Effects of Prenatal Exposure to Valproic Acid and Autism

INTRODUCTION

Autism is a neurodevelopmental Disorder, one of five disorders classified collectively as autism spectrum disorder, is diagnosable by the age of three. Diagnosed individuals may show many symptoms such as pervasive impairments in social interactions, deficits in verbal and nonverbal communication and, stereotyped, repetitive patterns of behaviors & interests (Zimmermann, Gaspary, Leite, Cognato, & Bonan, 2015). Autism is said to have a strong genetic & environmental connection. However, the etiology of autism has not yet been copiously understood or studied (Chen, et al., 2018).

Effects of prenatal exposure to valproic acid, one of the widely used antiepileptic drug for the treatment of seizures and bipolar disorder and overactivation of Protein Kinase C is being discussed in this paper (Liu, et al., 2018).

PRENATAL EXPOSURE TO VALPROIC ACID

Exposure to valproic acid during embryonic growth adjusts neural progenitor cell proliferation and could also cause behavioral disfigurements in adult organisms (Lee, Kim, Yun, & Lee, 2013). Embryos collected from Wild-type AB line spawning adult zebrafish within about 0.5 h of spawning had been exposed to a VPA stock solution of 500 mM for the initial step of this study. The working solution had been prepared by diluting stock solution instantly prior to the experiment. The six different concentrations of solutions where the embryos from 8 to 120 h post fertilization were continuously exposed were between 0 and 1500 ?M, with the 0 being the control (Chen, et al., 2018).

Developmental endpoints measured in this study comprised of malformations & mortality at 120 hours post fertilization. In this study the experiments contained of three biological triplicates with 20 embryos in each repeat. At the end embryos were moved to clean fish water for additional evaluations where the size of their head was measured at 4.5 days post fertilization and behavioral patterns were evaluated five days post fertilization with the use of Alcian blue stain and Image J software procedures (Chen, et al., 2018).

Zebrafish embryos exposed to Valproic acid were assessed for Embryonic and larval movement behaviors, spontaneous movement such as alternating tail bending or coiling, the response to touch in embryos that were manually dechorionated and the distance moved after touching were scored manually. Moreover, Larvae were adapted for 20 minutes before recording swimming for 10min visible light period, followed by a 10-minute dark (infrared light) period for evaluation of the average swim speed in light and dark atmospheres (Chen, et al., 2018). Basic tracking settings were used for larval movement tests such as their preference behavior of light and dark backgrounds and the number of times larva crossed between light & dark areas where data collected every 60sec for 6 minutes. Shoaling behaviors, Mirror attack behaviors and, Social contact were additional tests that was carried out (Chen, et al., 2018).

OVERACTIVATION OF PROTEIN KINASE C

AB strain zebrafish larvae soaked in PMA, a Phorbol 12-myristate- Subgroup of PKC enzyme between 48?hours and 72?post fertilization were used in this study. Once the chorions were detached at 12?hours post fertilization embryos were exposed to DMSO and PMA, then collected for subsequent analysis once 24 hours passed. RNA isolation and Reverse transcription quantitative PCR (RT-qPCR) was carried out on an ABI Viia 7 Real Time PCR System for further analysis at the end of the experimental procedures (Liu, et al., 2018).

The head size was demarcated by the otic vesicle & semicircle of eyes as posterior and lower frontier. Measurements just as in the previous study were estimated using ImageJ software. For the Behavioral assays larval fish that were placed in a 24-well plate inside a wooden box were monitored on automated video tracking system in two cycles of 5-minute light and 5-minute dark backgrounds. Western blotting, Immunostaining and, TUNEL labeling procedures had been used for additional valuations (Liu, et al., 2018).

RESULTS

It was confirmed that valproic acid exposures induced several malformations in the zebrafish. Uninflated swim bladder, pericardial edema and yolk sac edema had been observed. Consequently, the occurrence of abnormalities was found to be significantly diverse compared to control larvae at concentrations including and above 500 ?M (P < 0.001) with 100% of larvae displaying malformations at 1500 ?M. It was also verified that valproic acid did not produce larval mortality in any tested concentrations at 120 hours post fertilization (Chen, et al., 2018).

Most importantly it was found that valproic acid exposure in zebrafish produced a macrocephalic phenotypic head where the circumference of the head is greater than 2 standard deviations than average for a given age and sex. However, no changes in the body length were noted. Additionally, the lowest concentration of valproic acid (5 ?M, P < 0.05) found to produce an increased intraocular distance while all exposure groups (P < 0.05 for 50 and 500 ?M; P < 0.001 for 5 ?M) showed significantly increased lower jaw and certohyal cartilage lengths compared to the controls (Chen, et al., 2018).

The hypothesis of early-life PKC hyper-activation leading to mild developmental delay and reduced brain size resulting in neurogenic defects was verified in the study of protein kinase experimentation. It was also evaluated that zebrafish exposed to PMA were hypoactive in lighted backgrounds and hyperactive in the dark phased environments (Liu, et al., 2018).

DISCUSSION

Evaluating the studies, it is clear that non-teratogenic valproic acid exposure resulted in macrocephalic phenotypes in larval zebrafish, fabricating hyperactivity and impaired social behavior (Chen, et al., 2018). Deficits in social interaction, anxiety and stereotyped activities which are considered outcomes of a dysfunctional neural system could be compared with some of the core characteristics associated with autism spectrum disorders (Lee, Kim, Yun, & Lee, 2013). Increased head circumference and volume is also a clear visual abnormality that could be seen in some autistic individuals at a certain point of their life. PKC hyper-activation during early development could cause many pathological features such as developmental delays, motor abnormalities and exaggerated stress responses. Furthermore, neuropathogenic effects of developmental PKC hyper-activation was reinforced by behavioral changes (Liu, et al., 2018).

Several neurological and psychiatric syndromes are characterized by variations in the social realm (Zimmermann, Gaspary, Leite, Cognato, & Bonan, 2015). The optical lucidity and quick development, comparable stages and components of the central nervous system to higher vertebrates, all have made Zebrafish an ideal and advantageous model for many different research studies (Roper & Tanguay, 2018). Even though behavioral studies could be immeasurable accurately the identification of targetable molecular pathways underlying neurodevelopmental defects could create paths in the development of possible therapeutic strategies for autism as well as countless other disorders that affect the society.

References

Chen, J., Lei, L., Tian, L., Hou, F., Roper, C., Ge, X., . . . Huang, C. (2018). Developmental and behavioral alterations in zebrafish embryonically exposed to valproic acid (VPA): An aquatic model for autism. Neurotoxicology and Teratology,66, 8-16. doi:10.1016/j.ntt.2018.01.002 Kim, L., He, L., Maaswinkel, H., Zhu, L., Sirotkin, H., & Weng, W. (2014). Anxiety, hyperactivity and stereotypy in a zebrafish model of fragile X syndrome and autism spectrum disorder. Progress in Neuro-Psychopharmacology and Biological Psychiatry,55, 40-49. doi:10.1016/j.pnpbp.2014.03.007 Lee, Y., Kim, Y., Yun, J., & Lee, C. (2013). Valproic acid decreases the proliferation of telencephalic cells in zebrafish larvae. Neurotoxicology and Teratology,39, 91-99. doi:10.1016/j.ntt.2013.07.004 Liu, T., Shi, Y., Chan, M. T., Peng, G., Zhang, Q., Sun, X., . . . Cheng, C. H. (2018). Developmental protein kinase C hyper-activation results in microcephaly and behavioral abnormalities in zebrafish. Translational Psychiatry,8(1). doi:10.1038/s41398-018-0285-5 Roper, C., & Tanguay, R. L. (2018). Zebrafish as a Model for Developmental Biology and Toxicology. Handbook of Developmental Neurotoxicology,143-151. doi:10.1016/b978-0-12-809405-1.00012-2 Zimmermann, F. F., Gaspary, K. V., Leite, C. E., Cognato, G. D., & Bonan, C. D. (2015). Embryological exposure to valproic acid induces social interaction deficits in zebrafish (Danio rerio): A developmental behavior analysis. Neurotoxicology and Teratology,52, 36-41. doi:10.1016/j.ntt.2015.10.002

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