背景簡介：FMR1 (fragile X mental retardation 1) 基因位於人類的Xq27.3基因座，當其CGG核苷酸重複序列 (nucleotide repeat) 過長時，將會造成X-染色體脆折症 (fragile X syndrome, 簡稱 FXS)。現已開發出可用於研究FXS的斑馬魚 (Danio rerio) 突變品系，相較於囓齒類動物，斑馬魚具有快速發育、幼體透明和高繁殖力等研究優勢。前人的囓齒類動物研究發現，FMR1突變與發炎反應間存有關聯，特別是壓力條件下的細胞激素 (cytokine) 和c-Fos的表現。已知二甲基亞碸 (dimethyl sulfoxide，簡稱DMSO）為一種的消炎藥 (anti-inflammation drug)，具有多種免疫調節效果和臨床應用。 本計劃藉由建立清晰的FMR1突變行為和基因表達表型後，來探討DMSO當作治療FXS藥物的可能性。
研究方法：利用顯微鏡觀察受精後 (post-fertilization, dpf) 三天大幼魚的心率和體長，以評估DMSO處理對FMR1突變品系胚胎及幼體發育的影響。採用公開性的「深度學習軟件」，進行多動物同時自發性運動跟踪法 (multi-animal locomotor tracking)，分別對幼魚進行2分鐘 (n = 10) 及對成魚進行5分鐘 (n = 5) 的記錄及分析。通過觀察幼蟲的趨態性 (thigmotaxis) 和淺水內聚性 (shoal cohesion) 來評估幼魚的數焦慮行為 (anxiety-like behavior)，並使用新型水箱潛水模式 (novel tank) 對成魚進行焦慮評估。利用C-start反射評估幼魚的學習行為通過觀察幼蟲的趨態性 (thigmotaxis) 和淺水內聚性 (shoal cohesion) 來評估幼魚的焦慮程度，並使用新型水箱潛水模式 (novel tank) 對成魚進行焦慮評估。利用C-start反射評估幼魚的非聯結型學習行為 (non-associative learning)，而成魚則採用抑制性逃避模式 (inhibitory avoidance) 來評估學習反應。並以2項選擇模式 (two choice paradigm) 來評估成魚的社會興趣反應 (social interest paradigm)。最後透過定量聚合酶連鎖反應 (quantitative PCR) 評估全腦中FMR1和細胞激素 (cytokines) 的基因表現。
實驗結果：突變品系成魚的行為表型分析顯示，同型合子 (homozygotes) 出現過動的反應 (hyperactivity)，異型合子 (heterozygotes) 對陌生魚的社會興趣 (allospecific social interest) 增加，同型合子中的焦慮反應及恐懼學習(fear learning)減少。DMSO的長期投予最佳濃度為0.05％，可恢復突變品系幼魚的趨態性(thigmotaxis)和淺灘凝聚性 (shoal cohesion) 行為表型。在5-dpf時觀察到誘導的C-起始反射 (strike induced C-start) 的減少，暗示該濃度的DMSO對毛細胞可能具有潛在的毒性，然而在7-dpf的幼魚身上，並未呈現空間運動的異常。該濃度的DMSO投予能夠改善突變品系成魚的焦慮和學習缺陷等行為表型。儘管DMSO處理不能使FMR1的表現恢復到正常水平，但能顯著改善c-Fos及適度改善細胞激素因子 (IL-1β，IL-6和IL-10) 的表現。
結論：本計劃的結果顯示，1）FMR1突變品系的同型合子為適合的FXS動物模型，2）DMSO的使用可減少突變品系幼魚的異常行為，3）DMSO可降低突變品系成魚的異常行為，並使其腦中發炎反應基因 (inflammation genes) 表現減少。
討論：突變品系成魚的運動，焦慮和恐懼學習結果與以前的囓齒動物和斑馬魚FXS模型大致相同。但在社會興趣的結果有差異，前人報導將突變品系對同種 (cospecific) 的興趣大於同種異體 (allospecific)。在幼魚實驗中的一些新發現，包括FMR1突變體在5-dpf時明顯的非聯結型學習 (non-associative learning) 障礙，焦慮以及淺灘凝聚力的增加。過去的文獻推測的腦部發炎反應基因增加，本計劃發現FMR1 KO樣本腦中神經炎症基因被下調。進一步探討特次腦區的特異性表現 (region-specific expression)，特別是在端腦內側和外側大腦皮層 (telencephalic medial and lateral pallium) ，可能會得到與囓齒類動物一致的結果。

Introduction: The fragile X mental retardation 1 (FMR1) gene is prone to developing a series of CGG repeats at locus Xq27.3 in humans, when sufficiently long, will lead to Fragile X syndrome (FXS). Zebrafish (Danio rerio) mutant models have been developed to study FXS, as they offer several advantages over rodents such as rapid development, transparent larvae, and high fecundity. The link between FMR1 mutation and inflammation has been described in rodent models, particularly regarding cytokine and c-Fos expression under inflammatory or otherwise stressful conditions. Moreover, DMSO is a known anti-inflammatory drug known to possess several immunomodulatory effects and clinical applications. This project aimed to explore the potential of DMSO as a therapeutic agent in the treatment of FXS by establishing clear FMR1 mutant behavioral and gene expression phenotypes.
Methodology: The microscopic assessment of larval heart rate and body length were performed at three days post-fertilization (dpf) to evaluate the effect of FMR1 knockout and embryonic DMSO treatment on gross anatomical development. Multi-animal locomotor tracking was conducted for 2 minutes (larva; n=10) or 5 minutes (adult; n=5) using open-source, deep-learning software. Anxiety-like behavior was examined in larvae by observing thigmotaxis, shoal cohesion, and adults using the novel tank dive paradigm. Non-associative learning was evaluated in larvae by exploiting the C-start reflex and adults using the inhibitory avoidance paradigm. Social interest was evaluated in adults using a 2-choice paradigm. Whole-brain gene expression of the FMR1 and cytokines were evaluated by quantitative PCR.
Results: Identification of adult mutant behavioral phenotypes revealed hyperactivity among homozygotes, increased allospecific interest among heterozygotes, and decreased anxiety and fear learning in homozygotes. Optimal DMSO exposure was found to be a chronic dose at 0.05% concentration, which recovered mutant larval thigmotaxis and shoal cohesion phenotypes. While a potential toxic effect was observed as a loss of strike-induced C-start reflex at 5-dpf, no deficit in reactivity, locomotion or spatial orientation was observed at 7 dpf. This treatment protocol was also able to recover diminished anxiety fear learning in mutant adults. Although DMSO treatment did not recover target gene expression back to WT levels, a significant improvement was seen in c-Fos expression as well as a modest improvement among cytokines IL-1β, IL-6, and IL-10.
Conclusion: Evidence gathered during this project suggests that 1) FMR1 KO homozygotes are a suitable FXS knockout model, 2) Administration of DMSO improves abnormal behavior in larval mutants, and 3) DMSO improves abnormal behavior in adult mutants, for whom inflammation genes are strongly downregulated in the brain.
Discussion: Locomotor, anxiety, and fear learning results in adult mutants were consistent with previous rodent and zebrafish FXS models. However, social interest results differ from previous reports describing mutants as interested in conspecific over allospecific fish. My larval experiments demonstrate several novel findings, including an apparent non-associative learning deficit among FMR1 mutants at 5-dpf, increased anxiety, and an open-source method of quantifying shoal cohesion. Contrary to the hypothesized increase in brain inflammation based on past literature, neuroinflammation genes were strongly downregulated in whole-brain FMR1 KO samples. Further investigation of region-specific expression, particularly at the telencephalic medial and lateral pallium, may yield results in line with rodent results.

Anderson, J. L., Marí, A. R., Braasch, I., Amores, A., Hohenlohe, P., Batzel, P., & Postlethwait, J. H. (2012). Multiple Sex-Associated Regions and a Putative Sex Chromosome in Zebrafish Revealed by RAD Mapping and Population Genomics. PLoS ONE, 7(7). doi:10.1371/journal.pone.0040701

Ashwood, P., Nguyen, D. V., Hessl, D., Hagerman, R. J., & Tassone, F. (2010). Plasma cytokine profiles in Fragile X subjects: Is there a role for cytokines in the pathogenesis? Brain, Behavior, and Immunity, 24(6), 898-902. doi:10.1016/j.bbi.2010.01.008

Augustine, S., Gagnaire, B., Floriani, M., Adam-Guillermin, C., & Kooijman, S. (2011). Developmental energetics of zebrafish, Danio rerio. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 159(3), 275-283.doi:10.1016/j.cbpa.2011.03.016

Bencan, Z., Sledge, D., & Levin, E. D. (2009). Buspirone, chlordiazepoxide and diazepam effects in a zebrafish model of anxiety. Pharmacology Biochemistry and Behavior, 94(1), 75-80. doi:10.1016/j.pbb.2009.07.009

Bercik, P., Verdu, E., Foster, J., Macri, J., Potter, M., Huang, X., Malinowski, P., Jackson, W., Blennerhassett, P., Neufeld, K., Lu, J., Khan, W., Corthesy-Theulaz, I., Cherbut, C., Bergonzelli, G., and Collins, S. (2010). Chronic Gastrointestinal Inflammation Induces Anxiety-Like Behavior and Alters Central Nervous System Biochemistry in Mice. Gastroenterology, 139(6). doi:10.1053/j.gastro.2010.06.063

Birder, L. A., Kanai, A. J., & Groat, W. C. (1997). Dmso: Effect On Bladder Afferent Neurons And Nitric Oxide Release. Journal of Urology, 158(5), 1989-1995. doi:10.1016/s0022-5347(01)64199-5

Blank, M., Guerim, L., Cordeiro, R., & Vianna, M. (2009). A one-trial inhibitory avoidance task to zebrafish: Rapid acquisition of an NMDA-dependent long-term memory. Neurobiology of Learning and Memory, 92(4), 529-534.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248-254. doi:10.1016/0003-2697(76)90527-3

Broeder, M. J., Linde, H. V., Brouwer, J. R., Oostra, B. A., Willemsen, R., & Ketting, R. F. (2009). Generation and Characterization of Fmr1 Knockout Zebrafish. PLoS ONE, 4(11). doi:10.1371/journal.pone.0007910

Calder, P. C. (2017). Omega-3 fatty acids and inflammatory processes: From molecules to man. Biochemical Society Transactions, 45(5), 1105-1115. doi:10.1042/bst20160474

Christou, M., Kavaliauskis, A., Ropstad, E., & Fraser, T. W. (2020). DMSO effects larval zebrafish (Danio rerio) behavior, with additive and interaction effects when combined with positive controls. Science of The Total Environment, 709, 134490. doi:10.1016/j.scitotenv.2019.134490

Crawford, D. C., Acuña, J. M., & Sherman, S. L. (2001). FMR1 and the fragile X syndrome: Human genome epidemiology review. Genetics in Medicine, 3(5), 359-371. doi:10.1097/00125817-200109000-00006

Cheng, C., Huang, S., Wu, C., Gong, H., Ken, C., & Hu, S. (2015). Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection. Journal of Biomedical Science. 22:103. doi: 10.1186/s12929-015-0208-1

Dreosti, E., Lopes, G., Kampff, A., & Wilson, S. (2015). Development of social behavior in young zebrafish. Frontiers in Neural Circuits. 9:39. doi: 10.3389/fncir.2015.00039

Eaton, R. C., Farley, R. D., Kimmel, C. B., & Schabtach, E. (1977). Functional development in the mauthner cell system of embryos and larvae of the zebra fish. Journal of Neurobiology, 8(2), 151-172. doi:10.1002/neu.480080207

Egan, R., Bergner, C., Hart, P., Cachat, J., Canavello, P., Elegante, M., Elkhayat, S., Bartels, B., Tien, A., Tien, D., Mohnot, S., Beeson, E., Glasgow, E., Amri, H., Zukowska, A., and Kalueff, A. (2009). Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behavioural Brain Research, 205(1), 38-44. doi:10.1016/j.bbr.2009.06.022

Ferron, L., Novazzi, C. G., Pilch, K. S., Moreno, C., Ramgoolam, K., & Dolphin, A. C. (2020). FMRP regulates presynaptic localization of neuronal voltage gated calcium channels. Neurobiology of Disease, 138, 104779. doi:10.1016/j.nbd.2020.104779

Gerlai, R., Lee, V., & Blaser, R. (2006). Effects of acute and chronic ethanol exposure on the behavior of adult zebrafish (Danio rerio). Pharmacology Biochemistry and Behavior, 85(4), 752-761. doi:10.1016/j.pbb.2006.11.010

Goering, R., Hudish, L. I., Guzman, B. B., Raj, N., Bassell, G. J., Russ, H. A., . . . Taliaferro, J. M. (2020). FMRP promotes RNA localization to neuronal projections through interactions between its RGG domain and G-quadruplex RNA sequences. Elife, doi:10.1101/784728

Grigsby, J., Brega, A., Jacquemont, S., Loesch, D., Leehey, M., Goodrich, G., Hagerman, R., Epstein, J., Wilson, R., Cogswell, J., Jardini, T., Tassone, F., Dominguez, D., Hagerman, P. (2006). Impairment in the cognitive functioning of men with fragile X-associated tremor/ataxia syndrome (FXTAS). Journal of the Neurological Sciences, 248(1-2), 227-233. doi:10.1016/j.jns.2006.05.016

Hagerman, P. (2013). Fragile X-associated tremor/ataxia syndrome (FXTAS): Pathology and mechanisms. Acta Neuropathologica, 126(1), 1-19. doi:10.1007/s00401-013-1138-1

Hardingham, G., & Bading, H. (2010). Synaptic versus extrasynaptic NMDA receptor signalling: implications for neurodegenerative disorders. Nat Rev 11: 682–696.

Heras, F. J., Romero-Ferrero, F., Hinz, R. C., & Polavieja, G. G. (2018). Deep attention networks reveal the rules of collective motion in zebrafish. PLoS Computational Biology, doi:10.1101/400747

Hessl, D., Dyer-Friedman, J., Glaser, B., Wisbeck, J., Barajas, R. G., Taylor, A., & Reiss, A. L. (2001). The Influence of Environmental and Genetic Factors on Behavior Problems and Autistic Symptoms in Boys and Girls With Fragile X Syndrome. Pediatrics, 108(5). doi:10.1542/peds.108.5.e88

Hodges, S. L., Nolan, S. O., Tomac, L. A., Muhammad, I. D., Binder, M. S., Taube, J. H., & Lugo, J. N. (2020). Lipopolysaccharide-induced inflammation leads to acute elevations in pro-inflammatory cytokine expression in a mouse model of Fragile X syndrome. Physiology & Behavior, 215, 112776. doi:10.1016/j.physbeh.2019.112776

Hollebeeck, S., Raas, T., Piront, N., Schneider, Y., Toussaint, O., Larondelle, Y., & During, A. (2011). Dimethyl sulfoxide (DMSO) attenuates the inflammatory response in the in vitro intestinal Caco-2 cell model. Toxicology Letters, 206(3), 268-275. doi:10.1016/j.toxlet.2011.08.010

Hsu, M.T., (2017). The developmental abnormalities in social behavior and therapeutic effects of omega-3 polyunsaturated fatty acid in fmr1 knock-out zebrafish (Denio rerio) (Master’s thesis). National Taiwan Normal University, Taipei, Taiwan.

Huang, S.H., Wu, C.H., Chen, S.J., Sytwu, H.K., Lin, G.J. (2020). Immunomodulatory effects and potential clinical applications of dimethyl sulfoxide. Immunobiology, 225, https://doi.org/10.1016/j.imbio.2020.151906

Jacob, S. W., & Herschler, R. (1986). Pharmacology of DMSO. Cryobiology, 23(1), 14-27. doi:10.1016/0011-2240(86)90014-3

Jacob, S. W., & Torre, J. C. (2009). Pharmacology of dimethyl sulfoxide in cardiac and CNS damage. Pharmacological Reports, 61(2), 225-235. doi:10.1016/s1734-1140(09)70026-x

Johnston, I., Lee, H., Macqueen, D., Paranthaman, K., Kawashima, C., Anwar, A., Kinghom, J., Dalmay, T. (2009). Embryonic temperature affects muscle fibre recruitment in adult zebrafish: Genome-wide changes in gene and microRNA expression associated with the transition from hyperplastic to hypertrophic growth phenotypes. Journal of Experimental Biology, 212(12), 1781-1793. doi:10.1242/jeb.029918

Kais, B., Schneider, K., Keiter, S., Henn, K., Ackermann, C., & Braunbeck, T. (2013). DMSO modifies the permeability of the zebrafish (Danio rerio) chorion-Implications for the fish embryo test (FET). Aquatic Toxicology, 140-141, 229-238. doi:10.1016/j.aquatox.2013.05.022

Krasovska, V., & Dauring, L. (2018). Regulation of IL-6 Secretion by Astrocytes via TLR4 in the Fragile X Mouse Model, Frontiers in Molecular Neuroscience, Retrieved from https://www.frontiersin.org/articles/10.3389/fnmol.2018.00272/full

Kuzniewska, B., Cysewski, D., Wasilewski, M., Sakowska, P., Milek, J., Kulinski, T. M., Dziembowska, M. (2020). Mitochondrial protein biogenesis in the synapse is supported by local translation. EMBO Reports, 21(8). doi:10.15252/embr.201948882

Licznerski, P., Park, H., Rolyan, H., Chen, R., Mnatsakanyan, N., Miranda, P., . . . Jonas, E. A. (2020). ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome. Cell, 182(5). doi:10.1016/j.cell.2020.07.008

Loesch, D. Z., Bui, Q. M., Dissanayake, C., Clifford, S., Gould, E., Bulhak-Paterson, D., . . . Huggins, R. M. (2007). Molecular and cognitive predictors of the continuum of autistic behaviours in fragile X. Neuroscience & Biobehavioral Reviews, 31(3), 315-326. doi:10.1016/j.neubiorev.2006.09.007

Hsu, MT. (2017) The Developmental Abnormalities in Social Behavior and
Therapeutic Effects of Omega-3 Polyunsaturated Fatty Acid
in fmr1 Knock-Out Zebrafish (Denio rerio). (Master’s thesis). National Taiwan Normal University. Taipei, Taiwan.

MacGregor, W. (1967). The Chemical and Physical Properties of DMSO. Crown Zellerbach Corporation. Camas: Washington.

Manuel, R., Gorissen, M., Roca, C. P., Zethof, J., Vis, H. V., Flik, G., & Bos, R. V. (2014). Inhibitory Avoidance Learning in Zebrafish (Danio Rerio): Effects of Shock Intensity and Unraveling Differences in Task Performance. Zebrafish, 11(4), 341-352. doi:10.1089/zeb.2013.0970

Marquez-Legorreta, E., Constantin, L., Piber, M., Favre-Bulle, I. A., Taylor, M. A., Vanwalleghem, G. C., & Scott, E. (2019). Brain-wide visual habituation networks in wild type and fmr1 zebrafish. BMC Biology, doi:10.1101/722074

Martin, J. P., & Bell, J. (1943). A Pedigree Of Mental Defect Showing Sex-Linkage. Journal of Neurology, Neurosurgery & Psychiatry, 6(3-4), 154-157. doi:10.1136/jnnp.6.3-4.154

Mezzomo, N. J., Silveira, A., Giuliani, G. S., Quadros, V. A., & Rosemberg, D. B. (2016). The role of taurine on anxiety-like behaviors in zebrafish: A comparative study using the novel tank and the light–dark tasks. Neuroscience Letters, 613, 19-24. doi:10.1016/j.neulet.2015.12.037

Michopoulos, V., Powers, A., Gillespie, C., Ressler, K., & Jovanovic, T. (2017). Inflammation in Fear- and Anxiety-Based Disorders: PTSD, GAD, and Beyond. Neuropsychopharmacology Reviews. 42. 254-270.

Mocelin, R., Marcon, M., D’ambros, S., Mattos, J., Sachett, A., Siebel, A., Herrmann, A., & Piato, A. (2019). N-Acetylcysteine Reverses Anxiety and Oxidative Damage Induced by Unpredictable Chronic Stress in Zebrafish. Molecular Neurobiology. 56:1188–1195. https://doi.org/10.1007/s12035-018-1165-y

Mos, M. D., Huygen, F. J., Stricker, C. B., Dieleman, J. P., & Sturkenboom, M. C. (2009). The association between ACE inhibitors and the complex regional pain syndrome: Suggestions for a neuro-inflammatory pathogenesis of CRPS. Pain, 142(3), 218-224. doi:10.1016/j.pain.2008.12.032

Ng, M., Yang, Y., & Lu, K. (2013). Behavioral and Synaptic Circuit Features in a Zebrafish Model of Fragile X Syndrome. PLoS ONE, 8(3). doi:10.1371/journal.pone.0051456

Nielsen, D. M., Derber, W. J., Mcclellan, D. A., & Crnic, L. S. (2002). Alterations in the auditory startle response in Fmr1 targeted mutant mouse models of fragile X syndrome. Brain Research, 927(1), 8-17. doi:10.1016/s0006-8993(01)03309-1

Nielsen DM, Derber WJ, McClellan DA, Crnic LS (2002). Alterations in the auditory startle response in Fmr1 targeted mutant mouse models of fragile X syndrome. Brain Research 927: 8–17.

Nolin, S. L., Glicksman, A., Tortora, N., Allen, E., Macpherson, J., Mila, M., . . . Hadd, A. G. (2019). Expansions and contractions of the FMR1 CGG repeat in 5,508 transmissions of normal, intermediate, and premutation alleles. American Journal of Medical Genetics Part A, 179(7), 1148-1156. doi:10.1002/ajmg.a.61165

Ogryzko, N. V., Hoggett, E. E., Solaymani-Kohal, S., Tazzyman, S., Chico, T. J., Renshaw, S. A., & Wilson, H. L. (2013). Zebrafish tissue injury causes upregulation of interleukin-1 and caspase-dependent amplification of the inflammatory response. Disease Models & Mechanisms, 7(2), 259-264. doi:10.1242/dmm.013029

Oostra, B. A., & Willemsen, R. (2003). A fragile balance: FMR1 expression levels. Human Molecular Genetics, 12(Suppl 2). doi:10.1093/hmg/ddg298

Padje, S. V., Engels, B., Blonden, L., Severijnen, L., Verheijen, F., Oostra, B. A., & Willemsen, R. (2005). Characterisation of Fmrp in zebrafish: Evolutionary dynamics of the fmr1 gene. Development Genes and Evolution, 215(4), 198-206. doi:10.1007/s00427-005-0466-0

Petzold, A., Balciunas, D., Sivasubbu, S., Clark, K., Bedell, V., Westcot, S., Myers, S., Moulder, G., Thomas, M., and Ekker, S. (2009). Nicotine response genetics in the zebrafish. Proceedings of the National Academy of Sciences, 106(44), 18662-18667. doi:10.1073/pnas.0908247106

Pietropaolo, S., Goubran, M., Joffre, C., Aubert, A., Lemaire-Mayo, V., Crusio, W. E., & Layé, S. (2014). Dietary supplementation of omega-3 fatty acids rescues fragile X phenotypes in Fmr1-Ko mice. Psychoneuroendocrinology, 49, 119-129. doi:10.1016/j.psyneuen.2014.07.002

Price, D. K., Zhang, F., Ashley, J. C., & Warren, S. T. (1996). The ChickenFMR1Gene Is Highly Conserved with a CCT 5′-Untranslated Repeat and Encodes an RNA-Binding Protein. Genomics, 31(1), 3-12. doi:10.1006/geno.1996.0002

Ribas, L., Valdivieso, A., Díaz, N., & Piferrer, F. (2017). Appropriate rearing density in domesticated zebrafish to avoid masculinization: Links with the stress response. The Journal of Experimental Biology, 220(6), 1056-1064. doi:10.1242/jeb.144980

Richendrfer, H., Pelkowski, S., Colwill, R., & Creton, R. (2012). On the edge: Pharmacological evidence for anxiety-related behavior in zebrafish larvae. Behavioural Brain Research, 228(1), 99-106. doi:10.1016/j.bbr.2011.11.041

Roberts, A., Reichl, J., Song, M., Dearinger, A., Moridzadeh, N., Lu, E., Pearce, K., Esdin, J., Glanzman, D. (2011). Habituation of the C-Start Response in Larval Zebrafish Exhibits Several Distinct Phases and Sensitivity to NMDA Receptor Blockade. PLoS ONE, 6(12). doi:10.1371/journal.pone.0029132

Roberts, A., Chornak, J., Alzagatiti, J., Ly, D., Bill, B., Trinkeller, J., Pearce, K., Choe, R., Campbell, C., Wong, D., Deutsch, E., Hernandez, S., & Glanzman, D. (2019). Rapid habituation of a touch-induced escape response in Zebrafish (Danio rerio) Larvae. PLoS ONE. https://doi.org/10.1371/journal.pone.0214374

Romero-Ferrero, F., Bergomi, M. G., Hinz, R., Heras, F. J., & Polavieja, G. G. (2018). Idtracker.ai: Tracking all individuals in large collectives of unmarked animals. Doi:10.1101/280735

Romero-Ferrero, F., Bergomi, M. G., Hinz, R. C., Heras, F. J., & de Polavieja, G. G. (2019). idtracker.ai: tracking all individuals in small or large collectives of unmarked animals. Nature methods. 16, 179-182.

Schneider, A., Hagerman, R., & Hessl, D. (2009). Fragile X syndrome - From genes to cognition. Developmental Disabilities Research Reviews, 15(4), 333-342. doi:10.1002/ddrr.80

Schnörr, S., Steenbergen, P., Richardson, M., & Champagne, D. (2012). Measuring thigmotaxis in larval zebrafish. Behavioural Brain Research, 228(2), 367-374. doi:10.1016/j.bbr.2011.12.016

Shams, S., Chatterjee, D., Gerlai, R. (2015) Chronic social isolation affects thigmotaxis and whole-brain serotonin levels in adult zebrafish. Behavioural Brain Research. 292. 283-287.

Siomi, M., Siomi, H., Sauer, W., Srinivasan, S., Nussbaum, R., & Dreyfuss, G. (1995). FXR1, an autosomal homolog of the fragile X mental retardation gene. The EMBO Journal, 14(11), 2401-2408. doi:10.1002/j.1460-2075.1995.tb07237.x

Smith, L. E., Barker, E. T., Seltzer, M. M., Abbeduto, L., & Greenberg, J. S. (2012). Behavioral phenotype of fragile X syndrome in adolescence and adulthood. American Journal on Intellectual and Developmental Disabilities, 117(1), 1-17. doi:10.1352/1944-7558-117.1.1

Soma, L. R., Robinson, M. A., You, Y., Boston, R. C., & Rudy, J. (2018). Pharmacokinetics, disposition, and plasma concentrations of dimethyl sulfoxide (DMSO) in the horse following topical, oral, and intravenous administration. Journal of Veterinary Pharmacology and Therapeutics, 41(3), 384-392. doi:10.1111/jvp.12476

Teraoka, H., Sawai, M., Takase, K., Yamamoto, K., Nozaki, N., Okazaki, T., & Tsukada, K. (1991). Expression of c-fos and c-myc in Raji Burkitts lymphoma cells during the progression of DMSO-induced G1 cells into S phase. Experimental Cell Research, 195(1), 274-276. doi:10.1016/0014-4827(91)90528-3

Treit, D., & Fundytus, M. (1988). Thigmotaxis as a test for anxiolytic activity in rats. Pharmacology Biochemistry and Behavior, 31(4), 959-962. doi:10.1016/0091-3057(88)90413-3

Tsarouchas, T.M., Wehner, D., Cavone, L., Munir, T., Keatinge, M., Lambertus, M., Underhill, A., Barrett, T., Kassapis, E., Ogryzko, N., Feng, Y., van Ham, T.J., Becker, T., Becker, C.G. (2018). Dynamic control of proinflammatory cytokines Il-1β and Tnf-α by macrophages in zebrafish spinal cord regeneration. Nature communications 9:4670.

Tucker, B., Richards, R., & Lardelli, M. (2004). Expression of three zebrafish orthologs of human FMR1-related genes and their phylogenetic relationships. Development Genes and Evolution, 214(11), 567-574. doi:10.1007/s00427-004-0438-9

Varela, M., Dios, S., Novoa, B., & Figueras, A. (2012). Characterisation, expression and ontogeny of interleukin-6 and its receptors in zebrafish (Danio rerio). Developmental & Comparative Immunology, 37(1), 97-106. doi:10.1016/j.dci.2011.11.004

Verkerk, A., Pieretti, M., Sutcliffe, J., Fu, Y., Kuhl, D., Pizzuti, A., Reiner, O., Richards, S., Victoria, M., Zhang, P. (1991). Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell, 65(5), 905-914. doi:10.1016/0092-8674(91)90397-h

Wan, L., Dockendorff, T. C., Jongens, T. A., & Dreyfuss, G. (2000). Characterization of dFMR1, a Drosophila melanogaster Homolog of the Fragile X Mental Retardation Protein. Molecular and Cellular Biology, 20(22), 8536-8547. doi:10.1128/mcb.20.22.8536-8547.2000

Wei, H., Chadman, K., McCloskey, D., Sheikh, A., Malik, M., Brown, T., & Li, X. (2012). Brain IL-6 elevation causes neuronal circuitry imbalances and mediates autism-like behaviors. Biochemica et Biophysica Acta, 1822. Retrieved April 10, 2021, from https://pubmed.ncbi.nlm.nih.gov/22326556/

Welty F, Alfaddagh A, & Elajami T. (2015). Targeting inflammation in metabolic syndrome. Translational Research, 167: 257–280.

Wheeler, A., Raspa, M., Bann, C., Bishop, E., Hessl, D., Sacco, P., & Bailey, D. B. (2013). Anxiety, attention problems, hyperactivity, and the Aberrant Behavior Checklist in fragile X syndrome. American Journal of Medical Genetics Part A, 164(1), 141-155. doi:10.1002/ajmg.a.36232

Wu, Y., Hsu, M., Ng, M., Amstislavskaya, T. G., Tikhonova, M. A., Yang, Y., & Lu, K. (2017). Fragile X Mental Retardation-1 Knockout Zebrafish Shows Precocious Development in Social Behavior. Zebrafish, 14(5), 438-443. doi:10.1089/zeb.2017.1446