Targeted knockout of a chemokine-like gene increases anxiety and fear responses

Abstract

Emotional responses, such as fear and anxiety, are fundamentally important behavioral phenomena with strong fitness components in most animal species. Anxiety-related disorders continue to represent a major unmet medical need in our society, mostly because we still do not fully understand the mechanisms of these diseases. Animal models may speed up discovery of these mechanisms. The zebrafish is a highly promising model organism in this field. Here, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of one of its members, sam2 We show exclusive mRNA expression of sam2 in the CNS, predominantly in the dorsal habenula, telencephalon, and hypothalamus. We found knockout (KO) zebrafish to exhibit altered anxiety-related responses in the tank, scototaxis and shoaling assays, and increased crh mRNA expression in their hypothalamus compared with wild-type fish. To investigate generalizability of our findings to mammals, we developed a Sam2 KO mouse and compared it to wild-type littermates. Consistent with zebrafish findings, homozygous KO mice exhibited signs of elevated anxiety. We also found bath application of purified SAM2 protein to increase inhibitory postsynaptic transmission onto CRH neurons of the paraventricular nucleus. Finally, we identified a human homolog of SAM2, and were able to refine a candidate gene region encompassing SAM2, among 21 annotated genes, which is associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome. Taken together, these results suggest a crucial and evolutionarily conserved role of sam2 in regulating mechanisms associated with anxiety.

Keywords: anxiety; chemokine-like; fear; knockout; zebrafish.

Fig. 1.

Characterization of sam2-expressing cells in the adult zebrafish brain. (A and A′) Whole-mount two color in situ hybridization of a dissected zebrafish brain with sam2 and a dopaminergic neuron marker, th. Anterior is to the left; lateral (A) and dorsal view (A′). The sam2 expression region did not overlap with th⁺ neurons. Prominent sam2 expression is seen in the Vd and Hb as well as hypothalamic regions. (B–D) Section images of brain hybridized with sam2/vglut2b (B, sagittal section; C, cross-section) or sam2/gad1b (D). (E and F) Cross-section of Hb region stained with sam2 alone (E) or sam2/vglut2a (F) probes. AN, auditory nerve; D, area dorsalis telencephlali; Dc, central zone of area dorsalis telencephali; MO, medulla oblongata; OB, olfactory bulb; TeO, optic tectum; Vv, ventral nucleus of area ventral telencephali. (Scale bars, 100 µm.)

Fig. 2.

Generation of sam2 KO fish using ZFNs. (A) DNA sequencing analysis of two sam2 KO alleles (sam2^cnu1 and sam2^cnu2) with 5-bp and 17-bp deletion, respectively. Yellow mark, Left and Right ZFN-binding regions; red, spacer. (B and B′) Allele-specific genotyping of sam2^cnu1 and sam2^cnu2 KO fish by genomic PCR and digestion for BtgI site (CCGTGG) newly created in sam2^cnu1. (C and C′) Normal morphology of sam2^cnu1 KO embryo at 3 d. (D–E′) Projections of Hb efferent axons targeting the IPN in the Et(-1.0otpa:mmGFP)hd1 transgenic sam2^cnu1 KO fish were not visibly affected. Lateral views (D and E) and dorsal views (D′ and E′). dIPN, dorsal IPN; FR, fasciculus retroflexus; lFR, Left FR; lHb, Left Hb; MR, median raphe; rFR, Right FR; rHb, Right HbvIPN, ventral IPN. (Scale bar, 100 µm.)

Fig. 3.

Increase of anxiety-related behaviors in sam2^cnu1 KO zebrafish. (A–E) Novel tank tests of individually placed fish. The distance traveled (A) (sam2^+/+, n = 18; sam2^−/−, n = 28; Cohen’s d = 0.24; Mann–Whitney U = 181, P = 0.11), average velocity (B) (sam2^+/+, n = 18; sam2^−/−, n = 28; Cohen’s d = 0.32; Mann–Whitney U = 171, P = 0.07), and transition to upper half (C) (sam2^+/+, n = 18; sam2^−/−, n = 28; Cohen’s d = 0.19; Mann–Whitney U = 202, P = 0.27) were not significantly changed in sam2 KO fish. However, the frequency of erratic behavior (D) (sam2^+/+, n = 18; sam2^−/−, n = 28; Cohen’s d = 1.65; Mann–Whitney U = 41, P < 0.00001) and the number of freezing fish (E) (sam2^+/+, n = 18; sam2^−/−, n = 27; Cohen’s d = 0.62; P = 0.042, Student’s t test) were increased in sam2 KO fish. (F and G) Black/white preference (scototaxis) test. Distance traveled in white arena (F) (sam2^+/+, n = 18; sam2^−/−, n = 24; Cohen’s d = 1.06; Mann–Whitney U = 85, P = 0.0032) and frequency of transitions (G) (sam2^+/+, n = 18; sam2^−/−, n = 24; Cohen’s d = 1.42; Mann–Whitney U = 61.5, P = 0.00016) were measured. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (P > 0.05).

Fig. 4.

Anxiety-like behavior and increased social cohesion in sam2^cnu1 KO fish. Five fish (3-mo-old male siblings) were placed as a group in a novel tank. (A and A′) A snapshot of video tracking after 20-min novel tank test. (B and B′) Temporal 3D reconstructions of video tracking before habituation (early, 5–8 min, blue; Cohen’s d = −0.063) and after habituation (late, 13–16 min, red; Cohen’s d = −0.80) to the novel environment. (C and C′) Measurement of social cohesion as the mean individual gap. We measured the distance between the focal fish and one of its shoal members (lines a, b, c, and d; interindividual distances). P = 0.02 for control and P = 0.88 for sam2 KO fish (sam2^+/+, n = 5; sam2^−/−, n = 5). *P < 0.05; ns, not significant (P > 0.05).

Fig. 5.

Increase of anxiety-related behaviors in Sam2 KO mice. (A) Sam2 KO mice have normal body weight (Sam2^+/+, n = 25; Sam2^−/−, n = 10; Cohen’s d = 0.31; unpaired two-tailed, Mann–Whitney U = 124, P = 0.98). (B) Sam2 KO mice show normal total locomotion in an open-field test [Sam2^+/+, n = 11; Sam2^−/−, n = 6; two-way repeated-measures ANOVA, Time × Group interaction: F(5, 75) = 0.32, P = 0.89]. (C) Sam2 KO mice spent significantly less time in the open arms on an elevated plus maze (Sam2^+/+, n = 11; Sam2^−/−, n = 8; Cohen’s d = 0.74; unpaired one-tailed, Mann–Whitney U = 23, P = 0.045). (D) Sam2 KO mice show similar levels of freezing response during training days [Sam2^+/+, n = 11; Sam2^−/−, n = 7; two-way repeated-measures ANOVA, Time × Group interaction: F(3, 48) = 0.48, P = 0.69]. (E) Sam2 KO mice show higher freezing behavior in contextual test 24 h after training (Sam2^+/+, n = 11; Sam2^−/−, n = 7; Cohen’s d = 1.21; unpaired two-tailed, Mann–Whitney U = 15, P = 0.035). (F) Sam2 KO mice show higher freezing behavior in cued test 24 h after training (Sam2^+/+, n = 11; Sam2^−/−, n = 7; Cohen’s d = 1.75; unpaired two-tailed, Mann–Whitney U = 7, P = 0.0028). *P < 0.05; **P < 0.01; ns, not significant (P > 0.05).

Fig. 6.

Increase of mRNA expression of stress-related crhb in sam2^cnu1 KO fish and spontaneous inhibitory postsynaptic currents onto CRH neurons by SAM2. (A–D) Increase of crhb mRNA expression in sam2^cnu1 KO fish (sam2^+/+, n = 6; sam2^−/−, n = 6). Ventral views of the whole brain of control sam2^+/+ (A and C) and sam2^−/− KO fish (B and D). (A′ and B′) Higher magnifications of the boxed regions in A and B are the PPa in zebrafish, homologous to the mouse PVN. (Scale bars, 200 µm.) (E) Representative photomicrograph of the Cre-dependent TdTomato in the PVN CRH neurons. (Magnification: E, 10×.) (F) Representative voltage-clamp traces of sIPSCs in response to SAM2 application in PVN CRH neurons. (G) SAM2 application significantly increased sIPSC frequency. (H) SAM2 did not affect sIPSC amplitude. (I) Representative voltage-clamp trace of eIPSC onto CRH neurons in response to SAM2 application. (J) SAM2 did not affect the amplitude of eIPSCs. (K) SAM2 did not change the paired-pulse ratio. *P < 0.05; **P < 0.01.

Fig. 7.

CNV mapping at 12q14.1 with six human patients, implicating SAM2 as a candidate gene. Deletions and duplications are depicted in red and blue, respectively. (A) Alignment of three microdeletions and one microduplication at 12q14.1 has refined the candidate gene region to a 61 kb (arrows), which contains 56-kb 3′ end of SAM2. Notably, the microdeletion in a 6-y-old boy 290951 with autism spectrum disorder and intellectual disability contains SAM2, as well as USP15 and part of MON2. (B) Pedigree showing three family members affected by intellectual disability in three generations. We have confirmed the deletion in each family member; II-2 (del/+), III-2 (del/+), III-3 (^+/+), IV-1 (^+/+), IV-2 (^+/+), and IV-3 (del/+). (C) SAM2 copy number in the family as determined by qPCR. The proband IV-3 has inherited the deletion from the maternal grandfather II-2 through his mother III-2 and affected members have only one SAM2 copy. The patient’s siblings are normal having two SAM2 copies. (D) Transcript levels of SAM2 in patient, his mother, sister, and white male control as revealed by qRT-qPCR. SAM2 transcripts are reduced approximately by half in both the patient and his mother relative to control.