Hypoactivity, Abnormal Musculature, and Learning and Memory Defects in slc6a3 zebrash Mutant Larvae

Dopamine (DA) is one of the most common neurotransmitters in living organisms and is involved in the regulation of behavior, physiology, and disease. The dopamine transporter (DAT, encoded by the slc6a3 gene) re-uptakes dopamine from the synaptic cleft back into neurons, which plays a critical role in regulating dopamine signaling. Clinically, mutations in slc6a3 have been implicated in various diseases. Zebrash are good model organisms for studying the functions of slc6a3 and dopamine. Work using the zebrash slc6a3 mutant has previously been reported, but this mutant is not a complete loss-of-function mutant. In the present study, we knocked out the slc6a3 gene using CRISRP/Cas9 technology and obtained a zebrash mutant with complete loss of function of slc6a3. Behavioral assessments and quantitative reverse transcription (qRT)-PCR revealed musculature injury, decreased activity, and decreased learning and memory ability in the slc6a3 mutant. This study provides a new theoretical basis for understanding the function of slc6a3 and provides a new model organism for studying the molecular pathology underscoring dopamine metabolism-related diseases. It also provides a suitable model for high-throughput screening of small-molecule drugs targeting slc6a3.


Introduction
DA is a neurotransmitter that transmits information between neurons and regulates exercise, attention, learning, and emotional reactions [1][2][3][4] . Disorders of dopamine metabolism can lead to many physiological changes including attention de cit hyperactivity disorder (ADHD), learning defects, and depression [5,6] .
Recent studies have reported that disorders of dopamine metabolism are also associated with Parkinson's disease [7] . The main treatment for clinical Parkinson's disease is the drug L-dopa, which stimulates dopamine production [8,9] . In addition, dopamine is associated with schizophrenia, but its role in this disorder is not fully understood [10] . Further, addictive substances such as cocaine affect dopamine secretion, which can promote the development of addiction [11] .
The metabolism of dopamine includes the synthesis, release, re-absorption, and degradation of dopamine [12] . Presynaptic neurotransmitter re-uptake is mediated by the transmembrane dopamine transporter (DAT) and is the primary mechanism for terminating synaptic transmission [13] . The DAT is encoded by the slc6a3 gene and is a sodium and chloride ion-dependent transmembrane neurotransmitter transporter [14,15] . Since the time of dopamine activity in the synaptic cleft determines dopamine transmission, slc6a3 is critical for the maintenance of dopamine homeostasis and controlling the duration of dopamine signaling.
Clinical research has indicated that hereditary and pharmacological changes in slc6a3 affect human health. Polymorphisms of slc6a3 (rs28363170, rs393795) are associated with levodopa-induced dyskinesias in Parkinson's disease during treatment of Parkinson's disease [16,17] . Moreover, polymorphisms of slc6a3 are associated with extreme behavioral characteristics and diseases such as anger-type personality disorder, destructive behavior disorder, overeating, attention de cit, hyperactivity, and alcohol dependence [17][18][19][20] . In autism patients, deletion of a single amino of the slc6a3 gene was identi ed to alter transmembrane structural conformation of the protein [21] . Tissues in slc6a3 mutant mice exhibit a substantial decrease in DA content and excitatory motor activity [22] . The activity of slc6a3 is low in patients with general anxiety disorder [23] . In addition, slc6a3 is also a target of cocaine and alcohol [11] . Drug and alcohol abuse can reduce the expression of this gene, resulting in abnormal changes in behavior and mood.
Zebra sh are excellent model organisms that have been used successfully in the eld of biomedical science for many years. The slc6a3 zebra sh knockout mutant was successfully established in 2008 using zinc nger nucleases made by Oligomerized Pool ENgineering (OPEN) technology [24] . Subsequent studies have revealed that zebra sh slc6a3 mutants exhibit anxiety-like behavior [25] . As slc6a3 is mainly expressed in dopaminergic neurons, slc6a3:GFP has been established [26] , and green uorescence is mainly observed in ventral diencephalon (vDC) clusters, amacrine cells in the retina, olfactory bulb, pretectum, and caudal hypothalamus, similar to the results of in situ hybridization experiments [27] . The dopaminergic system of zebra sh is highly conserved compared to that of mice and humans; thus, dopamine-related studies are abundant in zebra sh. Zebra sh therefore serve as a good model to study DAT-related disorders and relevant mechanisms.
In this study, we rst constructed an slc6a3 mutant using CRISRP/Cas9 technology. We then used behavioral and gene expression assays to investigate the musculature, behavior, learning, and memory in the slc6a3 mutant.

Materials And Methods
Zebra sh cultivation and egg production Adult AB strain zebra sh were raised in a recirculating water system under a 14-/10-h light/dark cycle at 28°C and fed three times per day. To produce embryos, male and female zebra sh were paired in the evening; spawning occurred the next day within 1 h of the lights being switched on. The embryos were placed in 10-cm Petri dishes containing egg water with methylene blue (0.3 ppm) and raised in a lightcontrolled (14-/10-h light/dark) incubator at 28°C. At 6 days post-fertilization (dpf), samples were collected for quantitative reverse transcription (qRT)-PCR analysis.
Generation of slc6a3 mutant zebra sh slc6a3 mutants were generated using CRISPR-Cas9 technology. We designed the target site at the rst exon and synthesized gRNA in vitro. gRNA (50 pg) and Cas9-capRNA (300 pg) were co-injected into zebra sh embryos at the one-cell stage. The fragment containing the mutation was PCR-ampli ed with primers P1 and P2 (Table 1), and con rmed by DNA sequencing. Two types of base deletion existed; both resulted in a premature stop codon leading to a truncated protein of only 39 or 73 amino acids (aa). RNA isolation, cDNA synthesis and qRT-PCR Total RNA was extracted from more than 30 larvae with TRIzol® reagent (Invitrogen) and reverse transcribed into cDNAs using SuperScript™ III Reverse Transcriptase (Invitrogen). qRT-PCR was performed in an ABI StepOnePlus™ instrument (7500) using the TB Green® system (TaKaRa) with thermal cycling through 40 cycles at 95°C for 10 sec and 60°C for 30 sec. Each qRT-PCR analysis was performed in triplicate on three independent biological samples. The housekeeping gene β-actin was ampli ed as a control. All results were standardized to the expression level of β-actin. Relative mRNA expression levels were calculated using the 2 ΔΔCt method. Calculations were performed in Microsoft Excel. qRT-PCR primers for exon boundaries were designed using Primer Express 3.0 software and checked for selfannealing, heterodimerization, and hairpin structure formation with Oligo Analyzer 3.1. Primer sequences are listed in Table 1.

Touch-evoked escape response
Wild-type (WT) and slc6a3-/embryos were raised in the same standard conditions described above. At 48 and 96 hours post-fertilization (hpf), mechanical stimulation was performed using an insect pin attached to a micromanipulator. Light touches were applied to the side of the embryo at somites 14-16, at the level of the caudal part of the yolk tube. Each trial consisted of a recording of the spontaneous activity for 2 min, followed by three to ve stimulations, each separated by at least 1 s. Responses of larvae to pin stimulation were video captured.

Behavioral analysis
Embryos were raised to 72 hpf in the same standard conditions described above. All analyses were performed during the light portion of the 14 h light:10 h dark cycle at the same time of day. The larvae were transferred to clear 96-well plates with lids (one individual per well) and allowed to acclimate overnight. Analyses were performed in an isolated room maintained at 28.5°C. The movement of 4 dpf larvae was video captured and quanti ed using an infrared camera setup and tracking extension of the software system (Noldus Information Technology). In all behavioral protocols, animal color was set to black, and background-subtracted detection threshold was set to 20 sec. This value represented a greyscale pixel intensity value; any pixels darker than this threshold in the video were detected as the animal. The integration period (bin time) for movement slc6a3 mutant was set to 10 sec. slc6a3 mutant were processed and analyzed using Excel, and Fast Monitor (Noldus Information Technology).

Statistical analyses
Data are presented as means ± SD. Statistical differences between groups were determined using Student's t-test. All experiments were repeated at least three times. A p-value <0.05 was considered statistically signi cant.

Results
Construction of slc6a3 null mutant zebra sh The CRISPR Cas9 system was used to construct slc6a3 mutant zebra sh. The zebra sh slc6a3 gene includes 17 exons encoding 629 amino acids. The transcription initiation site ATG is located in the rst exon (Fig. 1A). We designed the gRNA locus behind ATG, then synthesized gRNA and Cas9 Capped mRNA in vitro, which were co-injected into zebra sh single-cell fertilized eggs. We then screened heritable zebra sh mutants in adulthood. The results revealed that two line slc6a3 mutants with four and ve base deletions were successfully screened (Fig. 1B). Bioinformatics analysis of the mutant sequences revealed that the predicted slc6a3 mutant sequences encoded 39 and 73 truncated polypeptides, respectively (Fig.  1C). qRT-PCR was used to quantify slc6a3 mRNA, revealing that the expression of the slc6a3 gene was signi cantly decreased in the slc6a3 mutant ( Fig. 2A). DA content was detected using ELISA assay. DA content in the slc6a3 mutant was signi cantly decreased (Fig. 2B).
Weak touch-evoked escape response in slc6a3 mutants Touch-evoked escape response assays are a method to assess muscle performance and function in zebra sh [28] . To explore the role of slc6a3 in muscle function, we performed touch-evoked escape response assays in WT and slc6a3 mutant zebra sh (Fig. 3A-H). At 48 hpf, WT zebra sh rapidly swam out of the eld of view after tip stimulation. In contrast, most slc6a3 mutant larvae remained in the eld of view despite swimming after stimulation (Fig. 3A-B and E-F). Similar results was observed in 96 hpf larvae (Fig. 3C-D and G-H).

Decreased locomotor activity in slc6a3 mutants
To investigate the effects of slc6a3 mutations on autonomic activity, behavioral experiments were performed to examine the activity of slc6a3 mutants. At 10 mins, the swimming distance of slc6a3 mutants was signi cantly lower than that of WTs (Fig. 4A). Analysis of the cumulative swimming distance revealed that the difference between slc6a3 mutants and WTs increased over time (Fig. 4B). Statistical analysis of total swimming distance revealed that the total swimming distance was signi cantly lower in slc6a3 mutants than in WTs (Fig. 4C). The speed of swimming is also an important indicator of the amount of activity. Swimming speed of slc6a3 mutants was signi cantly lower than that of WTs (Fig. 4D). These results indicated that the slc6a3 mutation reduced zebra sh locomotor activity.

De cits in learning and memory in slc6a3 mutants
To further investigate the role of slc6a3 in learning and memory, a single multiple light experiment was used to detect learning and memory ability in slc6a3 mutants. When the number of repeated light-dark cycles increased, the swimming speed of sh larvae began to decrease, and the speed of slc6a3 mutants was always lower than that of WT sh (Fig. 5A). In addition, the swimming speed of WT sh remained stable (platform) after a period of time, while the swimming speed of slc6a3 mutants continued to decrease (Fig. 5A). Data analysis of behavior indicated that slc6a3 mutant zebra sh took longer to reach the platform than did WT zebra sh (Fig. 5B). These results implied that learning and memory in slc6a3 mutants were impaired.

Alteration in expression patterns of learning and memory-related genes in slc6a3 mutants
To further investigate the reasons underlying learning and memory abnormalities in slc6a3 mutants, qRT-PCR was used to detect the expression of learning and memory-related genes in WT and slc6a3 mutants. There was no signi cant difference in the expression of creb1a and bdnf in slc6a3 mutants when compared with WT. fosl2 and hat1 were up-regulated in slc6a3 mutants when compared with WT.

Discussion
Numerous studies have used zebra sh as a model to study DA or drug addiction [25,[29][30][31] . These investigations have been facilitated by the evolution of the relatively primitive DA metabolism and response system in zebra sh. In this study, zebra sh slc6a3-de cient mutants were successfully established using CRISP/Cas9 technology. We observed that slc6a3 mutants exhibited weaker responses to stimulation, decreased motor activity, and impaired learning and memory.
Src6a3 belongs to the SLC6 gene family, which comprises a variety of neurotransmitter transporters [14] . The protein encoded by slc6a3 has a membrane structure composed of 12 transmembrane helices (TMs), an intracellular N-terminus, a C-terminus, and an extracellular glycosylation loop located between TM3 and TM4 [14] . Zebra sh slc6a3 mutants have been reported in previous studies, but the mutation site is located in the 12th exon [24,25] . This mutant protein still contains TM1-TM10, N-terminal, and glycosyl rings. Further, the C-terminus of slc6a3-encoded protein has multiple response sites, such as PCK and CamKII regulatory sites [32] . Thus, this mutant may not be a complete loss-of-function mutant. Subsequent studies have found that this mutant exhibits an anxious phenotype. In this study, we knocked out the slc6a3 gene using a speci c CRISPR system to obtain two mutants. The gRNA design site in our mutant was adjacent to the transcription start site. Thus, the mutant we obtained completely lacked TMs, glycosylation loop, and C-terminus, and was a null mutant with complete loss of function. This provides a suitable model organism for studying DA metabolism, slc6a3-related functions, and drug screening.
Touch-evoked escape response assays enable investigation of neuromuscular disorders in zebra sh in response to tactile stimuli [33] . After tactile stimuli presentation, slc6a3 mutants demonstrated signi cantly slower responses and did not exhibit escape responses. This indicated that the muscle tissue of zebra sh slc6a3 mutants was negatively affected. Autonomous swimming experiments revealed that the activity of slc6a3 mutants was signi cantly reduced. Whether the decrease in behavior was due to the negative effects of muscles remains unknown. Tactile responses of zebra sh are regulated by spinal nerve circuits, and Mauthner neurons are involved in escape responses to tactile stimuli in zebra sh [34] . The presence of DA receptors, expression of slc6a3 in the spinal cord, and whether behavioral abnormalities are due to the absence of slc6a3 in the spinal cord requires further investigation.
Zebra sh are excellent model organisms for studying learning and memory [35] . Many experimental techniques and methods are based on the behavior of adult sh such as the T-maze, but there are limited experiments based on sh larvae [36,37] . Previous studies have revealed that zebra sh larvae can produce memories of multiple light and dark stimuli [38] . In this study, we presented 3 min dark/1 min light and examined learning and memory based on the speed and time taken by zebra sh to reach the platform. Time to reach the platform was longer for slc6a3 mutants than for WT sh, indicating that the loss of slc6a3 affected learning and memory of zebra sh larvae. In mammals, DA regulates hippocampaldependent learning. In previous mouse slc6a3 mutants, it was reported that the decrease in slc6a3 expression in mice impaired learning in a running experiment [39] . In patients with Parkinson's disease, the expression level of slc6a3 is signi cantly reduced and is accompanied by learning and memory impairments [40] .
The process of learning and memory involves many neurochemical systems and mainly occurs at the synapse. In this study, gene expression of learning and memory-related genes was examined. The expression of creb1a and bdnf was not signi cantly altered in slc6a3 mutants. The expression of fosl2 and hat1 was signi cantly elevated in slc6a3 mutants. Creb1a encodes an effector molecule of the cAMP signaling pathway and is a classical learning and memory-related gene [41] . Bdnf encodes a neurotrophic factor in the brain that regulates the differentiation and growth of neurons [42] . No changes in expression of these two genes were observed, suggesting that slc6a3 does not affect these two genes in the context of learning and memory. Fosl2 is an early expression gene related to learning and memory, and hat1 encodes histone acetyltransferase 1 [43,44] . Expression of these genes in slc6a3 mutants was elevated, suggesting that the deletion of slc6a3 or disorders of DA signaling may affect functional changes in learning and memory via these genes, but the intrinsic mechanisms require further research.
To conclude, in this study, zebra sh slc6a3 mutants were successfully established by CRISP/Cas9 technology. Using behavior and qRT-PCR experiments, we demonstrate musculature injury, decreased activity, and decreased learning and memory in slc6a3 mutants. This study provides a new model organism for researching the molecular pathology underscoring DA metabolism-related diseases and for screening high-throughput small-molecule drugs targeting slc6a3.