion (perhaps call it a preference index instead, or flip the signs so more positive = more aversive).

4) In the aversion assay (3G, 4E, 5F): can the authors clarify if some form of multiple comparisons correction was done in calculating the p-values at each time bin?

5) The authors have performed ANOVA on the aversion indices shown in the supplementary figures, and report a significant effect of odor and odor x group interaction. Is there a significant effect of group alone? there is no explicit mention of the aversion index in the main text, and no interpretation in the figure legends. For clarity, the authors should elaborate how the statistical results from this 2nd analysis method ties in with / complements the statistical methods used in the main figures.

Reviewer #2 (Recommendations for the authors):

I thank the authors for improving on the manuscript, particularly for repeating the calcium imaging experiments and for improving the data presentation.

While significant effort has been put into improving the statistics, I have some additional questions about the analyses performed and their interpretation. Ultimately I will defer to the other reviewers regarding whether they are satisfied with the current methods.

For the main figures, I was expecting a two-way ANOVA to be performed for the time course data in the aversion assay (3G, 4E, 5F) to compare main effects of group and time and group x time interactions. I understand the authors are using a different methodology here (signed rank test) which has also been applied in other papers – however, can I clarify if some form of multiple comparisons correction was done in calculating the p-values at each time bin?

The authors have performed ANOVA on the aversion indices shown in the supplementary figures, and report a significant effect of odor and odor x group interaction. I might have missed something, but is there a significant effect of group alone? I also do not see any explicit mention of the aversion index in the main text, and no interpretation in the figure legends. For clarity, perhaps the authors could elaborate how the statistical results from this 2nd analysis method ties in with / complements the statistical methods used in the main figures?

Overall the behavioral differences to cadaverine shown in Figure 3G (Cre- vs Cre+) are relatively mild, and the aversion indices are significantly different from baseline in both Cre- and Cre+ condition (Figure 3—figure supplement 2B). The data is what it is, but given that this is a key experiment in the paper, a discussion regarding whether this is a limitation of the existing tools (e.g. insufficient neurons silenced) or a reflection of underlying biology (e.g. redundancy in circuits for avoidance, different circuits controlling duration vs magnitude of aversion) could be refreshing.

Reviewer #3 (Recommendations for the authors):

All my specific points were addressed by the authors. The complementary experiments and the modification provided in the text improved the paper and it is in my view suitable for publication.

https://doi.org/10.7554/eLife.72345.sa1

Author response

Essential revisions:

The three reviewers have appreciated the novelty and originality of the study, but note that improved visualization, quantifications and statistical analyses will be necessary to fully support the conclusions of the manuscript. Without performing these quantifications and statistical tests for all figures as detailed below, the magnitude and significance of reported effects are not clear, nor do they take into account the variability of the measures and the dependence of some of the measures.

We thank the editors and reviewers for their positive feedback and for the care they took to assess our data analysis, which we agree was problematic. We corrected all of the statistical tests that were used as suggested by the reviewers and consulted with an expert in biostatistics, Dr. Eugene Demidenko, a Professor in the Department of Biomedical Data Science at the Geisel School of Medicine at Dartmouth, to ensure we were performing the analyses correctly. We also repeated the calcium imaging experiments on 7 day old larvae so that the same neurons were individually monitored for both olfactory cues and vehicle controls.

1. Anatomy (Figure 1):

The authors should verify the specificity of the novel transgenic lines generated in the study to target lratd2a+ neurons in the right dHb (some left dHb expression is seen in Figure 1C-E). In addition, improving the quantification of c-fos and lratd2a overlap is necessary.

The lratd2a gene is asymmetrically expressed in the left and right dHb, with transcripts in more cells and far more abundant on the right than on the left as assayed by RNA in situ hybridization. Consistent with this finding, in the transgenic lines we generated, expression of the QF2 transcription factor under the control of endogenous lratd2a cis-regulatory sequences drives QUAS regulated reporter labeling in more neurons in the right dHb and at higher levels. We have added a new Figure showing the quantification of lratd2a-expressing neurons in the left and right dHb in the QF/QUAS transgenic larvae (Figure 1—figure supplement 2).

We now show magnified images of cells co-expressing fos and lratd2a in Figure 2 to improve visualization of the overlap in colorimetric double in situ hybridization. We attempted to perform these experiments with fluorescently labeled probes, but we were unable to achieve the sensitivity needed to detect both of these relatively low abundance transcripts in doubly labeled cells.

2. Calcium imaging and response to chemical stimuli (Figure 2):

The effects of the aversive chemical stimuli on the calcium activity of the lratd2a+ cells in the dorsal habenula are not clear in the traces presented nor quantified for all cells in all fish recorded. As a matter of fact, the traces of single examples show responses to water alone and not a very clear effect of the olfactory cues. The statistics are lacking to compare the response to water, cadaverine and chondroitin sulfate. The authors should determine whether the olfactory stimulus used induced a larger response than water in the same cells. They should assess the variability across fish, and the reliability of the effects. We advise to use linear mixed models which are suited to take into account the variability of cells within the same fish, as well as the effect of clutch or day of the experiment. In order to to take into account the effect of time and pairing within the same cell that receives the water stimuli before the olfactory ones, we recommend using a linear mixed model with a fixed effect of time (measuring the average or max DFF before, during and after the chemical stimulation) and treatment (olfactory cue added), pairing values within the same cell and same fish.

We agree with these suggestions and have performed new experiments in which we assess the change in GCaMP6f fluorescence in the same set of lratd2a right dHb neurons in response to the application of water or to odorant. We included the standard error of the mean to the traces to show the variability in the response among neurons (refer to Figure 2A-D).

3. Behavior (Figures 3 and 4):

The effects of genetic manipulations on the response to cadaverine and chondroitin sulfate should be quantified and compared across genotypes using a proper statistical tests. In the submitted manuscript, the authors only quantified an effect of the drug within each group using t-tests using only the last point before the drug was applied, instead of comparing between the groups the effects of different genotypes on the preference index at all time points (before and after the application of the olfactory cue). This is an issue for both figures, note that in Figure 4 the preference index during baseline might differ between the wild type and mutant group. The authors should use a two-way repeated measures analysis of variance (ANOVA), or Kruskal-Wallis test for non-parametric data, to assess differences between treatment groups over time for validation studies, and test the effect of the drug over time within each group and between conditions before and after. By doing so, the authors will determine whether there is an effect of time, whether there is an effect of genotype and if there is an effect of time, measure the effect of an interaction. Note that if the authors opt for the ANOVA, they should perform post hoc comparison tests using either the Tukey method (commonly used to make pairwise comparisons) by correcting for multiple testing or t-tests with Bonferroni correction for multiple comparison. By doing so, the authors will determine whether there is an effect of time, whether there is an effect of genotype and measure the effect of a possible interaction.

We have shown responses to odorants within groups and between groups (refer to Figures 2-5, Figure 3—figure supplement 1-2, Figure 4—figure supplement 1 and Figure 5—figure supplement 1). We quantified and compared the response to odorants between groups during 5 min before and after application of cadaverine so the effects of genetic manipulations can be assessed (refer to Figure 3—figure supplement 2, Figure 4—figure supplement 1 and Figure 5—figure supplement 1).

As correctly pointed out, we have now performed the appropriate statistical analyses (refer to Figure legends). For analyzing response to cadaverine within a group, we used the Wilcoxon signed-rank test and paired t-test and cited publications that used these tests to evaluate the results of comparable behavioral experiments (Koide et al., 2009; Wakisaka et al., 2017). For analyzing between groups, we used two-way ANOVA followed by Bonferroni's post hoc test.

Reviewer #1 (Recommendations for the authors):

Figure 1: Anatomy

To show that lratd2a+ neurons receive inputs from the olfactory bulb, the authors rely on gross low scale overlap of YFP+ axons on the soma of mApple-CAAX in the triple transgenic line lratd2a:QF2; QUAS:mApple-CAAX; lhx2a:YFP,

– A: First thing, add a quantification of (a) the number of neurons that are expressing lratd2a in the wild type fish at 7, 14, 21dpf and adult stage and (b) the number of neurons labeled in the lratd2a:QF2 transgenic line and the left and right side as it is central to the study – in the C panel, there seems to be expression of the transgene obtained by KI in the lratd2a locus (lratd2a:QF2)c601 in the left side.

We have added a new Figure showing the quantification of lratd2a-expressing neurons in the left and right dHb in QF/QUAS transgenic larvae (Figure 1—figure supplement 2). Because of the morphology of the adult dHb, it is more challenging to obtain accurate cell numbers for the adult brain.

– Next, add histological examinations or higher fluorescent images for panels D-D".

As suggested, we have added higher magnification single plane confocal images from transverse sections of the adult brain (refer to new Figure 1—figure supplement 3B).

– Finally, can you specify if the overlap has been observed at the level of single planes or projections as it should be to suggest direct connectivity? If so, add a quantification to illustrate the overlap based on single planes from optical sections obtained on confocal with high mag and resolution and close up.

We provided single plane confocal images of the axon terminals of lhx2a neurons labeled with the presynaptic marker synaptophysin (refer to new Figure 1—figure supplement 3A). We also quantified the overlap in labeling of axon terminal and lratd2a neurons in confocal optical sections and now provide this information in the Figure 1—figure supplement 3.

Figure 2: Calcium Imaging

– A: in order to understand whether the response of lratd2a+ neurons is specific to aversive cues: can you show the response to (a) water (as illustrated in B), (b) one other aversive cue as well as (c) one non aversive cues?

In this paper, we focused on the role of lratd2a right dHb neurons in mediating aversive cues as the dorsal habenulae has been implicated in fear-related behaviors (Agetsuma et al., 2010; Lee et al., 2010; Lee et al., 2019; Okamoto et al., 2012).

– A: for 14 dpf and response to chondroitin sulfate, please add the number of neurons missing.

– A: for 21 dpf and response to chondroitin sulfate, how come there are only 16 neurons measured instead of ~>35 ? Does it mean that the line is variegated and numbers can vary from 15 to 40 in the right habenula?

The previous Figure 2 was removed because we repeated calcium imaging experiments (i.e., determined the change in GCaMP6f fluorescence in the same set of lratd2a right dHb neurons in response to the application of water or to odorant). The new results are shown in Figure 2A-D.

– B: since spontaneous activity is large and we cannot conclude for single examples, panels need to show the variability across cells and not only single trace : (a) use mean and ste to show the intrinsic variability of the response across cells and (b) quantify the response by calculating the peak and subtracting the baseline averaged over a similar time window before stimulation.

As suggested, each trace now includes the standard error of the mean to show the variability in the response among neurons. The new figures represent the fluorescent changes above baseline, which are averaged over a similar time window before and after application of water controls and of odorants (refer to Figure 2A and B). We also provide the complete data set for all neurons imaged over a 5 sec interval for two applications of vehicle alone followed by two applications of an odorant, each administered 1 min apart (new Figures 2C and D).

– C: the response to cadaverine measured with cfos is not clearly overlapping with expression of lratd2a: the authors should perform double fluorescent in situ labeling to demonstrate the overlap at the cellular level.

Magnified images of cells co-expressing fos and lratd2a are now shown in Figure 2 to allow better visualization of the overlap in labeling. Double fluorescent in situ hybridization was not sensitive enough to detect these transcripts.

Figure 3: Behavior of adult zebrafish expressing botulinum toxin in a subset of neurons in the right dorsal habenula.

– A-E: the authors should validate that the intersectional genetics combining a KI line, the QF2/QUAS and Cre under the scl5a7a promoter is effective to target the lratd2a neurons only and which proportion of them. On these images, it appears that the KI line has expression on the left Habenula as well.

The lratd2a gene is asymmetrically expressed in the left and right dHb, but expression on the left is in fewer cells and at barely detectable levels. We quantified the number of dHb neurons labeled in the intersectional transgenic approach and included these data in the new (Figure 3—figure supplement 4B).

– G-J: The effect of the expression of the botulinum toxin is not clear at all (for the preference index: T-test a single time points on a subset of them can be misleading) statistics need to be improved : if the data is quantified and plotted every minute, we would expect to compare the conditions before baseline and establish that there is no difference in preference index before addition of cadaverine, and that a difference is observed after the addition and quantify for how long.

The statistics and choice of parameters needs to be sorted out and represented fully and fairly with consistency across compounds and figures.

As pointed out by the reviewer, we corrected the statistical analyses for all behavioral data (please refer to the revised Figure legends). For analyzing the response to cadaverine within groups, we used the Wilcoxon signed-rank test and paired t-test and cited papers (Koide et al., 2009; Wakisaka et al., 2017) and for analyzing the responses between groups, we used two-way ANOVA followed by Bonferroni's post hoc test.

For the alarm substance, the data is not represented the same as for cadaverine: in the single measure of before/after, there is no difference across genotypes in speed, onset time of the fast swim, or time between fast swimming and freezing. But would there be a difference for cadaverine using the same single measure of before/after ? Probably not.

Please refer to the responses below for clarification on the changes that we have now made on depicting data from cadaverine and alarm substance. We have added Figures to be more consistent.

Figure 4: Behavior of left-isomerized adult zebrafish.

– Expression of lratd2a is affected in the dorsal right and ventral right and left habenula so the mutant does not reveal only the role of lratd2a+ neurons in the dorsal right locus.

We had referred to this issue in the discussion: “Our findings also rule out a role for the ventral habenulae in the response to alarm substance, as the reaction to alarm substance was intact in transgenic adults in which lratd2a neurons were inactivated by BoTxBLC in the bilateral vHb as well as in the right dHb.” (page 15, lines 295-297)

– Same issue here for the behavior: the pre-condition appears possibly different for the homozygous mutant and control sibling. The authors should test whether there are any difference of preference index in the two groups before drug application, and after drug application.

We analyzed behavior during the entire experiment (i.e., 5 mins before and after addition of odorant). We have added a new figure that provides the data for all individual fish that were tested (refer to Figure 3—figure supplement 2, Figure 4—figure supplement 1 and Figure 5—figure supplement 1).

– Why are again different parameters plotted in H-G for the alarm substance compared to cadaverine? In addition instead of time onset and interval between fast and freeze, duration in the top of the tank is quantified. This choice looks arbitrary and all parameters should be chosen and kept the same for comparing the effects of cadaverine and alarm substance.

We agree that we could have been more consistent in presenting these results. We now show the same parameters for the response to alarm substance in BoTx-GFP and intersectional BoTx-GFP transgenic lines, and in tcf7l2 and bsx mutants (refer to Figures 3, 4, 5 and Figure 3—figure supplement 1).

Reviewer #2 (Recommendations for the authors):

1. If calcium imaging experiments were to be repeated, water and odorant cues should be alternated so a direct comparison can be made for individual neurons. Also, both sides of the habenula could be simultaneously imaged, with lratd2a neurons labeled with another (e.g. RFP) marker, to allow for comparison between left and right habenular responses.

We agree with the reviewers that it is important to compare responses to an odorants relative to the vehicle control in the same individual cells. We therefore include the results of the requested experiment performed on 7 dpf larvae (refer to new Figure 2A-D).

2. Can c-fos experiments be performed on the lratd2a transgenic background in adults to facilitate quantification?

Fluorescence in situ hybridization with the fos probe was not as sensitive as colorimetric in situ hybridization and we were not able to detect transcripts using this approach. We did not use a GFP probe to label lratd2a neurons in the transgenic background as we would still need to rely on a colorimetric reaction to detect fos transcripts.

3. While not necessary for this paper, chemogenetic approaches (e.g. TRPV1 from Prober lab) could be useful to activate the population.

We do not yet have this reagent under QUAS control, but it is a good idea to build this line for future work. We appreciate the suggestion.

Reviewer #3 (Recommendations for the authors):

The authors of this work describe how cholinergic neurons expressing the lratd2a gene of the right dHb increase their activity to aversive odorant guiding aversive behaviors. The design of the study is very elegant, especially exciting is the combination of genetic tools that allow to label, as well as manipulate synaptic function. The authors present elegantly their data, and my impression is that this work deserves publication. The author may want to consider the following points:

1. The concepts of aversion and avoidance are confusing. Avoidance implies a form of learning (see reviews from J LeDoux) after an individual learns to anticipate an upcoming aversive stimulus. If I correctly interpreted the authors use an on-line reading of escape/aversive behaviour after inclusion of cadaverine. This should be probably better defined throughout the text.

Thank you for this valuable feedback. We completely agree that avoidance could be a misleading term based on learning paradigms and have changed our language throughout to reflect aversion or repulsion, which gives a more accurate description of the observed behavioral response.

2. At one point in the results the authors make use of a genetic approach allowing to control synaptic function with Botulinun neurotoxin. They state that "Axons labeled by BoTxBLC-GFP terminated at the vIPN.…. suggesting that botulinum neurotoxin inhibits synaptic transmission within this restricted region of the vIPN". I understand the technology is used in published data, yet it would be elegant to show along with the behavioural results an assessment of collapsed synaptic function.

We didn’t examine neuronal activity at the axon terminals in the BoTxBLC-GFP transgenic line, however, we confirmed that expression of BoTxBLC-GFP inhibits the touch response (refer to Video 1). To measure transmission in dHb axon terminals at the vIPN effectively will require electrophysiology or more sensitive transgenic tools. We concur that this will be useful for future studies.

3. The authors use a labeling strategy that allows claiming that the cholinergic neurons innervate a precise area of the IPN, supporting previous data in literature. In their data set the authors however study the functional responses to cadaverine only in somata of these neurons. It would be extremely relevat in my opinion to show that calcium transients are also detectable in the Cholinergic axons in the IPN. This would corroborate the functional integration of aversive signal within this neuronal circuit, and not only within the right habenula.

A recent study (Zaupa et al., 2021) reported that “axon terminals of cholinergic and non-cholinergic habenular neurons exhibit stereotypic patterns of spontaneous activity that are negatively correlated and localize to discrete subregions of the target IPN” which has prompted us to consider the responses to odorants at the specific region of the IPN where the axons of lratd2a expressing neurons terminate. However, these axons are a small subset of the total cholinergic population and their terminals are challenging to visualize in vivo at larval stages. Such experiments will require us to adopt the explant approach used in Zaupa et al. or to develop transgenic lines to measure the responses of IPN neurons. As with the earlier point, we envision such strategies for our future experiments.

4. The authors may want to consider referring to the following reviews when citing habenula work in rats, mice, and humans in the context of physiology and disease: Hailan Hu et al., 2020; Lecca et al., 2014; Proulx et al., 2014.

The cited references describe functions of lateral/ventral habenulae. Our study focuses on the dorsal habenulae of zebrafish, which are equivalent to the medial not the lateral habenulae of rodents. We ruled out the involvement of vHb in this study as indicated in the discussion, thus it is not relevant to cite the published work on this other brain region.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Essential revisions:

1) Overall the behavioral differences to cadaverine shown in Figure 3G (Cre- vs Cre+) are relatively mild, especially as the aversion indices are significantly different from baseline in both Cre- and Cre+ condition (Figure 3—figure supplement 2B). Given that this is a key experiment in the paper, a discussion regarding whether this is a limitation of the existing tools (e.g. insufficient neurons silenced) or a reflection of underlying biology (e.g. redundancy in circuits for avoidance, different circuits controlling duration vs magnitude of aversion) would be beneficial.

As suggested, we have now elaborated on these points in the Discussion (page 14, line 281) as follows:

“As a group, zebrafish in which the synaptic activity of lratd2a neurons in the right dHb was inhibited by BoTxBLC-GFP did not exhibit repulsion to cadaverine, although some individuals showed a mild aversive response that was not sustained relative to sibling controls. […] More finely tuned techniques for temporal or spatial regulation of neuronal inactivation would help resolve these issues.”

2) Presentation of Figure 2c-d can be improved further – the same neurons presumably are being imaged "before" and "after", however the way the data is currently plotted makes it look like they are independent neurons.

We now present the data in revised figures that clearly indicate the results from the same individual neurons before and after addition of odorants or vehicle control (refer to Figures 2C and 2D).

3) It is counterintuitive that a negative aversion index means stronger aversion (perhaps call it a preference index instead, or flip the signs so more positive = more aversive).

We agree with this feedback. We changed the aversive index to a preference index and we explain how this was measured in the Methods and in the Results (refer to revised Figure 3—figure supplement 1 and Figures 3G, 4E, Figure 4—figure supplement 1, Figure 5F and Figure 5—figure supplement 1).

4) In the aversion assay (3G, 4E, 5F): can the authors clarify if some form of multiple comparisons correction was done in calculating the p-values at each time bin?

We now show new figures (3G, 4F and 5F) with the data analyzed by ANOVA followed by Bonferroni's correction. The previous Figures 3G, 4E and 5F were moved to Figure 3—figure supplement 1A, Figure 4—figure supplement 1A and Figure 5—figure supplement 1A. We believe that it is useful to include the statistical analyses of individuals within groups in supplemental figures as they reveal minute by minute behavioral differences not apparent in the ANOVA multiple comparisons.

5) The authors have performed ANOVA on the aversion indices shown in the supplementary figures, and report a significant effect of odor and odor x group interaction. Is there a significant effect of group alone?

As correctly pointed out by the reviewer, we had mistakenly omitted the effect of group alone although we had done these analyses. The effect of group values are now all provided in the figure legends (refer to legends for Figure 3—figure supplement 1B, Figure 4—figure supplement 1B and Figure 5—figure supplement 1B).

There is no explicit mention of the aversion index in the main text, and no interpretation in the figure legends.

We had previously included the description of the aversion index in the Material and Methods section of the main text. As suggested by Reviewer 2, we have modified this and now depict the results as a “preference index”. We have updated this information accordingly in the “Behavioral assays” section of the Materials and methods and we have also added the following sentence to the Results (page 7, line 141): “We used a preference index that is based on the position of an individual fish at a given time relative to the application site of the odorant (refer to Materials and methods).”

For clarity, the authors should elaborate how the statistical results from this 2nd analysis method ties in with / complements the statistical methods used in the main figures.

Depicting our results with different statistical approaches reveals different aspects of the aversive behavior we observed within and between groups of adults. We now have analyzed all data by ANOVA followed by Bonferroni's correction and provide the results in the main figures (3G, 4F and 5F). We moved the previous graphs to supplemental figures (Figure 3—figure supplement 1A, Figure 4—figure supplement 1A and Figure 5—figure supplement 1A). For all experiments, we show the raw data for the response to cadaverine over time and used the Wilcoxon signed-rank test to compare a set of matched samples (i.e., behavior before and after application of cadaverine by the same fish) within each group. We provide this information for all experimental paradigms in Figure 3—figure supplement 1A, Figure 4—figure supplement 1A and Figure 5—figure supplement 1A.

As recommended by the reviewer, we updated the following paragraphs in the Results section to describe how the results from different statistical methods tie in (page 9, line 174):

“To determine whether the lratd2a neurons in the right dHb contributed to the aversive response to cadaverine, we monitored the behavior within and between groups of adults that had or did not have the BoTxBLC-GFP transgene. Fish lacking the transgene showed a significantly reduced preference for the side of the tank where cadaverine had been applied (Figure 3G).

When the response of individual fish within each group was compared over time (Figure 3—figure supplement 1), adults with or without BoTxBLC-GFP initially avoided the side of the test tank where cadaverine had been introduced. However, aversion was sustained for 4 min in control fish, but not in those expressing BoTxBLC-GFP in lratd2a neurons. These findings, from statistical tests on individuals both within and between groups, suggest that lratd2a neurons in the right dHb are required for a prolonged aversive response to cadaverine.”

https://doi.org/10.7554/eLife.72345.sa2

Article and author information

Author details

  1. Jung-Hwa Choi

    Carnegie Institution for Science, Department of Embryology, Baltimore, United States
    Present address
    Geisel School of Medicine at Dartmouth, Department of Molecular and Systems Biology, Hanover, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Performed all of the experiments, Validation, Visualization, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0591-9708
  2. Erik R Duboue

    1. Jupiter Life Science Initiative, Florida Atlantic University, Jupiter, United States
    2. Wilkes Honors College, Florida Atlantic University, Jupiter, United States
    Contribution
    Methodology, Software, Writing – review and editing, Wrote MATLAB script for analyzing behavioral experiments
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3303-5149
  3. Michelle Macurak

    Carnegie Institution for Science, Department of Embryology, Baltimore, United States
    Contribution
    Methodology, Resources, Technical assistance, constructed sgRNAs for lratd2a and slc5a7a
    Competing interests
    No competing interests declared
  4. Jean-Michel Chanchu

    Carnegie Institution for Science, Department of Embryology, Baltimore, United States
    Contribution
    Methodology, Resources, Technical assistance, generated Tol2 constructs and transgenic lines
    Competing interests
    No competing interests declared
  5. Marnie E Halpern

    Carnegie Institution for Science, Department of Embryology, Baltimore, United States
    Present address
    Geisel School of Medicine at Dartmouth, Department of Molecular and Systems Biology, Hanover, United States
    Contribution
    Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review and editing
    For correspondence
    Marnie.E.Halpern@Dartmouth.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3634-9058

Funding

National Institutes of Health (R01HD078220)

  • Marnie E Halpern

National Institutes of Health (R37HD091280)

  • Marnie E Halpern

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are grateful to Bethany Malskis for animal care and to Dr. Krishan Ariyasiri and Dr. Eugene Demidenko for advice on statistical analyses. We thank Dr. Shin-ichi Higashijima for the Gbait-hsp70:Gal4 donor plasmid, Dr. Wenbiao Chen for pT3TS nCas9n plasmid, Dr. Koichi Kawakami for the UAS:zBoTXBLC-GFP construct, Paul Krieg for the Xla.Tubb promoter construct, Dr. Claire Wyart for GCaMP6f plasmid and Dr. Wolfgang Driever and Dr. Tatjana Piotrowski for providing bsxm1376 and tcf7l2zf55 mutant zebrafish, respectively. This work was supported by NIH grants R37HD091280 and R01HD078220 to MEH.

Ethics

All zebrafish protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Carnegie Institution for Science [Protocol #122] or Dartmouth College [Protocol #00002253(m3a)].

Senior Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Reviewing Editor

  1. Claire Wyart, Institut du Cerveau et la Moelle épinière, Hôpital Pitié-Salpêtrière, Sorbonne Universités, UPMC Univ Paris 06, Inserm, CNRS, France

Reviewers

  1. Caroline Lei Wee, Harvard University, United States
  2. Manuel Mameli, The University of Lausanne, Switzerland

Publication history

  1. Preprint posted: July 20, 2021 (view preprint)
  2. Received: July 20, 2021
  3. Accepted: December 7, 2021
  4. Accepted Manuscript published: December 8, 2021 (version 1)
  5. Version of Record published: December 21, 2021 (version 2)

Copyright

© 2021, Choi et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jung-Hwa Choi
  2. Erik R Duboue
  3. Michelle Macurak
  4. Jean-Michel Chanchu
  5. Marnie E Halpern
(2021)
Specialized neurons in the right habenula mediate response to aversive olfactory cues
eLife 10:e72345.
https://doi.org/10.7554/eLife.72345

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