The endocannabinoid gene <i>faah2a</i> modulates stress-associated behavior in zebrafish

<div><p>The ability to orchestrate appropriate physiological and behavioral responses to stress is important for survival, and is often dysfunctional in neuropsychiatric disorders that account for leading causes of global disability burden. Numerous studies have shown that the endocannabinoid neurotransmitter system is able to regulate stress responses and could serve as a therapeutic target for the management of these disorders. We used quantitative reverse transcriptase-polymerase chain reactions to show that genes encoding enzymes that synthesize (<i>abhd4</i>, <i>gde1</i>, <i>napepld)</i>, enzymes that degrade (<i>faah</i>, <i>faah2a</i>, <i>faah2b</i>), and receptors that bind (<i>cnr1</i>, <i>cnr2</i>, <i>gpr55-like</i>) endocannabinoids are expressed in zebrafish (<i>Danio rerio</i>). These genes are conserved in many other vertebrates, including humans, but fatty acid amide hydrolase 2 has been lost in mice and rats. We engineered transcription activator-like effector nucleases to create zebrafish with mutations in <i>cnr1</i> and <i>faah2a</i> to test the role of these genes in modulating stress-associated behavior. We showed that disruption of <i>cnr1</i> potentiated locomotor responses to hyperosmotic stress. The increased response to stress was consistent with rodent literature and served to validate the use of zebrafish in this field. Moreover, we showed for the first time that disruption of <i>faah2a</i> attenuated the locomotor responses to hyperosmotic stress. This later finding suggests that FAAH2 may be an important mediator of stress responses in non-rodent vertebrates. Accordingly, FAAH and FAAH2 modulators could provide distinct therapeutic options for stress-aggravated disorders.</p></div

Temporal expression patterns of eCB receptor genes.

<p>The time points on all graphs are represented as means ± 95% CI (0.25–1 dpf: 20 larvae/n, n = 3; 2–7 dpf: 10 larvae/n, n = 3). * Indicates that a group is significantly different from the 5 dpf group (Sidak's multiple comparisons test, <i>P <</i> 0.05). (A) The fold change of <i>cnr1</i> transcript levels relative to 5 dpf as determined by qRT-PCR. (B) The fold change of <i>cnr2</i> transcript levels relative to 5 dpf as determined by qRT-PCR. (C) The fold change of <i>loc793909</i> transcript levels relative to 5 dpf as determined by qRT-PCR.</p

TALEN-mediated mutagenesis of <i>faah2a</i>.

<p>(A) The <i>faah2a</i> TALEN target site was designed in exon 3 so that mutagenesis would disrupt all predicted splice variants. NCBI Accessions: Gene ID, 436973; DNA, NC_007112.6; mRNA (i), NM_001002700.2; Protein (i), NP_001002700.1. The target site is just upstream of the sequence encoding a lysine in the predicted serine 228 (S) / serine 204 (S) / lysine 129 (K) catalytic triad. (B) An alignment of wild type and mutant <i>faah2a</i> sequences reveals the TALEN-induced indel in the target BsrI restriction enzyme site.</p

<i>faah2a</i> modulates stress-associated behavior.

<p>(A) The rolling means of distances travelled by wild type (WT), heterozygous <i>faah2a</i> (HET) mutant, and homozygous <i>faah2a</i> (HOM) mutant zebrafish. The pre-treatment baseline locomotor activity was recorded from −15–0 min, and the post-treatment locomotor activity was recorded from 0–31 min. At time 0 the zebrafish were treated with either E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity at each second is represented as a mean of the distance travelled during the preceding 60 s. (B) The means of distances travelled after the zebrafish were treated with E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity of each group is represented as a mean of the distance travelled per min during the 5–25 min time bin ± 95% CI. Groups with all different letters above the columns are statistically different from each other, while groups with a conserved letter above the columns are not statistically different from each other (Tukey's honest significant difference test, <i>P <</i> 0.05).</p

<i>cnr1</i> modulates stress-associated behavior.

<p>(A) The rolling means of distances travelled by wild type (WT), heterozygous <i>cnr1</i> (HET) mutant, and homozygous <i>cnr1</i> (HOM) mutant zebrafish. The pre-treatment baseline locomotor activity was recorded from −15–0 min, and the post-treatment locomotor activity was recorded from 0–31 min. At time 0 the zebrafish were treated with either E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity at each second is represented as a mean of the distance travelled during the preceding 60 s. (B) The means of distances travelled after the zebrafish were treated with E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity of each group is represented as a mean of the distance travelled per min during the 5–25 min time bin ± 95% CI. Groups with all different letters above the columns are statistically different from each other, while groups with a conserved letter above the columns are not statistically different from each other (Tukey's honest significant difference test, <i>P <</i> 0.05).</p