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Identification of slit3 as a locus affecting nicotine preference in zebrafish and human smoking behaviour

  1. Judit García-González
  2. Alistair J Brock
  3. Matthew O Parker
  4. Riva J Riley
  5. David Joliffe
  6. Ari Sudwarts
  7. Muy-Teck Teh
  8. Elisabeth M Busch-Nentwich
  9. Derek L Stemple
  10. Adrian R Martineau
  11. Jaakko Kaprio
  12. Teemu Palviainen
  13. Valerie Kuan
  14. Robert T Walton  Is a corresponding author
  15. Caroline H Brennan  Is a corresponding author
  1. School of Biological and Chemical Sciences, Queen Mary, University of London, United Kingdom
  2. School of Pharmacy and Biomedical Science, University of Portsmouth, United Kingdom
  3. Barts and The London School of Medicine and Dentistry, Blizard Institute, United Kingdom
  4. Centre for Immunobiology and Regenerative Medicine, Institute of Dentistry, Barts and The London School of Medicine and Dentistry, United Kingdom
  5. Wellcome Trust Sanger Institute, United Kingdom
  6. Cambridge Institute of Therapeutic Immunology & Infectious Disease (CITIID), Jeffrey Cheah Biomedical Centre, University of Cambridge, United Kingdom
  7. Institute for Molecular Medicine FIMM, HiLIFE, Finland
  8. Department of Public Health, Faculty of Medicine, University of Helsinki, Finland
  9. Institute of Cardiovascular Science, University College London, United Kingdom
Research Article
Cite this article as: eLife 2020;9:e51295 doi: 10.7554/eLife.51295
10 figures, 3 tables and 2 additional files


10 μM nicotine induced place preference in zebrafish is sensitive to inhibition by therapeutics effective in humans.

(A) Varenicline (nicotine partial agonist) and (B) Bupropion (norepinephrine and dopamine reuptake inhibitor with nicotine antagonist properties when metabolised). Bars represent mean and error bars represent + SEM. Asterisk (*) represents significance at p<0.05.

Figure 1—source data 1

Inhibition of nicotine CPP by varenicline and bupropion.

Nicotine CPP is heritable.

(A) Breeding and selection to assess heritability of nicotine-induced place preference in ENU-mutagenized zebrafish. To test whether nicotine preference is heritable, fish in the upper (blue circle) and lower (red circle) 10% of the change in preference distribution curve were inbred and screened for CPP (Second generation CPP assay). A similar approach was used for the third generation CPP assay. (B) CPP for nicotine is heritable. Mean preference change is increasingly distinct for the second and third generation CPP analysis. Plot represents mean and ± SEM. First generation (corresponding to the F3 families used for the screen) (n = 120): mean = 0.11; SD = 0.17. Second generation: Offspring of fish from upper 10% of the first generation screen (n = 92): mean = 0.17; SD = 0.14. Offspring of fish from lower 10% of the first generation screen (n = 64): mean = 0.08; SD = 0.15. Third generation. Offspring of fish from upper 10% of the second generation screen (n = 69): mean = 0.21; SD = 0.10. Offspring of fish from lower 10% of the second generation screen (n = 67): mean = 0.01; SD = 0.09.

AJBQM1 and AJBQM2 families show increased and decreased nicotine place preference.

AJBQM1 and AJBQM2 siblings, not included in the screen (n = 10 for AJBQM1; n = 14 for AJBQM2), AJBQM1 significantly differed from the parental strain, Tupfel longfin (TLF) wild type (w/t) saline control (n = 17) and wild type nicotine exposed fish (n = 7). AJBQM2 differed from wild type nicotine exposed fish but not wild type saline controls. Different superscript letters indicate significant difference between groups (p<0.05), same superscript letters indicate no significant differences between groups. Bars indicate Mean + SEM.

slit3 mutations segregate with nicotine place preference.

(A) Segregation of slit3sa1569 mutation with nicotine seeking. CPP change scores for individual un-mutagenized TLF wild type fish (n = 7) and AJBQM1 fish (n = 10). Following CPP analysis, fish were genotyped for 25 loss of function mutations contained within the family. Black dots indicate slit3sa1569/+ heterozygous mutant fish. White dots indicate slit3sa1569+/+ fish. Heterozygosity for slit3sa1569 segregates with increased nicotine seeking behaviour. (B) Position of ENU-induced mutations in zebrafish Slit3 protein. slit3sa1569 (A > G transition) disrupts a splice site in intron seven affecting translation at amino acid 176. slit3sa202 (G > T transversion) introduces a stop codon at amino acid 163. Both mutations truncate the protein before the leucine rich repeat domain 2 (D2), which interacts with membrane bound ROBO during SLIT-ROBO signalling. (C) Nicotine preference of slit3sa202 line. slit3sa202/+ fish (n = 18) show increased nicotine preference compared to wild type TLF controls (n = 8) (p=0.001) and wild type siblings slit3+/+ (n = 14) (p<0.05). Bars indicate mean + SEM. (D) Segregation of slit3sa202 allele with nicotine seeking. CPP change scores for individual un-mutagenised TLF wild type parent strain fish (n = 8) and slit3sa202 fish (n = 21). Black dots indicate slit3sa202/+ heterozygous mutant fish, white dots indicate slit3sa202+/+ fish. Mutations in slit3sa202 co-segregate with nicotine preference. Heterozygous slit3+/sa202 present increased place preference compared to slit3sa202+/+ siblings (n = 11).

Figure 5 with 1 supplement
Fluorescent immunohistochemistry in three-day old wild type slit3sa1569+/+ and homozygous mutant slit3sa1569/sa1569.

(A–D) Anti-5-HT, (E–M) anti-TH: (A) 5-HT-labelled neurons in wild type zebrafish brain. Circles indicate regions used for quantification of cell number in rostral hypothalamus (RH), inferior hypothalamic lobes (HL-R, HL-L) and raphe nucleus (RN). (B) Anti-5-HT labelled cells in slit3 wild type brain, (C) Anti-5-HT labelled cells in slit3sa1569 homozygous mutant brain. (D) Quantification of anti-5HT labelled cell number in wild type and slit3 mutant brains No significant differences were observed between wild type and slit3 mutant larvae. (E) Unprocessed maximum intensity projection of anti-TH-labelled whole mounted wild type zebrafish brain. Circles indicate areas used for quantification, or in the case of LC-R and LC-L, landmarks used as reference to determine the extension of the medial longitudinal catecholaminergic tract (MLC) used when quantifying the number of anti-TH labelled projections to the midline (panels L, M). (F) Cell quantification for diencephalic dopaminergic cluster (DDC). No significant differences were observed between wild type and slit3 mutant larvae. (G) Cell quantification for medulla oblongata interfascicular zone and vagal area, and area postrema (MO, AP). No significant differences were observed between wild type and slit3 mutant larvae. (H–K) Anti-TH labelled wild types and slit3sa1569. Zoomed-in visualization of diencephalic dopaminergic cluster (H–I) and medulla oblongata interfascicular zone and vagal area (J–K). (L–M) Quantification of catecholaminergic projections projecting to the midline. Examples of projections are indicated with yellow arrows. Projections were assessed from posterior to anterior using the locus coerulus and posterior extent of the raphe nucleus as landmarks (Panel L, yellow line) and from dorsal to ventral (Panel M, stacks 1–3). Figure 5—figure supplement 1 shows individual planes. n = 5 samples per genotype group.

Figure 5—source data 1

Quantification of anti-5HT and anti-TH labelled cell number and anti-TH labelled axon projections.

Figure 5—figure supplement 1
Fluorescent immunohistochemistry in three-day old wild type and homozygous mutant slit3sa1569 labelled with tyrosine hydroxylase (A) and tubulin (B–F).

(A) Example of three Z-planes used to quantify catecholaminergic projections crossing the midline. (Stacks 1–3). (B) Anti-tubulin staining of slbpty77e/ty77e mutant larvae, known to have fewer neurons and axonal defects (Turner et al., 2019). (C–D) Anti-tubulin staining of wild type and homozygous mutant slit3sa1569. (E–F) Zoom-in of C-D for midline in the ventral forebrain, where slit3 is known to be expressed (Miyasaka et al., 2005).

Figure 6 with 1 supplement
Habituation response in the presence and absence of amisulpride.

(A–B) Response and habituation to 10 stimuli with two seconds interval between stimuli in wild type, drug free zebrafish. Mean distances travelled were measured in one second time bins. Line indicates 4.6 mm, which corresponds to mean basal distance moved per second plus 2 standard deviations of the mean and was used to define respondents. The percentage of fish responding to the stimuli decreases with stimulus/tap number (Main effects of tap number p<0.05) 68% respond to the first tap; 16% respond to the last tap. Respondents are defined as fish moving more than 4.6 mm. (C) Proportion of responders across the ten stimuli in drug free individuals from each genotype: there was no significant effect of genotype on response across taps (p=0.34) or responsiveness (p=0.35) in drug free fish. (D) Mean percentage of responders across the ten stimuli (± SEM). Data are stratified by slit3sa1569 genotype and amisulpride dose normalised to response in absence of drug. The effect of amisulpride on habituation varies by genotype. (E, F) Proportion of individuals responding in each amisulpride dose condition in wild type and homozygous mutant fish, respectively. The interaction between amisulpride dose and stimulus event number had a significant effect on the proportion of responsive individuals in wild type individuals (p<0.05) but not homozygous mutants (p=0.16).

Figure 6—source code 1

Zebrafish habituation to acoustic startle R code.

Figure 6—source data 1

Response to acoustic startle in the presence and absence of amisulpride.

Figure 6—figure supplement 1
Average distance moved before (Figure 1A) and during startle stimuli (Figure 1B) in wild type and slit3sa1569 mutant five-day-old zebrafish larvae.

(A) Basal distance moved as function of amisulpride dose and slit3 genotype. The effect of dose and genotype and their interaction was tested in a linear mixed model. Timepoint, well where the fish were placed and plate were also included as fixed factors and the fish ID as random factor. (B) Distance moved during taps as function of amisulpride dose and slit3 genotype. Dose and genotype and their interaction were examined in a linear mixed model including stimulus number, well, plate used and distance moved before stimuli as fixed factors and Fish ID as random factor. Zebrafish larvae did not differ in the distance travelled before or during startle stimuli as a function of amisulpride dose nor genotype (p>0.05). Bars represent estimated marginal means ± SEM (n = 42–48 fish per group).

CPP induced by 5 μM nicotine is blocked by 0.5 mg/L dopamine/serotonin antagonist amisulpride in wild type slit3sa1569+/+ fish but not in slit3sa1569 homozygous mutants.

Bars represent mean (+ SEM). (n = 11–14 fish per group). *Two-way ANOVA followed by post-hoc Tukey tests (p<0.05).

Quantitative real-time PCR analysis of five-day-old wild type slit3sa1569+/+, slit3sa1569/+ heterozygous and slit3sa1569/sa1569 homozygous mutant larvae.

Quantitative PCR analysis of gene expression was performed for members of (A) dopaminergic signalling pathway, (B) adrenoreceptors, (C) serotonin signalling pathway and (D) nicotinic cholinergic receptors. (Total n=30, 3 samples per experimental group with n=10 embryos per sample). Only htr1aa ([F(2,6)=44], p=0.0003) showed a significant difference across genotypes after correcting for multiple testing. *Two-way ANOVA followed by post-hoc Tukey test (p < 0.05).

Figure 8—source data 1

Gene expression data for slit3 mutant and wild type zebrafish.

Linkage disequilibrium (LD) plot of SLIT3 SNPs in human smoking association analysis.

Numbers within each square indicate D’ values (white: D’<1, LOD <2; blue: D’=1, LOD <2; pink: D’<1, LOD ≥2; and bright red: D’=1, LOD ≥2). Top part of the figure shows domain organization of the SLIT protein based on the UCSC Genome Browser (http://genome.ucsc.edu/) in relation to the SNP location. LRR: leucin-rich repeats. EGF: epidermal growth factor domains. LamG; Laminin G domain. Some intron numbers were added for reference.

Zebrafish breeding scheme to generate F3 families.

F2 ENU-mutagenized zebrafish, heterozygous for multiple mutations across the entire genome were obtained from the Wellcome Sanger Institute as part of the Zebrafish Mutation Project. At QMUL, heterozygous F2 fish were incrossed to generate 30 F3 families, each containing 10–20 nonsense or essential splice site mutations and about 1500 additional exonic and intronic point mutations. F3 Families were arbitrarily numbered 1–30. Expected mutation rate and type of mutations in coding regions are specified on the right hand side.


Table 1
Associations of SLIT3 SNPs with level of tobacco consumption for the London study groups (n = 863).

Regression coefficients, confidence intervals and p-values from linear regression of cigarettes smoked per day (CPD) on minor allele count for smokers from COPD, asthma and general cohorts, adjusted for age, sex and cohort. β coefficient represents effect of each additional minor allele. Benjamini-Hochberg cut-off at q-value 0.1 = 0.01053. Associations of SLIT3 SNPs with tobacco consumption in a subset of heavy smokers (≥20 cigs/day). Adjusted for age, sex and cohort. (q-value 0.1 = 0.01579). Associations of SLIT3 SNPs in a subset of light smokers (<20 cigs/day). Adjusted for age, sex and cohort (q-value 0.1 = 0.00526). Association analysis of SLIT3 SNPs with smoking cessation. Logistic regression of current smokers vs ever smokers controlling for age, sex and cohort. Odds ratio >1 indicates minor allele increases odds of persistent smoking relative to major allele. SE: standard error, L95: lower limit of 95% confidence interval, U95: upper limit. For all panels, associations in bold remained significant after adjustment for multiple comparisons using a Benjamini-Hochberg procedure to control false discovery rate at 10%.

Tobacco consumptionTobacco consumption -
heavy smokers
(≥20 cigs/day)
Tobacco consumption - light
(<20 cigs/day)
Smoking cessation
SNPP valueβSE95%P valueβSE95%P valueβSE95%ORSEL95U95P value
rs100367270.629−0.3880.802(−1.960, 1.183)0.448−0.6530.860(−2.337, 1.032)0.940−0.0510.686(−1.396, 1.293)0.9470.1600.6931.2950.734
rs111345270.2181.0140.822(−0.596, 2.625)0.327−0.8670.883(−2.599, 0.864)0.2610.7950.705(−0.586, 2.176)0.6650.1650.4820.9180.013
rs117425670.135−1.1660.779(−2.691, 0.361)0.005−2.3460.825(−3.962,–0.730)0.0041.8880.644(0.6258, 3.151)1.5860.1631.1532.1830.005
rs117490010.0591.9721.044(−0.074, 4.018)0.8730.1771.103(−1.985, 2.338)0.2061.2000.944(−0.651, 3.051)0.9530.2060.6371.4260.817
rs125157250.592−0.4060.756(−1.888, 1.076)0.488−0.5650.813(−2.159, 1.029)0.278−0.6880.631(−1.925, 0.550)1.0280.1510.7651.3810.855
rs125210410.904−0.1050.865(−1.801, 1.591)0.996−0.0050.942(−1.851, 1.841)0.059−1.3540.710(−2.746, 0.038)1.5540.1781.0962.2050.013
rs126544480.001−4.2411.307(−6.803, –1.680)0.0003−4.8301.334(−7.444, –2.216)0.410−1.0341.251(−3.486, 1.417)1.6250.2790.9412.8080.082
rs13455880.240−1.2681.078(−3.380, 0.845)0.253−1.3341.164(−3.616, 0.948)0.869−0.1500.907(−1.927, 1.627)1.4170.2220.9182.1890.116
rs14217630.272−0.9820.894(−2.735, 0.770)0.978−0.0270.959(−1.908, 1.853)0.162−1.0740.764(−2.571, 0.424)0.9170.1760.6491.2940.622
rs15590510.961−0.0400.819(−1.644, 1.564)0.4580.6560.882(−1.073, 2.384)0.5070.4550.685(−0.880, 1.797)0.9190.1630.6681.2650.606
rs176651580.1311.3380.884(−0.394, 3.070)0.2361.1140.939(−0.727, 2.955)0.0341.6200.758(0.1354, 3.106)0.7230.1720.5161.0130.060
rs177345030.002−3.9871.299(−6.534, –1.441)0.001−4.4581.325(−7.055, –1.861)0.410−1.0341.251(−3.486, 1.417)1.6160.2750.9422.7730.081
rs29387740.1401.1010.745(−0.359, 2.562)0.5280.4960.786(−1.044, 2.036)0.015−1.6550.674(−2.976, –0.333)0.7530.1480.5631.0070.056
rs2959940.7140.2830.770(−1.227, 1.793)0.6430.3780.813(−1.215, 1.971)0.238−0.7960.672(−2.114, 0.521)0.7990.1540.5911.0820.147
rs2978860.6200.4420.890(−1.303, 2.187)0.961−0.0480.986(−1.979, 1.884)0.4890.4880.704(−0.891, 1.867)1.1080.1770.7841.5680.561
rs37339750.909−0.0990.860(−1.784, 1.587)0.9820.0220.934(−1.809, 1.852)0.059−1.3540.710(−2.746, 0.038)1.4880.1761.0542.1010.024
rs42823390.669−0.4341.013(−2.419, 1.552)0.942−0.0801.103(−2.241, 2.081)0.238−1.0060.849(−2.670, 0.658)0.9840.2030.6611.4640.936
rs77286040.7010.2860.744(−1.173, 1.745)0.3210.8270.832(−0.803, 2.457)0.6540.2620.583(−0.880, 1.404)0.9350.1490.6981.2530.653
rs96880320.948−0.0500.766(−1.551, 1.451)0.7700.2460.839(−1.398, 1.890)0.080−1.0760.610(−2.272, 0.119)1.0660.1560.7861.4460.680
Table 2
Associations between detailed nicotine dependence phenotypes and SLIT3 genotype in a Finnish twin cohort (n = 1715).

Associations of SLIT3 SNPs with DSM-IV nicotine diagnosis, symptoms, Fagerström scores (FTND), cigarettes smoked each day (CPD), age of onset of weekly smoking, sensation felt after smoking first cigarette and time to first cigarette in the morning. The three SNPs that were linked to smoking behaviour in the London cohorts are shown in bold.

DSM-IV ND diagnosisDSM-IV ND symptomsFTND (≥4)FTND score
SNPβSEP valueβSEP valueβSEP valueβSEP value

max CPDAge of onset of weekly smokingFirst time sensationFTND time to first cigarette
SNPβSEP valueβSEP valueβSEP valueβSEP valueβSEP value
Key resources table
Reagent type
(species) or
DesignationSource or
Genetic reagent (Danio rerio)slit3Sanger Institutesa202Generated by ENU mutagenesis. Soon to be available from ZIRC zebrafish resource centre
Genetic reagent (Danio rerio)slit3Sanger Institutesa1569Generated by ENU mutagenesis. Soon to be available from ZIRC zebrafish resource centre
Strain, strain background (Danio rerio)TupfelSanger InstituteTubingen longfinWild type strain, now available from the ZIRC zebrafish resource centre
Antibodyanti-5-HT (Rabbit polyclonal)SigmaCat#S5545,IHC (1/200)
Antibodyanti-tyrosine hydroxylase (mouse monoclonal)AbcamCat# AB152IHC (1/1000))
Chemical compoundAmisulprideTocrisC21320.05–0.5 mg/L
Chemical compoundvareniclinesigmaPZ000410-20micromolar
Chemical compoundbupropionSigmaB12771–10 micromolar
Chemical compoundNicotine hemisulphateSigmaN10195–10 micromolar

Additional files

Supplementary file 1

Supplementary methods and results for zebrafish genotyping and gene expression analysis, and human association analyses.

Supplementary methods: (A) Loss of function mutations present in AJBQM1 and AJBQM2 founders; (B) Gene identifiers and primer sequences used for zebrafish gene expression analysis; (C) Human association study sample characteristics; (D) Primer and reporter sequences used for human genotyping. Supplementary results: (H) Results of site specific PCR genotyping of AJBQM1 and AJBQM2; (F) Logistic regression analysis for human association analysis on smoking initiation; (G) Logistic regression analysis for human association analysis on persistent smoking; (H) Full qPCR gene expression results for slit3 wildtype and slit3sa1569/sa1569 homozygous mutants.

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