fmr1基因剔除影響斑馬魚社會行為發育與Omega-3多元不飽和脂肪酸之治療效果

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(1)fmr1 Omega-3 The Developmental Abnormalities in Social Behavior and Therapeutic Effects of Omega-3 Polyunsaturated Fatty Acid in fmr1 Knock-Out Zebrafish (Denio rerio). Mao-Ting Hsu. Kwok-Tung Lu. 105. 07.

(2) Table of content Table of content ......................................................................................... 1 Abbreviation table ...................................................................................... 3 .................................................................................................... 5 Abstract ...................................................................................................... 8 Introductions ............................................................................................ 11 1. Zebrafish .................................................................................... 11 2. Fragile X syndrome (FXS) ......................................................... 11 3. Animal models for studying FXS .............................................. 14 4. Social behavior ........................................................................... 16 5. Omega-3 Polyunsaturated Fatty Acids ....................................... 18 6. Aims ........................................................................................... 20 Materials and Methods ............................................................................. 21 1. Animals ...................................................................................... 21 2. Genotyping ................................................................................. 21 3. Western blot analysis ................................................................. 22 4. Behavioral analysis .................................................................... 23 5. Gene expression quantitative analysis ....................................... 27 6. Analyzing the fatty acids composition in fmr1 KO zebrafish .... 30 7. Behavior analysis after n-3 PUFAs supplement ........................ 32 Results ...................................................................................................... 33 1. Qualitative analysis of the genotype .......................................... 33 2. Shoaling behavior and shoaling preference in wildtype and fmr1 KO zebrafish at 14 and 28 dpf ........................................... 33 3. Locomotor activity in larval zebrafish ....................................... 36 4. Evaluate the anxiety-like behavior of fmr1 KO zebrafish by using novel tank task .................................................................. 36 5. Analyzing the locomotor activity of wild-type and fmr1 KO zebrafish ...................................................................... 37 6. The anxiety-like behavior in wildtype and fmr1 KO zebrafish.. 37 7. The shoaling behavior and shoaling preference of wildtype and fmr1 KO zebrafish ............................................................... 38 8. Inhibitory avoidance learning in wildtype and fmr1 KO zebrafish without dietary treatment ............................................ 38.

(3) 9.. 10. 11. 12. 13. 14. 15. 16.. 17. 18.. 19.. 20.. Decrease in glutamate receptor but increase in 5-hydroxytryptamine receptor mRNA content in fmr1 KO zebrafish telencephalon .............................................................. 39 Low polyunsaturated fatty acid content in fmr1 KO comparing with WT zebrafish ...................................................................... 40 Fatty acid composition in different dose of n-3 PUFAs diets .... 40 Linseed oil enrichment incrementally elevated the composition of total PUFAs with duration of treatment ................................. 41 n-3 PUFAs supplement elevated the composition of total PUFAs after 4 weeks of dietary treatment ......................... 42 Locomotor activity in wildtype and fmr1 KO zebrafish after 4 weeks of dietary treatment ...................................................... 43 The anxiety-like behavior in wildtype and fmr1 KO zebrafish after 4 weeks of dietary treatment .............................................. 44 The shoaling behavior and shoaling preference of wildtype and fmr1 KO zebrafish after 4 weeks dietary treatment with 4% linseed oil plus 4% fish oil ................................................... 44 Inhibitory avoidance learning in wildtype and fmr1 KO zebrafish after 4 weeks of dietary treatment .............................. 45 Comparison of fatty acid composition between normal diet and 4% linseed oil + 4% fish oil diet in wildtype and fmr1 KO zebrafish ...................................................................... 46 No significant difference in gene expression of omega-3 synthesis enzymes in liver between wildtype and fmr1 KO zebrafish ...................................................................... 48 Glutamate and 5-hydroxytryptamine receptor expression in wildtype and fmr1 KO zebrafish telencephalon after 4 weeks n-3PUFAs supplement ............................................................... 49. Discussions .............................................................................................. 50 References ................................................................................................ 58 Tables and figures .................................................................................... 71. 2.

(4) Abbreviation table. FXS. Fragile x syndrome. FMRP. Fragile x mental retardation protein. PUFAs. Polyunsaturated fatty acids. n-3 PUFAs. Omega-3 polyunsaturated fatty acids. n-6 PUFAs. Omega-6 polyunsaturated fatty acids. ALA. α –Linolenic acid. SDA. Stearidonic acid. ETA. Eicosatetraenoic acid. EPA. Eicosapentaenoic acid. DHA. Docosahexaenic acid. DPA. Docosapentaenoic acid. LA. Linoleic acid. GLA. γ-linoleic acid. DGLA. Dihomo-γ-linolenic acid. ARA. Arachidonic acid. GC-MS. Gas chromatography-Mass spectrometry. NMDA. N-methyl-D-aspartate. 3.

(5) . AMPA. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. HTR2. 5-hydroxytryptamine (serotonin) receptor 2. FADS. Fatty acid desaturase. ELOVL. ELOVL fatty acid elongase. GRIN. Glutamate receptor, ionotropic, N-methyl D-aspartate. GRIA. Glutamate receptor, ionotropic, AMPA. IA. Inhibitory avoidance. 4.

(6) X. (fragile X syndrome, FXS) FXS. 1/4000. 1/8000. fmr1 CGG. fmr1. mental retardation protein, FMRP). (promoter) (fragile x. FXS. fmr1. fmr1. fmr1 fmr1. (novel tank. 5.

(7) task). fmr1 14. 28. ω-3 fatty acids, n-3 PUFAs). (n-3 polyunsaturated. fmr1 (docosahexaenic acid, DHA). (eicosapentaenoic acid, EPA) DHA. EPA ω-3 fmr1 ω-3. grin1b. htr2a. htr2cl1. -. ω-3 ω-3. 6.

(8) ω-3. X. X. fmr1. 7. ω-3.

(9) Abstract Fragile X syndrome (FXS) is the most generally hereditary form of human mental retardation. Previous researches showed that the onset ratio of FXS is approximately 1/4000 in male and 1/8000 in female. It frequently induced by triplet repeat expansion (CGG) mutation in fragile X mental retardation 1 (fmr1) gene promoter, and resulted in absence of the fragile x mental retardation protein (FMRP) expression. The common symptoms of fragile X patients include learning disabilities, inattention, hyperactivity, anxiety, autistic behaviors, social impairments, as well as other behavioral abnormalities. Recently, zebrafish is considered as an ideal animal model for studying human neurological disorder, due to the progression of genetic techniques and accumulated knowledge on the developmental biology of zebrafish. Our previous results demonstrated the behavioral abnormalities in fmr1 knock out zebrafish such as hyperactivity, abnormal anxiety level, fear memory impairment and autism-like behavior. The present study was aimed to study the functional role of fmr1 gene on the development of social behavior. For achieving this goal, behavioral experiment including shoaling behavior, shoaling preference, locomotor activity monitoring and novel tank task were applied. In addition, we also evaluated the possible therapeutic effect of dietary supplement with polyunsaturated fatty acid on the behavioral abnormalities in fmr1 KO zebrafish. Our results demonstrated the precocious development of shoaling preference behavior is found in fmr1 KO zebrafish which might be resulted from the elevated anxiety level in fmr1 KO zebrafish, but do not. 8.

(10) affect the development of shoaling preference on conspecific zebrafish. We determined the relation between shoaling preference behavior and anxiety level by novel tank test, a well-established behavioral test for anxiety-like behavior in zebrafish. Results indicated the shoaling behavior appeared after 14 dpf, and the level of shoaling in fmr1 KO zebrafish is higher than the wildtype control. Furthermore, the locomotor activity was elevated in fmr1 KO zebrafish at 28 dpf, and they expressed higher anxiety level in novel tank test. These results suggest that the change of shoaling behavior in fmr1 KO zebrafish may be resulted from hyperactivity and increase of anxiety. We also evaluated the possible therapeutic effects of omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as ALA, EPA, and DHA, on behavioral abnormalities in fmr1 KO fish. It is well-known that DHA and EPA are essential nutrients which can reduce the mortality of premature born infants, and they have been proved to enhance mental function in both aging and Alzheimer patients. Studies also suggest the dietary supplement of n-3 PUFAs can reduce the behavioral abnormalities including social-relative problems, autistic, and attention deficit. Recently, n-3 PUFAs supplementation was proved to rescue the behavioral abnormalities, such as alterations in emotionality, social interaction and non-spatial memory in fmr1 KO mice. In our experiments, it indicated that the telencephalic gene expression of grin1b declined in fmr1 KO zebrafish, but htr2a and htr2cl1 elevated after fmr1 loss-off-function. According to our gas chromatography-mass spectrometry (GC-MS) results, a reduction in total PUFAs of the fmr1 KO zebrafish body was found which raised the possibility of using n-3 PUFAs as an adjunctive. 9.

(11) therapy for FXS. Our results demonstrated that after 4 weeks of n-3 PUFAs dietary treatment can partially rescue abnormal behaviors, such as elevated anxiety level and avoidance learning impairment. However, in the liver gene expression of omega-3 synthesis enzymes, there was no significantly difference between wild-type and fmr1 KO zebrafish. We suggested that the lack of PUFAs may account for the abnormal behaviors in fmr1 KO zebrafish, and the n-3 PUFAs supplementation is a potential therapy agent for FXS patients. Keywords: zebrafish, fragile X syndrome, autism, fragile X mental retardation 1, social behavior, omega-3 polyunsaturated fatty acids. 10.

(12) Introductions 1.. Zebrafish Zebrafish (Denio rerio) is a tropical freshwater teleost originated. from south-east Asia. It was introduced as an experimental animal model by Dr. George Streisinger of University of Oregon in the early 1980’s (Streisinger et al., 1981). Not only the simplification of physiological structure and function compared with the conventional rodent models, Zebrafish offers many irreplaceable advantages, such as large number in offspring, easily breed, low cost in maintaining, adosculation, which makes it become an ideal model for studying human disease. Due to its transparence during the development of embryo, it becomes relatively easier than the rodent models for observing the process of organogenesis. Moreover, the hatching of zebrafish is less than 3 days, which can shorten observation time and easy for transgenerational study. Most importantly, its gene sequences were decoded completely, which made it easier to explore and determine the functional role of specific genes during embryonic development (Westerfield, 2007) and physiological function (van 't Padje, 2007). Its popularity was raised during the past decades and zebrafish was approved as a preclinical animal model for biomedical studies and preclinical drug screening by the Food and Drug Administration (FDA) of the United State (The CDER Safety Research Interest Group, 2015) in 2013, which firmed up our confidence of using zebrafish model to study the pathogenesis of heritable human neurological diseases and preclinical drug screening. 2.. Fragile X syndrome (FXS). 11.

(13) Fragile X syndrome (FXS) is the most generally hereditary mental retardation in human. It is reported that the onset ratio of FXS in human is approximately 1/4000 in male and 1/8000 in female since it is an x-linked hereditary disease in human (Turner et al., 1996; Garber et al., 2006) which can explain the higher onset ratio in male. The first clinical report of FXS was published by James Purdon Martin and Julia Bell in the mid-20th century (Martin and Bell, 1943). It had been proven that FXS is resulted from the mutation of the fragile X mental retardation protein (FMRP) which is encoded by fragile X mental retardation-1 (fmr1) in 1991 (Verkerk et al., 1991). The mutation in fmr1 gene resulted from an abnormal triplet repeat expansion (CGG) which located on the long arm of the X chromosome (xq 27.3) (Fu et al., 1991; Oberle et al., 1991; Verkerk et al., 1991), inhibits fmr1 transcription and blocked the expression of FMRP. According to the different numbers of CGG repeat within the 5’ untranslated region (UTR) in fmr1 gene, it can be characterized into at least two different clinically neurological disorders including FXS and fragile X associated tremor/ataxia syndrome (FXTAS). The repeat number of CGG excess 200 resulted in full deficiency of FMRP expression in human and pre-mutation is obtained when CGG repeat number is around 55 to 200 which resulted in FXTAS. Clinical studies showed FXTAS has minor syndrome compared with FXS, certain types of motor symptoms (tremor or ataxia) were occurred in aging male (Baba and Uitti, 2005; Hagerman, 2006). We used fmr1 knockout zebrafish in the present study for mimicking the FXS and clarify the influence of FMRP dysfunctions in the development of social behavior.. 12.

(14) FMRP is a cytoplasmic mRNA-binding protein which has three different domains, including two hnRNP-K-homology (KH) domains and an arginine-glycine-glycine (RGG) box which are involved in the modulation of protein/RNA interaction (Ashley et al., 1993; Siomi et al., 1993b; Siomi et al., 1993a). The amino acid sequences alignment in the functional domain such as nuclear localization signal (NLS), the nuclear export signal (NES), RGG box and two KH domains of the FMRP is highly conserved among species. It is estimated about 4% of developmental genes are interacted and regulated by FMRP (Ashley et al., 1993). FMRP is widely expressed in various tissues, such as brain and testis (Devys et al., 1993; Hinds et al., 1993). Especially, in the brain, the FMRP was expressed in the neurons of the hippocampus, amygdala and the Purkinje cells of the cerebellum (Abitbol et al., 1993; Devys et al., 1993). It had been reported that FMRP is essential for regulation of dendritic mRNA localization and/or synaptic protein synthesis (Feng et al., 1997; Darnell et al., 2001; Zhang et al., 2001; Bassell and Warren, 2008; Kelleher and Bear, 2008). It is also suggested that FMRP plays an important role in the regulation of mRNA binding, which may regulate gene expression by influencing the miRNA pathway (Lee et al., 1993). For example, FMRP would bind with some miRNAs (Ishizuka et al., 2002) and regulated associations with Ago2 (Nakamoto et al., 2005; Cheever and Ceman, 2009) and Dicer (Cheever and Ceman, 2009). These results suggested that FMRP may elicit its function via influencing miRNA pathways.. 13.

(15) Developmental, physical and behavioral abnormalities were found in human FXS patients. The most common physical characteristics in adult FXS patients including elongated face, prominent ears, a high arched palate and macroorchidism (Hagerman, 2006; Reiss and Hall, 2007; McLennan et al., 2011), while children with this disease usually have some emotional and behavioral disabilities such as attention deficit, anxiety, hyperactivity, aggressiveness, learning disabilities, hand flapping and hand biting. There are approximately 5% cases showed highly correlated between FXS and autism spectrum disorders (ASDs) (Brown et al., 1982; Schaefer and Mendelsohn, 2008; Budimirovic and Kaufmann, 2011; McLennan et al., 2011). Because the pathogenesis of FXS is unclear, there are no effective therapeutic strategies available. It is important for elucidating its mechanism which leads to the development of new treatment for FXS patients.. 3.. Animal models for studying FXS It is about 95% and 97% identical in fmr1 coding DNA and amino. acid sequences between human and mice, respectively, previous studies were focused on mice models for studying the pathogenesis of FXS. In addition, unlike the drosophila, zebrafish and frog model which do not have CGG repeat in the 5’ UTR sequences of fmr1 mRNA (van 't Padje et al., 2005; van 't Padje, 2007). The fmr1 has CGG repeat in the 5’UTR which is similar to human (Ashley et al., 1993). Previous microscopic analysis showed increasing spine density and immature spine morphologies were found in the hippocampus, cerebellum and neocortex of fmr1 KO mice (Comery et al., 1997; Nimchinsky et al., 2001;. 14.

(16) Grossman et al., 2006). There are some studies showed that fmr1 KO mice expressed some abnormal phenotypes including maroorchidism, seizures, hyperactivity, reduced anxiety, reduced social interaction, object recognition deficits, and learning impairment (Bakker et al., 1994; Liu et al., 2011; Pietropaolo et al., 2014). Furthermore, electrophysiological studies of the fmr1 KO mice demonstrated neuroplasticity abnormalities such as impairment of long-term potentiation (LTP) formation in the anterior cingulate cortex (ACC), lateral amygdala (Zhao et al., 2005), and elevated group I metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) in the cerebellum (Koekkoek et al., 2005) and hippocampus (Huber et al., 2002). Recently, a drug widely used for treating high cholesterol lovastatin was applied in fmr1 KO mice, which could. prevent. excessive. protein. synthesis. and. mGluR-induced. epileptogenesis in the hippocampus of fmr1 KO mice (Osterweil et al., 2013). This result implied that fmr1 gene may involve in the metabolism of cholesterol and account for the pathogenesis of FXS in human. In the zebrafish FMRP, there are 569 residues of amino acid sequence, which has 72% identity with human. Previous studies demonstrated that the zebrafish FMRP (zFMRP) was widely expressed throughout embryos at 3 hours- post-fertilization (hpf), and the most abundant expression of FMRP was in the brain at 72-hpf embryos. In addition, they showed that FMRP is highly expressed in the telencephalon, diencephalon, metencephalon, spinal cord, cerebellum, and testes (van 't Padje et al., 2005). They suggested that zFMRP might play an important role in the development of central nervous system.. 15.

(17) Furthermore, morpholino antisense oligonucleotide which blocked the translation of fmr1, significantly inhibited the expression of zFMRP and used for evaluating the consequence of fmr1 loss-of-function. Results showed fmr1 might regulate the functions of axonal branching, guidance and fasciculation which may account for the abnormalities of synaptic morphology in FXS patients (Tucker et al., 2006). Controversy, the fmr1 KO zebrafish generated by using TILLING (targeted induced local lesions in genomes) did not show any clinical features comparing to the morpholino knockdown paradigm (den Broeder et al., 2009). In addition, scientists also studied the possible behavioral abnormalities in fmr1 KO zebrafish.. Results. showed. behavioral. abnormalities. in. fmr1. loss-off-functions zebrafishes including lower anxiety level, increase locomotion, inhibitory avoidance learning impairment (Ng et al., 2013), and reduction of novelty-induced anxiety (Kim et al., 2014). Furthermore, in vitro electrophysiological recordings from telencephalic slice revealed markedly reduction in long-term potentiation (LTP) and enhancement in long-term depression (LTD) (Ng et al., 2013) in fmr1 KO zebrafish. These results are not only consistent with the results obtained from human and mouse which explained the possible mechanism of learning and memory impairment in fmr1 KO zebrafish, but also suggest zebrafish is an appropriate preclinical animal model for studying the FXS. 4.. Social behavior Both genetic and environment factors are essential for the. development of social behavior in animals. Those who have impairment of social behavior are often diagnosed with social and communication deficits from early childhood, such as autism, which is regarded to. 16.

(18) neurodevelopmental disorder (American Psychiatric et al., 2013). Interestingly, according to early studies by using rodent models, several candidate genes correlating with social behavior were found, such as oxytocin (Young et al., 2002; Francis et al., 2014), arginine vasopressin (Francis et al., 2014), monoamine oxidase-A (Shih and Chen, 1999) and fragile X (Churchill et al., 2002; Mineur et al., 2002). For example, FXS animal models are widely used to investigate social behavioral disabilities because of this form of mental retardation is observed in autism patients. Compared with the wildtype mice, there are significant increase of anxiety response and decrease in the explorative behavior in the fmr1 KO mouse (McNaughton et al., 2008; Mines et al., 2010; Bhattacharya et al., 2012; Santos et al., 2014), but no significant differences in social preference behavior (Mines et al., 2010; Bhattacharya et al., 2012; Santos et al., 2014). However, recent studies found that fmr1 KO mice preferred interact with stranger mice under normal situation, and their sociability were increased after a correction for hyperactivity (Sorensen et al., 2015). Therefore,. the. exact. social. behavioral. influences. of. fmr1. loss-off-functions require further investigation. Social behaviors in zebrafish such as shoaling, simply aggregation, and schooling, synchronizing movement during shoaling, are well defined (Miller and Gerlai, 2012; Gerlai, 2014). In the last decade, the number of study by using zebrafish model increased rapidly. Previous study by using Albino fish showed zebrafish preferred to interact with the companions has similar body strips (Engeszer et al., 2004). The preference on body strips was developed in the early juvenile and last for. 17.

(19) life long (Engeszer et al., 2007; Mahabir et al., 2013). Our previous study was the first research by using shoaling and shoaling preference behavior paradigm to explore the development of social behavior in fmr1 loss-off-functions zebrafish. Our results indicated that fmr1 KO zebrafish had the precocious development of shoaling preference behavior, which might be resulted from elevated anxiety level in fmr1 KO zebrafish (Hsu et al., 2014). Collectively, these findings suggest the fmr1 KO zebrafish is an ideal model for studying the essential genes account for the development of social behavior.. 5.. Omega-3 Polyunsaturated Fatty Acids The beneficial effect of long-chain omega-3 fatty acid, which may. prevent or treating human neurological diseases recently obtained extensive attention. In omega-3 polyunsaturated fatty acids (n-3 PUFAs) synthesis pathway, the long-chain n-3 PUFAs, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are synthesized from the others short-chain omega-3 PUFAs, such as α-linolenic acid (ALA), through a series of desaturation, elongation and ultimately β-oxidation biochemical reactions (Fig 1) (Moore et al., 1995; Gregory et al., 2011; Dyall, 2015). Clinical studies used n-3 PUFAs as an alternative and adjunctive treatment for neurodevelopmental disorders and desirable results were found. Dietary supplement of DHA and EPA have been proved to beneficial to restore higher mental function in aging patients and reduce the mortality of premature infants (Kidd, 2007). It is well known that DHA is essential for the development of sensory, cognitive, and motor neural systems in human (McCann and Ames, 2005).. 18.

(20) Significant decrease in plasma omega-3/omega-6 ratios was found in autism children (Bell et al., 2010) and certain types of behavioral abnormalities, such as antisocial disorder (Ooi et al., 2015), autism (Meiri et al., 2009; Ooi et al., 2015), and attention deficit disorder (Johnson et al., 2012; Ooi et al., 2015) could be reduced through the supplementary treatment of n-3 PUFAs. It was also reported that n-3 PUFAs treatment attenuated the clinical severity score in Rett syndrome (RTT) (De Felice et al., 2012). Furthermore, n-3 PUFAs have anti-inflammatory properties (Laye, 2010) which can restore and sustain synaptic function and plasticity in patients suffered by neurodegenerative disease (Zhang et al., 2011). Results also indicated that n-3 PUFA supplementation might rescue the neuroinflammatory imbalances and behavioral abnormalities, such as alterations in emotionality, social interaction and non-spatial memory in fmr1 KO mice (Pietropaolo et al., 2014) which raised the possibility that that n-3 PUFAs supplement might be use for treating FXS patients.. 19.

(21) 6.. Aims The fmr1 KO zebrafish model with the impairment in FMRP. expression,. demonstrated. similar. pathological,. physiological. and. behavioral abnormalities, and abnormal phenotypes as human FXS patients. Therefore, we turned our research interest for studying the possible role of FMRP on the development of social behavior and tried to clarify the relationship between social behavior and anxiety level and ensured development stage shoaling behavior. Furthermore, we wanted to study the possible therapeutic effect of the dietary supplement of linseed oil, a highly short-chain n-3 PUFAs, and fish oil, which has abundant long-chain n-3 PUFAs. We anticipated those dietary supplement may rescue the behavioral abnormalities including hyperactivity, abnormal anxiety-level, inhibitory avoidance learning impairment and shoaling preference behavior difference in fmr1 KO zebrafish. Results obtained from the present study will not only expand our knowledge of fmr1 function but also beneficial for the development of novel therapeutic strategies to the FXS patient.. 20.

(22) Materials and Methods 1.. Animals The fmr1 knockout (KO) zebrafish, caring the mutation in the. fmr1hu2787 allele, was acquired from the Wellcome Trust Sanger Institute Zebrafish Mutant Resource. The mutation caused a premature termination at codon position 113 via a C432T change in allele (den Broeder et al., 2009). The behavioral tests were used adult male fish or larvae fish to analyze. Tupfel long fin strain (TL) was used as the control background. Animals of the present study were maintained according to standard guideline (Westerfield, 2007) The experimental procedures were approved and supervised by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan Normal University.. 2.. Genotyping Transgenic zebrafish were subjected for genotyping for confirmation.. Tissues were collected from the fish fin and the genomic DNAs were extracted by using commercialized DNA purification kit (Genemark, Taipei, Taiwan). Quantification of DNA was performed by using a nano spectrophotometer for determine the optical density (OD) at 260 nm and 280 nm (NanoDrop Technologies, Inc. Wilmington, DL).. A derived. cleaved amplified polymorphic sequences (dCAPs) assay was applied. The fmr1 gene sequence was amplified by using forward primer and reverse primer (Table 1) which product size was 222-bp. The PCR cocktail contains 200ng genomic DNA, 0.5 mM dNTP, 1µM of forward and reverse primer, 1unit Prozyme DNA polymerase (Protech Enterprise,. 21.

(23) Taipei, Taiwan) and 1X PCR buffer. The condition of PCR was summarized as following. Cycle. 1. 40. 1. Time. 4 min. 30→30→20 (sec). 20 sec. Temperature (°C). 94. 94→60→72. 72. The amplified sequences were further confirmed by using specific restriction enzymes. Briefly, the PCR product was cleaved by 1 unit RsaI restriction enzyme and the condition was 1X restriction enzyme buffer for overnight (New England BioLabs® Inc., UK). The amplified cDNA products were subjected to restriction enzyme digestion. The cDNA derived from WT DNA template contained two fragments (193-bp and 29-bp). Conversely, the amplified sequence was not split if the template DNA was acquired from hu2787 mutation strain. The PCR products were verified by using electrophoresis in 3% agarose gel which was prepared from TBE buffer (Ng et al., 2013).. 3.. Western blot analysis The fmr1 KO zebrafish were further confirmed by using western. blotting to evaluate the expression of FMRP. Zebrafish were sacrificed and brains were dissected out, homogenized and centrifuged for protein extraction. We followed the procedure of NuPAGE® Technical Guide for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Invitrogen, USA). After electrophoresis, proteins were transferred to PVDF membrane (Millipore, Bedford, MA). The non-specific binding. 22.

(24) was blocked with 5% nonfat milk, 0.05% Tween and 1X PBS in room temperature for 1 hour. Rabbit polyclonal anti-FMRP (GTX125996, 1:1000; Genetex, Irvine, USA) antibody and mouse polyclonal anti-beta-actin. antibody. were applied. in. 4. °C. for overnight.. Chemiluminescence assay was used (Bioman Scientific Co. Ltd., Taiwan) to visualize the signals and protein contents were determined by a LAS3000 digital imaging system (Fujifilm, Tokyo, Japan).. 4.. Behavioral analysis Several behavioral paradigms were applied to determine the fmr1 KO. effects on development of social behavior and possible therapeutic effects of n-3 PUFAs supplement. For example, the locomotor activity was used to analyze the spontaneous motor activity; novel tank was applied to quantify anxiety-like behavior; social score was detected by shoaling and shoaling preference behavior; and inhibitory avoidance task (IA) was also applied for studying the formation of associative memory. The detail procedures of each paradigm were summarized as following.. 4.1.. Locomotor activity For larva, animals were placed into a 6 cm petri dish with water. depth kept in 0.4 cm height for larva behavioral test. For adult fish, a transparent acrylic cylinder (20 cm in height and 24 cm in diameter) with water depth kept with 3.5 cm was used. EthoVision video tracking system (Noldus Information Technology, Leesburg, VA, USA) was applied for data acquisition and analyzing. The recording has two stages including a. 23.

(25) 5 min adaptation and following with a 10 minutes recording. The total moving distance within the 10 minutes recording was used for analyzing the locomotor activity of the zebrafish.. 4.2. Novel tank test In this behavioral test, zebrafish was placed into a transparent rectangular tank (6 cm length, 2 cm width and 6 cm in height), which water depth kept with 4 cm for larval fish test or a transparent trapezoidal tank (26 x 22 x 6x 11.5 cm, top x bottom x width x height) and water depth kept with 11.5 cm for adult fish analysis. Furthermore, two tanks were divided into upper and lower water zone. Each zone contained 1/2 of the total volume in the two different tanks. During the experiment, fish were freely swimming in the tank for 15 minutes, and we divided three different 5 minutes from total detect duration. We calculated the duration in upper zone and transferred to percentage, which value indicates anxiety level in fish (Egan et al., 2009). 4.3. Social behavior To explore the development of social behavior, we followed behavioral procedure from our previous study (Hsu et al., 2014). There were three compartments in the behavior chamber for determining the shoaling and shoaling preference behavior in zebrafish (Fig 2). Compartments were separated by transparent acrylic plates, and each side compartment was placed with different group of fishes, either zebrafish and/or medaka. Animals were placed into the center area (6.4 x 3.0 x 2.5. 24.

(26) x 1.0, length x width x height x depth) and time spent in each side compartment was quantified. Briefly, the center compartment was further divided into three different zones. Both side zones, which closed to both side compartments, occupied 1/5 of the volume of center compartment and the center zone occupied 3/5 of the volume of center area. We analyzed larval fish from 6 days post fertilization (dpf) to 28 dpf. The experimental designs used here including group of zebrafish on both side (ZF-ZF), groups of medaka on both end (MK-MK), or place the zebrafish in one end with medaka in the other end (ZF-MK). The social activity was detected by using an EthoVision video tracking system (Noldus Information Technology, Leesburg, VA, USA) for 10 minutes, and we calculated the duration of swimming time in three different zones and transferred the time to percentage of shoaling “(duration in both side zones)*100 / total duration” and/or shoaling preference score “duration in closing zebrafish zone/ total duration for shoaling”. The n-3 PUFAs diet supplement studies were aimed to clarify whether supplement had rescue effects on social behavior abnormalities in fmr1 KO zebrafish. The apparatus was designed for testing adult fish, with center area size (24 x 12 x 12 x 4.5 cm, length x width x height x depth). In addition, the duration of experiment was 25 minutes. The first 10 minutes is called acclimation phase, both screens were blocked by white acrylic board for avoiding the visual contact between the subject and other group of fishes. After that, both acrylic boards were removed and the fish could observe the different groups of fishes (ZF-MK) in each end. We detected the time of the zebrafish spend on the three different. 25.

(27) zones by EthoVision video tracking system (Noldus Information Technology, Leesburg, VA, USA) The testing time was set for 15 minutes. In the same way for larval study, we calculated the percentage of shoaling and shoaling preference score for qualifying the therapeutic effects of n-3 PUFAs supplement (Engeszer et al., 2004; Engeszer et al., 2007). 4.4.. Inhibitory avoidance (IA) In this research, the apparatus (28 x 12 x 17, length x width x height). was divided into two different zones, a shallow water zone (2 cm in depth) and a deep water zone (8 cm in depth). They were separated with a white, opaque guillotine door between shallow and deep compartment (Fig. 3). Based on the natural preference of zebrafish for a deep environment comparing to a shadow one (Darland and Dowling, 2001), the procedure contained a training phase and a testing phase. Animals were allowed to acclimate to the behavior chamber via a habituation session. They were placed in the shallow chamber for 5 min; the white, opaque guillotine door was then removed, and the fish were allowed to swim freely between the two compartments for another 5 min. In the training session, the fish were placed in the shallow compartment, allowing them to swim for 1 min before the guillotine door was opened. Once the fish entered the deep compartment, the guillotine door was closed, and a mild electric shock was applied to the deep compartment for 5 s. Animals were then tested for the avoidance learning 24 hours later. During the testing phase, we used the similar procedure with training phase but omitting the electrical shock. The duration of fish moved from shallow to deep. 26.

(28) chamber escape latency) was recorded and analyzed. The maximum duration for testing is 300 seconds.. 5.. Gene expression quantitative analysis. 5.1.. RNA extraction Zebrafish was sacrificed with ice water, and we removed their. telencephalon or liver and put in a microcentrifuge tube. 500 µl TRIzol® Reagent (Invitrogen, USA) was added into the tube and we homogenized tissue by using a pestle. 100 µl chloroform (Sigma, USA) was added into the sample and completely mixed which kept on the ice for 10 min. Then, they were put into a centrifuge (Eppendorf, Germany) and centrifuged at 13000 rpm for 30 minutes, and the temperature was kept on 4 °C. Next, the aqueous phase was drawn to another clear tube, and we added 250 µl Isopropanol into the samples and gently mixed them. They were stored into a refrigerator keeping on -20 °C overnight. After that, the samples were centrifuged at 13000 rpm for 15 minutes, and the parameter of temperature was set on 4 °C. We removed supernatant and added 500 µl 75% ethanol (EtOH) into the tube. The samples were centrifuged for 5 minutes, the parameters were kept on 13000 rpm and 4 °C. After finishing centrifugation, we removed EtOH and dried them by using an oven which set on 60 °C until pallet transferred to transparent. Finally, the samples were dissolved through adding 20 µl DEPC water, and we quantified each sample by using NanoDrop ND-1000 (Thermo Scientific, USA) and stored at -80 °C (Alimuddin et al., 2005; Cheng et al., 2015).. 27.

(29) 5.2.. Reverse transcription We used the High-Capacity cDNA Reverse Transcription Kits. (Applied Biosystems, USA) to transfer RNA into complimentary DNA (cDNA). There were two steps in the experiment. The first formula was displayed on below table: Items. Volume (µl). RNA sample (2 µg). X. 10X RT Buffer. 2.0. 10X RT Random Primers. 2.0. 25X dNTP mix (100 mM). 0.8. MultiScribeTM Reverse Transcriptase. 1.0. Nuclease-free H2O. 14.2-X. Total volume. 20. The volume was kept 20 µl in each sample, and we put them into the PCR machine. The protocol was set at 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, and kept temperature at 4 °C. Finally, we stored cDNA (20X, 100 ng/µl) at a -20 °C refrigerator.. 5.3.. Quantitative real-time polymerase chain reaction (q-PCR) In order to investigate the difference of gene expression in. telencephalon and liver between WT and fmr1 KO fish, we selected fmr1, grin1b (glutamate receptor, ionotropic, N-methyl D-aspartate 1b), gria2a (glutamate. receptor,. (5-hydroxytryptamine. ionotropic, receptor,. 2a. AMPA, and. 2a), 2b),. htr2a, htr2cl1,. htr2b htr2cl2. (5-hydroxytryptamine receptor 2C, G-protein-coupled-like 1 and 2),. 28.

(30) fads2 (fatty acid desaturase 2), elovl5, elovl4a, elovl4b (ELOVL fatty acid elongase 5, 4a, and 4b) to be our target genes. eef1a1l1 (eukaryotic translation elongation factor 1 alpha 1, like 1) was used to be an internal control. Primer designs were placed on table 1. The cDNA was diluted to 1X concentration (5 ng/µl). The q-PCR synthesis mix reagents included 4 µL diluted cDNA, 5 µL 2X SYBR green dye, and 1 µL each primer pair (forward and reverse, 10 µM). The method was analyzed by using a LightCycler480 system (Monocolor hydrolysis UPL-probe, Roche Applied Science). The reaction contained four steps set on next page protocol: Cycle. Temp (°C). Duration (sec). Ramp Rate. Incubation. 1. 95. 20. 4.4. Amplification. 45. 95. 3. 4.4. 60. 30. 2.2. 95. 5. 4.4. 65. 60. 2.2. 97. 0. 0.11. 40. 30. 2.2. Melting Curve. Cooling. 1. 1. In order to analyze the relative expression value for each gene. First, threshold cycle (CT) of target gene subtracted CT of internal control gene (ΔCT). Next, the ΔCT in each sample subtracted the average CT of the control sample (WT) called ΔΔCT. Finally, the relative fold expression levels were calculated by the formula 2T-ΔΔC and presented on the result.. 29.

(31) 6. Analyzing the fatty acids composition in fmr1 KO zebrafish To analysis the composition of fatty acids in zebrafish, a protocol developed by Cheng et al in 2015 was applied. Briefly, we sacrificed the zebrafish and total fatty acids of whole fish body were extracted. Folch’s lipid extraction method were used to extract lipid content with mixing organic solvent (chloroform: methanol 2:1), containing butylated hydroxyanisole (BHA) 0.05 mg (Sigma, USA), and aqueous solution, containing 10 mL of 30 mM magnesium chloride (MgCl2) during overnight (Folch et al., 1957). Then, the organic layer was collected and evaporated with a vacuo concentrator (Eyela, Japan). One mL of 50% potassium hydroxide (KOH) (Merck, Germany), 15 mL 90% alcohol (Merck), and boiling stones were added and incubated them at 90°C for 40 min to saponify crude lipid. After saponification, the solvent was kept to cool temperature and purified the samples with 30 mL water and 30 mL ethyl ether (repeated three times). Ten mL of 2 N hydrogen chloride (HCl) (Merck, Germany), 30 mL ethyl ether, and 2 drops of methyl orange (Merck, Germany) were added into samples and were gently shaken to reduce saponified lipid. After 1 min to steady the samples, the organic layers were collected and washed to neutral pH value using water, and evaporated through vacuo concentrator. The purified lipid samples were methyl esterified by addiction of 5 mL of boron trifluoride methanol (BF3 – MeOH) 7% from the 14% solution (Sigma, USA) and boiling stones at 90°C for 20 min. we were then added 5 mL hexane (Sigma, USA) into the samples for 1 min. The organic layers were separated using saturated saline, and absorbed excess water through. 30.

(32) sodium sulfate (Na2SO4) (Merck, Germany). The fatty acid methyl esters were dried and diluted with 400 µL highly pure hexane. Finally, Agilent 5975C Series GC-MSD (Agilent, USA) was used to analyze our lipid samples. The analytic condition was modified from previous study (Abu and Oluwatowoju, 2009), and the Agilent column was 30 mm*0.25 mm with a film thickness of 0.25 mm. We calculated their percentage of fatty composition to analyze differences between dietary treatments.. 6.1.. Fatty acids composition in zebrafish body The lipid of zebrafish body was extracted and subjected for analyzing. the content of fatty acid difference between WT and fmr1 KO zebrafish. The percentage of fatty acid was determined by using GS-MS system.. 6.2.. Fatty acids composition in different n-3 PUFAs dose The dose of n-3 PUFAs in diet were modified from previous study. which significantly rescued behavioral abnormalities in fmr1 KO mice (Pietropaolo et al., 2014). Different doses of n-3 PUFAs (8% linseed oil, 4% linseed oil + 4% fish oil, and 8% fish oil) and Antemia nauplii were supplemented. The control diet was used as the commercial diet (Taikong, Taiwan), its composition of ingredient was displayed on table 2. Different dose of n-3 PUFAs diets were adjusted their type of lipid from commercial diet. The fatty acid composition among different groups were extracted and analyzed by using GC-MS system.. 31.

(33) 7.. Behavior analysis after n-3 PUFAs supplement In order to investigate the possible therapeutic effects of n-3 PUFAs. supplement in adult fmr1 KO zebrafish, we chose 4% linseed oil + 4% fish oil supplement diet to test therapeutic effects on their abnormal behaviors including hyperactivity, higher anxiety level, higher level of shoaling preference, and impairment on fear learning. The experimental procedure and grouping were described on below flow chart.. 32.

(34) Results 1. Qualitative analysis of the genotype The genotyping results showed three different genotypes including wild-type (WT), heterozygous strain (+/-) and homozygous (+/+). In the WT strain (+/+), its PCR product has an endonuclease cleavage site, two fragments, 193-bp and 29-bp (not showed in this figure) were obtained after enzyme digestion, in contrast, the PCR product in fmr1 KO strain (-/-) do not have cleavage site so its fragment size kept in 222-bp, and heterozygous strain (+/-) contained both 222-bp, 193-bp, and 29-bp fragments (Fig 4A). Moreover, the expression of FMRP (72 kDa) was not detected in the fmr1 KO zebrafish brain, the β-actin (42 kDa) was used as the internal control (Fig 4B) in this experiment.. 2. Shoaling behavior and shoaling preference in wildtype and fmr1 KO zebrafish at 14 and 28 dpf There were three experimental design including a zebrafish-zebrafish (ZF-ZF), medaka-medaka (MK-MK), and zebrafish-medaka (ZF-MK). The expectancy value is 40 % which means no shoaling preference behavior were found. In ZF-ZF design (both side areas were placed with zebrafish), we used one sample t test to compare the expectancy value (40%). According to statistics result (WT-6 dpf, p = 0.9195; WT-14 dpf, p = 0.0073*; WT-28 dpf, p < 0.0001*; KO-6 dpf, p = 0.4563; KO-14 dpf p = 0.0004*; KO-28 dpf < 0.0001*), it showed the development of shoaling behavior begin on 14 dpf in both wildtype and fmr1 KO zebrafish. Moreover, we used the two-way ANOVA to analyze the influence of. 33.

(35) genotype and time. The results indicated that there were significant effects in both genotype (F = 6.5017, p = 0.0131*) and age (F = 49.4885, p < 0.0001*) but there was no interaction between them (F = 1.4261, p = 0.2476). We used Dunnett-Hsu for post-hoc analysis in genotype effect, and used WT as the control group. It demonstrated that the percentage of shoaling in fmr1 KO was significantly higher than WT (p = 0.0131*). On the other hand, Tukey-Kramer post hoc analysis (α = 0.05) was used. A significant effect of age on the development of shoaling behavior was found. In MK-MK design (both sides were placed with medaka), the statistical methods were the same as mentioned in figure 5A. The statistic results were (WT-6 dpf p = 0.0028*, WT-14 dpf p = 0.0415*, WT-28 dpf p = 0.0006*, KO-6 dpf p = 0.2705, KO-14 dpf p < 0.0001*, KO-28 dpf <0.0001*), both wildtype and fmr1 KO zebrafish showed shoaling behavior developed in 14 dpf, however, the value in WT-6 dpf was lower than 40%. In two-way ANOVA analysis, the results indicated that there was significant effect in genotype (F = 17.1915, p < 0.0001*) and age (F = 38.2841, p < 0.0001*) but there were significant effects on both genotype and time (F = 2.1615, p = 0.1232). We used Dunnett-Hsu post-hoc for analyzing. It demonstrated that the percentage of shoaling in fmr1 KO was significantly higher than WT (p < 0.0001*). Tukey-Kramer post hoc (α = 0.05) was used to analyze the duration effects. The result showed the shoaling behavior increased incrementally with time (Fig 5B). Our results demonstrated that shoaling behavior is found in 2 week-age-old larval of both group. In the subsequent experiment, we examined the possible differences of shoaling preference development. 34.

(36) between WT and fmr1 KO zebrafish. We excluded the 6 dpf group since no shoaling behavior was detected at this stage. An alternative design was used with placed zebrafish in one end and medaka on the other end (ZF-MK). Results showed both WT and fmr1 KO zebrafish displayed shoaling behavior in 14 and 28 dpf. The statistic values are summarized as following: WT-14 dpf p = 0.0003*, WT-28 dpf p < 0.0001*, KO-14 dpf p < 0.0001*, KO-28 dpf p < 0.0001*. Moreover, it also indicated a significant effect in genotype (F = 5.8615, p = 0.0201*), age (F = 14.8873, p = 0.0004*), and there was no interaction between genotype and age was found (F = 0.0083, p = 0.928). The percentage of shoaling in both genotypes in 28 dpf was significantly higher than WT fish in 14 dpf (Fig 6A). Furthermore, we also explored the shoaling preference behavior in wildtype and fmr1 KO zebrafish. Comparing with expectancy value is 0.5, the statistical result (WT-14 dpf p = 0.6589, WT-28 dpf p = 0.1688, KO-14 dpf p = 0.5938, KO-28 dpf < 0.0001*) demonstrated that a precocious development in shoaling preference behavior in fmr1 KO fish. In two-way ANOVA, the result showed there was no significant difference on the effect of genotype (F = 0.7369, p = 0.3958) but there was significant effect on age (F = 0.0027, p = 0.0027*), and a significant interaction between genotypes and age was found (F = 4.1172, p = 0.0491) using two-way ANOVA. In addition, we used Tukey-Kramer (α = 0.05) for analyzing (Fig 6B). The preference score in fmr1 KO fish was significant higher than WT in 28 dpf. The results were consistency with our previous study (Hsu et al., 2014).. 35.

(37) 3. Locomotor activity in larval zebrafish To determine locomotor activity in larval zebrafish of different genotypes, we evaluated distance moved of WT and fmr1 KO zebrafish at the age of 14 dpf and 28 dpf. Two-way ANOVA was used for determining the influence of genotype and development age. Result showed significant differences on the effects of genotype (F = 16.7401, p = 0.0002*) and age (F = 188.6066, p < 0.0001*), but there was no interaction between two factors (F = 3.2568, p = 0.078). Therefore, we used Tukey-Kramer (α = 0.05) for post-hoc analysis. Results indicated that fmr1 KO zebrafish had increase in moving distance compared with the corresponding WT (28 dpf) (Fig 7).. 4. Evaluate the anxiety-like behavior of fmr1 KO zebrafish by using novel tank task For determining the possible role of fmr1 on the anxiety behavior of zebrafish, larval zebrafish at 14 and 28 dpf were subject to novel tank task. The percentage of time spent in upper zone was evaluated. Result showed significant increase in time spent in upper zone was found in the fmr1 KO fish at 14 dpf. Data were analyzed by using two-way ANOVA, in 14 dpf, it showed significant differences on the effects of genotype (F = 40.6103, p < 0.0001*) but there was no significant difference in the effect of age (F = 2.6381, p = 0.0765). Moreover, there was no interaction between them (F = 0.1502, p = 0.8607). Therefore, we used Dunnett-Hsu post-hoc test for further analysis. The result indicated that percentage of duration in upper zone in fmr1 KO fish was significantly higher than WT. 36.

(38) (p < 0.0001*) (Fig 8A). However, a different result was obtained in 28 dpf. The percentage of time spent in upper zone was significant lower in fmr1 KO fish than WT fish, two-way ANOVA indicated a significant difference on the effects of type (F = 13.9610, p = 0.0003*) and age (F = 6.0588, p = 0.0034*), but there was no interaction between them (F = 0.1576, p = 0.8544). The result suggested that fmr1 KO zebrafish displayed higher anxiety-like behavior (Fig 8B).. 5. Analyzing the locomotor activity of wild-type and fmr1 KO zebrafish It was evidenced that FXS patients performed hyperactivity. We would like to determine whether fmr1 KO zebrafish displays hyperactivity phenotype. We recorded and analyzed the total moving distance for evaluating the locomotor activity of fmr1 KO zebrafish. Data were analyzed by independent t test, and a significant increase in locomotor activity was found in the fmr1 KO zebrafish compared with the wild-type animals (p = 0.0417*) (Fig 9). This result suggested fmr1 KO zebrafish expressed hyperactivity phenotype.. 6. The anxiety-like behavior in wildtype and fmr1 KO zebrafish Novel tank test is widely used for studying the anxiety-like before in zebrafish. Previous results used dark-light task which demonstrated a decrease in anxiety-like behavior in fmr1 KO zebrafish (Ng et al., 2013). We used novel tank test to quantity the anxiety-like behavior of WT and fmr1 KO zebrafish before dietary treatment. Data was analyzed by. 37.

(39) independent t-test. Results indicated that fmr1 KO zebrafish spent less time in the upper zone compared with the WT (***p = 0.0002) (Fig 10) which suggested an increase of anxiety-like behavior in fmr1 KO zebrafish.. 7. The shoaling behavior and shoaling preference of wildtype and fmr1 KO zebrafish We tested shoaling behavior and shoaling preference to determine the possible social behavior deficiency of fmr1 KO zebrafish. Results showed either WT or fmr1 KO zebrafish developed shoaling behavior (Fig 10A). Significant differences were from in WT and fmr1 KO zebrafish comparing with the expectancy value which is 40 % (please refer to the materials and methods for detail). Data was analyzed by one sample t test (WT p = 0.0003*, KO p < 0.0001*). Moreover, independent t test indicated that there was no significant difference among groups (Fig 11A). In shoaling preference, comparing with expectancy value is 0.5, the results showed both groups developed shoaling preference (WT p = 0.0171*, KO p < 0.0001*) and the preference score in fmr1 KO was significantly higher than WT (p = 0.0454*) (Fig 11B).. 8. Inhibitory avoidance learning in wildtype and fmr1 KO zebrafish without dietary treatment For evaluating the possible therapeutic effect of fatty acid dietary treatment on the cognitive function of fmr1 KO zebrafish, single trial. 38.

(40) inhibitory avoidance test was applied in this study. The escape latency of each group was recorded and analyzed by using Wilcoxon signed-rank test. A significant avoidance learning was found in WT zebrafish, there was significant difference between training and test phase (p = 0.0065*). On the other hand, in fmr1 KO zebrafish, there was no significant difference between training and test phase (p = 0.4263) (Fig 12) which showed consistency with our previous founding that fmr1 KO resulted in deficit of avoidance learning (Ng et al., 2013).. 9. Decrease. in. glutamate. receptor. but. increase. in. 5-hydroxytryptamine receptor mRNA content in fmr1 KO zebrafish telencephalon To investigate the difference of telencephalic mRNA content between WT and fmr1 KO fish, we focused on fmr1, gria2a, grin1b, htr2a, htr2b, htr2cl1, and htr2cl2. Independent t test was used to analyze the mRNA content between WT and fmr1 KO zebrafish. In three months old zebrafish, the results indicated the mRNA content of fmr1 (p < 0.0001*) and grin1b (p = 0.0265*) were significantly higher but htr2a (p = 0.0011*) and htr2cl2 (p < 0.0001*) were significantly lower than WT in fmr1 KO zebrafish. Moreover, there was no significant difference was found in gria2a (p = 0.5543), htr2b (p = 0.0724), and htr2cl1 (p = 0.1728) among genotypes (Fig 13A). However, there was no significantly differences in grin1b (p = 0.9837), gria2a (p = 0.1172), htr2a (p = 0.1554), htr2b (p = 0.7102), htr2cl1 (p = 0.1915), and htr2cl2 (p = 0.8648) between WT and fmr1 KO zebrafish’s telencephalon in older than four. 39.

(41) months old zebrafish except for fmr1 (p < 0.0001*) (Fig 13B). In conclusion, fmr1 KO zebrafish showed reduction in mRNA content of NMDAR and increase in HTR2AR and HTR2CL2 in telencephalon than WT zebrafish.. 10. Low polyunsaturated fatty acid content in fmr1 KO comparing with WT zebrafish Recent study showed n-3 PUFA dietary supplementation could rescue the behavioral abnormalities in fmr1 KO mice (Pietropaolo et al., 2014). We speculated the similar effect might also occur in fmr1 KO zebrafish. We test this hypothesis by using GC-MS to compare fatty acid content between WT and fmr1 KO zebrafish (n = 6 in each group). According to the independent t test, in fmr1 KO zebrafish, there was significant difference in total PUFAs (p = 0.0437*) but no significant difference in the percentage of DHA (p = 0.3158), EPA (p = 0.0747), and total n-3 PUFAs (p = 0.1151). It demonstrated that total PUFAs were decreased in fmr1 KO zebrafish compared with WT group (Table 3).. 11. Fatty acid composition in different dose of n-3 PUFAs diets To investigate the fatty acid content of different diets including Artemia nauplii, normal diet, 8% linseed oil, 4%linseed oil plus 4% fish oil, and f% fish oil, we calculated and analyzed their fatty acids composition. One-way ANOVA and Tukey-Kramer test were used to analyze different composition of fatty acid. The detailed statistical value was described in table 4. We specially discussed total PUFAs and n-3. 40.

(42) PUFAs including ALA, EPA, and DHA by GC-MS. Result showed dietary elicit significant effect on the total PUFAs content (F = 26.3165, p < 0.0001*), it might indicate that the normal diet contained lower total PUFAs than other diets. In the content of n-3 PUFAs, there was significant dietary effect (F = 158.7976, p < 0.0001*) and n-3 PUFAs content increased during dose of fish oil elevated. In the content of ALA, there was significant dietary effect (F = 1255.948, p < 0.0001*) and Artemia nauplii might be included higher ALA than others diets. In content of EPA, there was dietary effects (F = 77.9483, p < 0.0001*), it demonstrated that EPA composition was elevated during dose of fish oil was raised and Artemia nauplii might be lower containment in different doets. In content of DHA, there was dietary effects (F = 326.6223, p < 0.0001*), the result showed that its composition was increased during dose of fish oil was elevated and DHA was no detected on Artemia nauplii. Therefore, 8% linseed oil diet was chosen in which specifically increase the content of total PUFAs. (Table 4). 12. Linseed oil enrichment incrementally elevated the composition of total PUFAs with duration of treatment In order to find the optimal duration of treatment, we selected commercial and 8% linseed oil supplement diets to be the experimental diets. Zebrafish were sacrificed and extracted their lipid after 1, 2, 4 weeks of dietary treatment, and calculated the composition of total PUFAs. One-way ANOVA and Dunnett’s test were used to be statistics method, and 0 week was as the control in each group. In WT zebrafish,. 41.

(43) there was no duration effect (F = 3.9638, p = 0.0530) and it did not increase with duration (1 week p = 0.8679, 2 weeks p = 0.0680, and 4 weeks p = 0.0611) after normal diet treatment, and there was temporal effects (F = 15.9973, p = 0.0010*) and increased with the duration of treatment (1 week p = 0.0125*, 2 weeks p = 0.0036*, and 4 weeks p = 0.0004*) after 8% linseed oil plus. In fmr1 KO zebrafish, there was temporal effects (F = 12.0138, p = 0.0025*) and increased with the duration of dietary supplement (1 week p = 0.6760, 2 weeks p = 0.1118, and 4 weeks p = 0.0051*) through normal diet treatment, and there was duration effects (F = 431.0834, p < 0.0001*) and increased with the rise time (1 week p < 0.0001*, 2 weeks p < 0.0001*, and 4 weeks p < 0.0001*) after 8% linseed oil supplement (Fig 14). In conclusion, we chose 4 weeks as the diet supplement duration because the total PUFAs peaked after 4 weeks dietary treatment.. 13. n-3 PUFAs supplement elevated the composition of total PUFAs after 4 weeks of dietary treatment For determining the optimal combination of n-3 PUFAs supplement, 8% linseed oil, 4% linseed oil + 4% fish oil, and 8% fish oil supplement were evaluated, and commercial diet was taken as control group. Zebrafish were sacrificed after 4 weeks of dietary treatment. Fatty acids were extracted and subject for GC-MC for analyzing the composition of total PUFAs. Data were analyzed by using two-way ANOVA and Tukey-Kramer test (α = 0.05), and statistical difference was represented by using different alphabets. According to the statistic results, there were. 42.

(44) genotypic effect (F = 21.0599, p < 0.0001*), dietary effect (F = 35.4458, p < 0.0001*), and interaction among factors (F = 8.4994, p = 0.0002*). Tukey-Kramer test indicated that normal diet groups contained lower composition of total PUFAs than others group except for 8% fish oil plus in fmr1 KO zebrafish (Fig 15). Base on those results, we chosen 4% linseed oil + 4% fish as supplement diet because it might increase total PUFAs content unlike 8% fish oil diet treatment which did not elevate total PUFAs in fmr1 KO zebrafish.. 14. Locomotor activity in wildtype and fmr1 KO zebrafish after 4 weeks of dietary treatment In our previous research, the fmr1 KO zebrafish expressed hyperactivity phenotype (Ng et al., 2013), since we found that total PUFAs was decreased in fmr1 KO zebrafish, we would like to determine the possible therapeutic effect of dietary treatment with n-3 PUFAs on the hyperactivity. In two-way ANOVA analysis, there was genotypic effect (F = 6.017 p = 0.0165), neither dietary effect (F = 0.6944, p = 0.4073) nor interaction between genotypic and dietary effects (F = 2.6281, p = 0.1091) was found. Moreover, we used Tukey-Kramer post-hoc for further confirmation, results showed that the total moving distance in fmr1 KO zebrafish was higher than WT after normal diet treatment and there was no significant effect on locomotor activity after 4% linseed oil + 4% fish oil treatment in fmr1 KO zebrafish (Fig 16).. 43.

(45) 15. The anxiety-like behavior in wildtype and fmr1 KO zebrafish after 4 weeks of dietary treatment The novel tank task was applied again for determining the possible therapeutic effect of n-3 PUFAs dietary treatment on the anxiety-like behavior in fmr1 KO zebrafish, two-way ANOVA and Tukey-Kramer post hoc were used. Results indicated that there were both genotypic effect (F = 6.5351 p = 0.0131*) and dietary effect (F = 46.4145, p < 0.0001*), but no interaction between genotypic and dietary effects (F = 0.4048, p = 0.527). Moreover, the post-hoc demonstrated that fmr1 KO zebrafish spent less time in the upper zone than WT zebrafish treated with normal diet, and the time spent in upper zone was increased after 4 weeks dietary treatment with 4% linseed oil plus 4% fish oil (Fig 17).. 16. The shoaling behavior and shoaling preference of wildtype and fmr1 KO zebrafish after 4 weeks dietary treatment with 4% linseed oil plus 4% fish oil To evaluate the possible rescue effect of n-3 PUFAs dietary supplement in social behavior of fmr1 KO zebrafish, we evaluated shoaling behavior and shoaling preference after 4 weeks of dietary treatment. First, we analyzed their percentage of shoaling by using one sample t-test. Comparing with expectancy value 40% (dash line in Fig 21A), results were (WT-normal p = 0.0031*, WT-4% linseed oil + 4% fish oil p = 0.0004*, KO-normal p = 0.0006*, KO-4% linseed oil + 4% fish oil p = 0.0007*). These data demonstrated that shoaling had developed in each group. Moreover, the two-way ANOVA analysis. 44.

(46) indicated that there was no treatment effect among genotypes (F = 0.8499, p = 0.3603), diets (F = 0.056, p = 0.8138), and no interaction between genotypes and diets (F = 0.1047, p = 0.7474) (Fig 18A). After the shoaling was confirmed, we explored its preference behavior after n-3 dietary treatment. Comparing with expected value (0.5), the statistical result (WT-normal p = 0.0408*, WT-4% linseed oil + 4% fish oil p = 0.0043*, KO-normal p < 0.0001*, KO-4% linseed oil + 4% fish oil p = 0.0162*) demonstrated that preference behavior had developed in each group. In two-way ANOVA, results showed that there was treatment effect in genotype (F = 4.2861, p = 0.0437*) and interaction between genotype and duration (F = 4.605, p = 0.0369*), but there was no dietary effect (F = 0.2448, p = 0.623). Moreover, the result of Tukey-Kramer test indicated that fmr1 KO zebrafish had higher shoaling preference score than WT zebrafish in normal diets. However, there was no significant difference after 4% linseed oil + 4% fish oil treatment in fmr1 KO zebrafish (Fig 18B).. 17. Inhibitory avoidance learning in wildtype and fmr1 KO zebrafish after 4 weeks of dietary treatment To determine the possible rescue effect of dietary treatment on the memory impairment in fmr1 KO zebrafish, we tested the inhibitory avoidance learning in both normal diet or 4% linseed oil plus 4% fish oil diet treated WT and fmr1 KO zebrafish. Results were analyzed by using Wilcoxon signed-rank test. In WT zebrafish, there were a significant difference in escape latency between training and test phase in both. 45.

(47) normal diet (p = 0.0002*) and 4% linseed oil plus 4% fish oil treated groups (p = 0.004*) (Fig 19A). In fmr1 KO zebrafish, no significant difference in escape latency between training and test phase was found in normal diet treated groups (p = 0.7148). There was significant difference in escape latency between training and test phase after 4% linseed oil plus 4% fish oil dietary treatment (p = 0.0322*) (Fig 19B). Collectively, these results implied that n-3 PUFAs dietary supplement might rescue the impairment of learning in fmr1 KO zebrafish.. 18. Comparison of fatty acid composition between normal diet and 4% linseed oil + 4% fish oil diet in wildtype and fmr1 KO zebrafish For evaluating the possible therapeutic effect of n-3 PUFAs diet supplement on behavioral abnormalities in fmr1 KO zebrafish, we analyzed the composition of fatty acid extracted from the whole body of zebrafish in two genotypes with normal diet or 4% linseed oil + 4% fish oil diet. Data were analyzed by using two-way ANOVA and Tukey-Kramer test (α = 0.05), and statistical difference was represented by using different alphabets. The detailed statistical value was displayed on table 5. We focused on a part of fatty acids such as total n-6 PUFAs, linoleic acid (LA), EPA, DHA, total n-3 PUFAs, total PUFAs, and n-6/n-3 ratio. In LA content, there were genotypic effect (F = 67.5282, p < 0.0001*), dietary effect (F = 130.1256, p < 0.0001*), and interaction among factors (F = 33.0921, p < 0.0001*). The result indicated that there was significant reduction in fmr1 KO zebrafish compared with WT. 46.

(48) zebrafish after Artemia nauplii and normal diet supplement, but there was no significant difference between two genotypes after n-3 PUFAs diet supplement. In the analysis on total n-6 PUFAs composition, there were genotypic effect (F = 88.9852, p < 0.0001*), dietary effect (F = 146.5937, p < 0.0001*), and interaction among factors (F = 42.5286, p < 0.0001*). According to Tukey-Kramer test, it indicated that there was significant reduction in fmr1 KO zebrafish compared with WT zebrafish after Artemia nauplii and normal diet supplement, but there was no significant difference between two genotypes after n-3 PUFAs diet supplement. In n-3 PUFAs such as EPA, there were genotypic effect (F = 29.5142, p < 0.0001*), dietary effect (F = 15.0622, p < 0.0001*), and interaction among factors (F = 22.0013, p < 0.0001*). The result indicated that there was significant increase in fmr1 KO zebrafish compared with WT zebrafish after Artemia nauplii, but there was no significant difference between two genotypes after normal and n-3 PUFAs diet supplement. In the DHA composition, there were no genotypic effect (F = 2.8184, p = 0.1036) and interaction between factors (F = 0.7764, p = 0.4691), but there was dietary effect (F = 84.4901, p < 0.0001*). The result indicated that there was significant increase after normal and n-3 PUFAs diet enrichment. In the analysis on total n-3 PUFAs composition, there were dietary effect (F = 8.3591, p = 0.0013*) and interaction between factors (F = 8.3263, p = 0.0013*), but there was no genotypic effect (F = 2.7236, p = 0.1093). It indicated that there was significant increase in fmr1 KO zebrafish compared with WT zebrafish after Artemia nauplii, and there was significant increased after n-3 PUFAs diet supplement. In total PUFAs content, there were genotypic effect (F = 37.6372, p < 0.0001*), dietary. 47.

(49) effect (F = 135.7849, p < 0.0001*), and interaction between factors (F = 10.3935, p = 0.0004*). The result indicated that there was significant reduction in fmr1 KO zebrafish compared with WT zebrafish after Artemia nauplii and normal diet supplement, but there was no significant difference between two genotypes after n-3 PUFAs diet supplement. In total PUFAs content, there were genotypic effect (F = 37.6372, p < 0.0001*), dietary effect (F = 135.7849, p < 0.0001*), and interaction between factors (F = 10.3935, p = 0.0004*). The result indicated that there was significant reduction in fmr1 KO zebrafish compared with WT zebrafish after Artemia nauplii and normal diet supplement, but there was no significant difference between two genotypes after n-3 PUFAs diet supplement. In n-6/n-3 ratio, there were genotypic effect (F = 59.7822, p < 0.0001*), dietary effect (F = 45.4193, p < 0.0001*), and interaction between factors (F = 38.0256, p < 0.0001*). The result indicated that there was significant reduction in fmr1 KO zebrafish compared with WT zebrafish after Artemia nauplii supplement, but there was no significant difference between two genotypes after normal and n-3 PUFAs diets supplement (Table 5).. 19. No significant difference in gene expression of omega-3 synthesis enzymes in liver between wildtype and fmr1 KO zebrafish In order to investigate the reason of lower total PUFAs content in fmr1 KO zebrafish, we hypothesize that the expression of n-3 synthesis enzymes, such as fads2, elovl4b, and elovl5, might be lower in fmr1 KO zebrafish telencephalon. We used real-time PCR to analyze the gene. 48.

(50) expression in liver, and independent t test was used to calculate the difference between WT and fmr1 KO zebrafish. The results indicated that there was no significant difference between two genotypes in fads2 (p = 0.1195), elovl4b (p = 0.7811), and elovl5 (p = 0.4485) (Fig 20). 20. Glutamate and 5-hydroxytryptamine receptor expression in wildtype and fmr1 KO zebrafish telencephalon after 4 weeks n-3PUFAs supplement For determining the manipulation of gene expression after n-3 PUFAs diet supplement, we analyzed the telencephalic gene expression in WT and fmr1 KO zebrafish after n-3 PUFAs enrichment. We focused on grin1b, gria2a, htr2a, htr2b, htr2cl1, and htr2cl2 which had been analyzed in previous experiment. Data were analyzed by using two-way ANOVA and Tukey-Kramer test (α = 0.05), and statistical difference was represented by using different alphabets. However, there was no genotypic effect, dietary effect, and interaction in each target genes. The detailed analytic data was presented in table 6.. 49.

(51) Discussions The present study was aimed to study the role of fmr1 on the development of social behaviors particularly the shoaling behavior and the shoaling preference in zebrafish. We also evaluated the possible therapeutic effect of dietary supplement of polyunsaturated fatty acids on the social behavioral deficit. It is a nice extension of our previous study showed that an reduction in anxiety-like behavior (Ng et al., 2013) and a precocious development of shoaling preference in fmr1 KO zebrafish (Hsu et al., 2014). We speculated that the elevated anxiety-like behavior and precocious development of shoaling preference is highly correlated. Our previous study did not use appropriate control groups for testing the shoaling behavior of fmr1 KO zebrafish which made it impossible to discriminate the confounding effect of the anxiety on social behavior. In addition, the previous studies solely performed dark-light task for evaluating the anxiety level of fmr1 KO zebrafish which was highly depends on visual function and spontaneous activity make it impossible for obtaining conclusive results. Most importantly, those experiments did not focus on the development of shoaling behaviors therefore unable to clarify the role of anxiety on precocious development of shoaling preference behavior in fmr1 KO zebrafish. Therefore, we want to explore the fmr1 KO fish’s development of shoaling and its correlation with anxiety. Our first result is consistent to previous study indicated that the development of shoaling behavior begins at around 14 day post fertilization (dpf) in both wildtype and fmr1 KO zebrafish. In the present. 50.

(52) study, we also showed the development of shoaling behavior response to the conspecific (WT zebrafishes) and different species (medaka in this experiment) (Fig 5A and 5B) (Engeszer et al., 2007; Dreosti et al., 2015). Results showed the shoaling behavior was elevated in fmr1 KO zebrafish comparing with the WT, it is similar to the previous study by Sorensen showing that an increasing of sociability in fmr1 KO mice which may resulted from hyperactivity of the KO mice (Sorensen et al., 2015). For determine whether the increase in shoaling behavior is resulted by hyperactivity and misinterpreted as increase in sociability, we evaluate the shoaling preference in fmr1 KO zebrafish. Since zebrafish and medaka were applied in different end of the behavioral chamber, it should have no difference in shoaling behavior between each end and the time spent in each side zone would be the same. Result showed a precocious development in shoaling preference was found in fmr1 KO zebrafish which begin at 28 dpf (Fig 6A and 6B), it showed consistency with our previous study (Hsu et al., 2014) and resembles the change of social behavior in fmr1 KO mice model which indicated the social preference remains intact but with impairment in the social response regard to novel objects (McNaughton et al., 2008; Mines et al., 2010; Bhattacharya et al., 2012; Santos et al., 2014; Sorensen et al., 2015). We suggested that the partially impairment in social behavior avoiding interaction with unfamiliar object, individual or species may be accounted for the change of shoaling preference behavior in fmr1 KO zebrafish. In addition, our result also demonstrated that it has higher locomotor activity in fmr1 KO zebrafish at 28 dpf but not at 14 dpf (Fig 7). Collectively, these results are supporting our assumption that a precocious development in social. 51.

(53) behavior is one of behavioral phenotype in fmr1 KO adult zebrafish (Ng et al., 2013). We used novel tank task for determining the anxiety-like behavior in fmr1 KO zebrafish. Unlike the conventional dark-light test that only allowed a two dimensional activity monitoring (horizontal movement) and was easy to be confounded by hyperactivity, novel tank task monitored the three dimensional activity of the zebrafish and used the time spent in upper tank as an index for describing the anxiety in fish, therefore the possible confounding effect of hyperactivity can be excluded. Furthermore, it is the first study for using novel tank task to determine the anxiety-like behavior in larvae and juvenile zebrafish. Results showed fmr1 KO zebrafish spent more time in the upper tank comparing with the corresponding WT in 14 dpf. In contrast, an inverse result was obtained in 28 dpf which fmr1 KO zebrafish showed a decrease in time spent in upper tank compared with the correspondence wildtype control. (Fig 8A and 8B). It is widely accepted that adult zebrafish prefer to stay in the lower zone of the tank when placed in a novel environment, the time spent in the upper zone is inverse proportional to the anxiety level. Therefore, can be used as an index for evaluating the anxiety level of the zebrafish, spent more time in the upper zone represent less anxiety and vice versa (Egan et al., 2009; Maximino et al., 2013), As we mentioned earlier, we are the first lab used novel tank task for studying anxiety level in larval zebrafish. Previous study by using dark-light task displayed opponent preference on anxiety-like behavior between larval and adult zebrafish (Sackerman et al., 2010; Steenbergen et al., 2011). In the present study, we found a. 52.

(54) significant increase in anxiety response in fmr1 KO zebrafish which compared with wildtype group. We speculate the difference might result from the confounding effect of hyperactivity in dark-light task was excluded in novel tank task. Recently, Maximino reported dark-light task and novel tanks task might detect different types of anxiety-like behavior in zebrafish which is similar to the freezing behavior and fear-potentiated startle response for evaluating contextual fear and cue conditioning in rodents (Maximino et al., 2012). One of our major goal of the study was exploring the possible therapeutic effects of n-3 PUFAs on abnormal behaviors in fmr1 KO fish, such as hyperactivity (Fig 9), increase in anxiety-like behavior (Fig 10), higher level of shoaling preference (Fig 11), and inhibitory avoidance learning impairment (Fig 12) (Ng et al., 2013; Hsu et al., 2014). According to real-time PCR, we also discovered that there is a significant reduction of grin1b in fmr1 KO telencephalon, it suggested that low expression on NMDAR and high expression on HTR2A and HTR2CL2 might cause their behavioral abnormalities (Fig 13A). At the beginning, we determine the total PUFAs of the zebrafish by GS-MS and found significant reduction of total PUFAs in fmr1 KO zebrafish (Table 3). It raised the possibility that decrease in total PUFAs may account to the behavioral abnormalities. Previous study showed dietary supplement of PUFAs might reduce the severity of social behavior deficit in Autisms. Therefore, we would like to evaluate the possible therapeutic effects of n-3 PUFAs dietary supplement on fmr1 KO zebrafish. The combination of PUFAs chosen was based on our pilot study showing 4 weeks of dietary treatment obtained optimal effect on enhancing zebrafish body. 53.