INTRODUCTION & HISTORY
Ever thought about how researchers and pharma companies test drugs and understand various pathologies on different animal species? What makes the animals lucrative and efficient when compared to the rest? Certain factors like gene homogeneity and similar tissue and organ growth are assessed. Organisms like fruit flies, mice, Zebrafishes and monkeys are prevalently used in science to analyze, study a disease model and reach better conclusions.
Zebrafish is one such creature that is been in use since the 1960s when a German scientist Streisinger aspired to have a simpler model organism than a mouse to study the early stages of the nervous system better.
Due to the transparent nature of zebrafish larvae, he believed that it is possible to study the neural development of the vertebrate, as the knowledge was limited due to techniques and incompatible animal models.
FEATURES
Zebrafish can regenerate multiple tissues including the fin, heart, retina, spinal cord and brain. Moreover, zebrafish ventral telencephalon is found to be homologous with the human striatum and basal ganglia, hippocampus and amygdala display a great degree of homogeneity with the human brain. Apart from this, behaviors and phenotypes demonstrated by zebrafish can be compared with the human species.
In terms of genetic factors, genome sequencing showed that humans and zebrafish shared 70% of the same genome, with 80% of genes located in the same chromosome and same order, which confirms greater amounts of similarity between the two species.
Zebrafish can lay 200-3000 eggs per week and reaches sexual maturity by the end of 3-4 months, which makes things much simpler.
Gene manipulation can be done by CRISPR-Cas9, that in turn leads to finding new approaches to tackle neurodevelopmental, cardiac and cell division pathologies.
ON FIELD APPLICATIONS
NEURODEGENERATIVE DISEASES
As the model is tiny, screening for neuroactive compounds and neurotransmitter levels is easy, Genetic manipulation of species can be done with help of CRISPR-Cas9, TALENS
The Alzheimer’s disease-related genes PSEN1 and PSEN2 have clear zebrafish orthologs psen1 and psen2, and the degree of similarity between the neurochemical and neuroanatomic pathways between the zebrafish and humans is high.
The medial, dorsal and lateral pallium of the zebrafish resembles the amygdala, isocortex and hippocampus in vertebrates respectively.
The zebrafish encephalon comprises the forebrain, midbrain and hindbrain (diencephalon, telencephalon and cerebellum), like the mammalian brain, the zebrafish brain uses the main excitatory glutamatergic and inhibitory GABAergic neurotransmitter circuits along with the presence of muscarine cholinergic receptors, besides they do possess GABA, glutamate, serotonin, dopamine, histamine, acetylcholine neurotransmitters, enzymes off synthesis and for metabolism.
At the cellular level, cell types like astrocytes, microglia, oligodendrocytes, cerebellar Purkinje cell, Myelin and motor neuron are found in human brain cells.
SLEEP STUDIES:
The interactions among the proteins aiding the circadian rhythm in the zebrafish are similar to those in mammals, moreover, the cholinergic neurotransmitter system modulates drug-induced reward activity in the zebrafish making it easy to study the brain on various types of addiction, this establishes zebrafish as an ideal model for studying the biology of behavior in vertebrates.
ADHD
ADHD patients have abnormal circadian rhythms, scientists worked on the zebrafish that has a mutation in the key circadian gene -period1b(per-1b), that mimics the sleep cycle in ADHD patients, it has been proven as an effective model, the per-1b variants were active thrice as the wild ones.
Other essential factors like impulsivity, tackling, focus, sensitivity, sense of learning and memory were taken into consideration.
PARKINSON’S:
Mature zebrafishes were exposed to rotenone to induce Parkinson’s, later on, they are administered with Oleandrin and Mitoquinone for the time duration of 30 days, the biochemical parameters were checked regularly by spectrophotometric methods, gene expression was determined by PCR techniques, Synuclein levels were measured by immunohistochemical staining, Neurotransmitter levels were checked by tandem mass spectrometry equipment and liquid chromatography.
The results were affirmative, mitoquinone enhanced the oxidant-antioxidant balance and neurotransmitter levels, and it also mitigated the gene expressions of Parkinson’s that rotenone disturbed. Mitochondrial functions were enhanced after exposure to mitoquinone and Oleandrin, this was proven by Thiazolyl Blue Tetrazolium bromide assay.
SCHIZOPHRENIA:
DISC1 is an example of rare schizophrenia-associated gene DISC1 mutants and morphants in zebrafish models have been reported with high distortion in brain morphology in early developmental stages, altered hypothalamic growth and stunted growth in ventricles, it has helped scientists to understand how pathology affects the patient.
STROKE:
Due to the transparent body, and a high percentage of similarities with the human nervous system, zebrafish are widely used to create stroke models (both ischemic and hemorrhagic).
Zebrafishes are highly compatible when compared to other model systems, testing methods like in-vivo imaging, behavioral conducts, and drug screening are performed to examine the pre-clinical studies and aftermath effects of a drug administered.
BEHAVIORAL NEUROSCIENCE:
Employing zebrafish in behavioral neuroscience, neurotoxicology and pharmaceutical trials has increased recently as the model shares basic neurodevelopmental patterns, neural structures and similar or equivalent behavioral patterns.
Tests like tank diving are performed to examine zebrafish for conditional-associated color-developed anxiety. Light and dark conditioning are performed to understand behavioral patterns.
Memory functions, retaining it back and learning processes can be tracked in this model as these species are capable of object discrimination, learning, avoidance learning, spatial learning and associative learning.
Zebrafishes are acquainted with an item to test whether they are ready to perceive the article post 1 or 24 hours of introduction, zebrafish telencephalon and thalamus are significant brain regions that are engaged with handling visual segregation.
Another task of planning to assess avoidance learning and memory is the avoidance learning test that requires the zebrafish to figure out how to tackle an electric shock by refraining to swim into a dark compartment, the experiment uncovers that the zebrafish can procure avoidance learning and interaction by withholding in its long-term memory.
Apart from the similarity of neural developmental patterns and certain brain structures, both humans and zebrafishes are social species. To test certain parameters like aggressiveness, anxiety, possible mate preferences and kinship recognition. A few tests like the mirror test, male-male interactions test, and shoaling test are performed to carefully analyze how they interact in activities that require social interactions among peers.
CANCER RESEARCH
Zebrafish is an ideal model to research cancer, certain studies like cancer imaging, adult cancer models and cancer-related developmental phenotypes area benefitted hugely, they can develop cancer spontaneously, after mutagen exposure and through transgenesis.
Methods like in vivo imaging, and chemical and genetic screening helps track the pathogenesis and tumor development. The zebrafish xenograft model (zPDX) is a low-cost and efficient tool that needs a small sample size and yields short-term clinical treatment.
Tumor microenvironment can be better observed with help of vascular fluorescence imaging and observation under microscopes for growth metastasis. The orthotopic zebrafish tumor model has emerged in studies of multiple types
CARDIOMYOPATHY MODEL :
Zebrafish are used to investigate the effects of environmental factors on cardiac development. PM2.5 is fine particulate matter, high levels of which are found in urban developmental areas with degraded air quality or high levels of pollution. Exposure of zebrafish embryos to PM2.5 led to developmental heart defects, including heart malformations and bradycardia. This model has been exploited to identify compounds, which help stabilize the impact of PM2.5 on the heart, like folic acid.
Protective effects of folic acid on developmental heart defects have also been experimented with in a zebrafish model of fetal alcohol spectrum disorder which demonstrated the negative impact of prolonged and continuous alcohol exposure during early development on heart morphology and growth.
CONTRADICTIONS AND SHORTCOMINGS
Every coin has two sides and zebrafishes are no exception, AD Model in zebrafish also suggests that Aβ(Amyloid-Beta) may play a role in maintaining cerebrovascular functions and deficit of Aβ promoting the decline in vessel length and cerebrovascular branching in the developing hindbrain of embryos, another research reported that Aβ is involved in the regulation of angiogenesis in the human umbilical cord vein and in the zebrafish hindbrain. This role of Aβ in the zebrafish stands in complete invalidation to its functionality in humans where it leads to memory impairments and progressive decline in cognitive processes, thus the role of Aβ in teleost and mammals are contradictory and cannot be relied on the whole.
The presence of duplicate orthologs for Alzheimer’s disease in Zebrafish may have differential results.
CONCLUSION
Model organisms are used widely to study a disease, develop new pathways to administer drugs and in translational research. There is no model organism with advantage-specific features (at least as of now), utilizing of models present up to date in research knowing their shortcomings and exploiting the required characteristics will help find new approaches to a pathological condition, advanced disease model and to view drug effects and recovery. Finding a foolproof model is highly impossible, utilizing present-day tools and modifying them to near precision is the wise choice
REFERENCES:
1. Whalley, K. A zebrafish model of ADHD. Nat Rev Neurosci 16, 188 (2015). https://doi.org/10.1038/nrn3936
2. International journal of neurosciences volume 130-2020 issue 6
3. Neuroprotective effects of mitoquinone and oleandrin on Parkinson’s disease model in zebrafish
İsmail Ünal,Esin Çalışkan-Ak,Ünsal V. Üstündağ,Perihan S. Ateş,Ahmet A. Alturfan, Meric A. Altinoz
4. De Rienzo, G., Bishop, J. A., Mao, Y., Pan, L., Ma, T. P., Moens, C. B., et al. (2011). Disc1 regulates both β-catenin-mediated and noncanonical Wnt signaling during vertebrate embryogenesis
doi: 10.1096/fj.11-186239
5. Zebrafish as a tractable model of human cardiovascular diseases
https://www.nature.com/articles/s41420-018-0109-7
6. Saleem, S., Kannan, R.R. Zebrafish: an emerging real-time model system to study Alzheimer’s disease and neurospecific drug discovery. Cell Death Discov. 4, 45 (2018).
https://doi.org/10.1038/s41420-018-0109-7
7. Zebrafish Embryos and Larvae: A New Generation of Disease Models and Drug Screens
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