Parkinson's disease (PD) is a complex neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra region of the brain. This degeneration results in a wide range of motor and non-motor symptoms, including bradykinesia, rigidity, tremors, and postural instability. PD not only affects motor function but also leads to cognitive and psychiatric impairments, significantly reducing the quality of life for those afflicted.

1. ROTENONE INDUCED PARKINSON’S
DISEASE IN ZEBRA FISH
Presented by: A. Hemanth Babu(M pharm-pharmacology)

2. WHAT IS
ZEBRA FISH ?
• The zebrafish (Danio rerio) is a
small tropical freshwater fish that
has become a popular model
organism in scientific research,
notably in developmental biology,
genetics, and neuroscience.
Zebrafish are indigenous to South
Asia, mainly in the Ganges basin.
They are distinguished by their
striking blue and yellow stripes,
which resemble those of a zebra,
hence the name "zebrafish.”

3. GENDER BIAS:
CONTENTS MALE ZEBRA FISH FEMALE ZEBRA FISH
BODY SHAPE Slightly smaller Bloated and larger
COLOURISATION More vibrant and bold colour Less intense colour
FIN(DORSAL FIN) Long and pointed Short and rounded
GENITAL PAPILLIA Anal vent is modified and extended
(gonopodium )
Ovapositor(deposit eggs ) present
near to anal fin
SEXUAL BEHAIVOUR More active (chasing) Not much active as males

4. WHY
ZEBRA FISH
MODEL :
Like mammals, zebrafish possess a blood brain barrier, and permeability tests indicate
that its physiological properties are conserved between zebrafish and humans (Cuoghi
and Mola, 2007; Wager and Russell, 2013).
In addition to their physiological benefits, zebrafish also exhibit sophisticated cognitive
behaviours, such as learning and retaining associations, and they manifest well-
documented anxiety behaviours (Lieschke and Currie, 2007).
One of the key advantages of zebrafish is their suitability for genetic manipulation,
which has led to the development of thousands of mutant, transgenic and otherwise
genetically- altered strains (Ruzicka et al., 2019)
Zebrafish had been found to have similar dopaminergic signalling pathways to
mammals, and transcription factors have been shown to play evolutionarily conserved
roles in the development of zebrafish dopaminergic neurons (Xi et al., 2011).
Dopaminergic neurons in zebrafish are sensitive to oxidative stress, which is one of the
main causes of their death in PD (McCormack et al., 2006; Rappold et al., 2011; also
see above)

5. • Zebrafish are an essential model organism for several
reasons:
• 1. Genetic Similarities: Zebrafish share a high degree of
genetic similarity with humans, with many of their genes
having equivalents in humans. This makes them valuable for
studying genetic and developmental processes relevant to
human biology The dopaminergic system in embryonic
zebrafish is also well characterized. Dopaminergic neurons
are first detected at 18h post fertilization (hpf) in a cluster in
the ventral diencephalon, and by 72 hpf, the organization of
the central nervous system is complete and subsequent
development only adds increased numbers of neurons
(Kimmel et al., 1995; Wullimann and Rink, 2001).
• 2. Transparent Embryos: Zebrafish embryos are
transparent, which allows scientists to observe the
development of internal organs and tissues in real time. This
transparency makes them particularly useful for
developmental biology research.
ADVANTAGES OF SELECTING ZEBRA FISH
FOR STUDY

6. 3. Rapid Reproduction: Zebrafish reproduce quickly, with each pair of fish producing
hundreds of eggs per week. This rapid reproduction enables scientists to conduct
experiments on a large scale.
4. External Development: Zebrafish embryos develop externally, making it easy to
manipulate and study their development. Researchers can microinject substances or
modify genes during early embryonic stages.
5. Accessibility: Zebrafish are relatively easy to maintain in a laboratory setting and are
cost-effective to study compared to other animal models, such as mice or rats. zebrafish
are becoming increasingly popular for use in high-throughput screens due to their prolific
reproduction and cost-efficient size. Based on these factors, potential therapeutics and
genes can be quickly identified in a fraction of the time required for rodents
ANIMAL ZEBRA FISH
AGE 3-5 MONTHS
TEMPARATURE 28°C +/- 2°C
SURVIVAL TEMPARATURE 10 – 35 °C
pH 7.0 – 8.0
LIFE SPAN UPTO 5 YEARS

7. MY STUDY: ROTENONE INDUCED PARKINSON’S DISEASE
Rotenone is an isoflavone compound that naturally occurs in the jicama
vine plant as well as many Fabaceae plants. It has broad-spectrum
insecticide and pesticide activity and is also toxic to fish. Rotenone is
soluble in organic solvents such as ethanol, DMSO, and chloroform,
which should be purged with an inert gas. The solubility of rotenone in
ethanol is approximately 5 mg/ml and approximately 50 mg/ml in
DMSO and chloroform.
1. Inhibiting Mitochondrial Complex I: It disrupts the cell's energy
production by inhibiting a crucial component called
mitochondrial complex I.
2. Inducing Oxidative Stress: This inhibition leads to the production
of harmful reactive oxygen species (ROS) and free radicals that
damage cells.
3. Dopaminergic Neuron Degeneration: The selective loss of
dopaminergic neurons in the brain occurs, mirroring Parkinson's
disease.
4. Triggering Neuroinflammation: Rotenone causes an
inflammatory response in the brain, which is linked to
Parkinson's progression.
5. Behavioural and Motor Deficits: Animals exposed to rotenone
exhibit symptoms such as slowness of movement, tremors, and
postural instability, akin to human Parkinson's patients.
CHEMICAL FORMULA: C23H22O6
DOSE (2-3 micro gm/litre)

8. BEHAVIOURAL STUDY :
Examination tank was filled with fresh aerated tank water.
It consisted of a 5‐L tank (30 × 15 × 10 cm, length ×
height × width) with spacing of 5cm *5cm
All behavioural evaluations were done using a web
camera (Microsoft life cam). A behavioral study was done
for 1 h and parameters were measured at 15-minute time
intervals, viz., 0, 15, 30, 45, and 60. Since after 1 h all
fishes recovered from the effect of haloperidol, hence,
examination time was standardized to 1 h.
Recording was done for 5 min at every time interval and
average readings during 5 min were calculated for every
individual fish at each time point.

9. LOCOMOTOR ACTIVITY :
locomotor activity was monitored in a 2-L tank (18 × 9 ×
7 cm). A tower system was used to capture a video
recording of each fish for 5 min. Swimming activity was
categorized into three speeds: fast (>20 mm/s), slow (>2
mm/s and <20 mm/s), and freezing (<2mm/s). Movement
distance and duration at each speed were calculated using
software (Viewpoint Life Sciences, France)
LIGHT / DARK BOX TEST:
The light/dark box test was performed as previously
described (Gebauer et al. 2011). The tank (18 × 9 × 7 cm)
was separated into equally sized dark and light
compartments with a plastic barrier. The barrier was lifted
up 1 cm to allow fish to swim between compartments, and
the water level was kept at a depth of 3 cm. Fish were
individually placed into the light compartment and
allowed to swim freely for 6 min, during which a video
recording was obtained using the tower system. Latency
to enter the dark compartment, time spent in the light
compartment

10. OLFACTORY PRESENCE TEST :
Olfactory preference for a mixture of amino acids was assessed according to a previously described method
(Koide et al. 2009). Briefly, fish were individually placed in a tank (18 × 9 × 7 cm) and video recorded for 16
min using the tower system. After a 6-min pre-stimulation, 0.6 ml amino acid mixture (Cys and Met, 0.1
mM) was delivered into a corner of the tank at a rate of 1.5 ml/min. Time spent in the amino acid (TA) and
non-amino acid side (TC) was analyzed, and swim paths were tracked using software. Preference index (PI)
for each minute was calculated as: PI = (TA-TC)/(TA+TC).

11. WESTERN BLOTTING :
• Sample Preparation: They started by homogenizing the zebrafish brain tissue in a
lysis buffer containing specific components (150 mM NaCl, 25 mM Tris, 5 mM EDTA,
1% Nonidet P-40; pH 7.5) along with protease inhibitor cocktail tablets to prevent
protein degradation.
• Centrifugation: The homogenized brain samples were then centrifuged at 13,200 g
for 15 minutes at a temperature of 4 °C. This step separates the protein of interest
from other cellular components.
• Protein Quantification: The protein concentration in each resulting supernatant was
determined using a bicinchoninic acid assay (BCA assay). This helped in accurately
measuring the amount of TH protein.
• Gel Electrophoresis: 40 microgram aliquots of the protein samples were loaded onto
8% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). This process separates
proteins by size.
CHEMICAL ASSESMENTS

12. • membrane Transfer: The separated proteins were then transferred from the gel onto
polyvinylidene fluoride (PVDF) membranes. This transfer step is crucial for further
analysis.
• Blocking and Antibody Incubation: The PVDF membranes were blocked using 5% fat-
free dry milk in a buffer solution. Subsequently, they were incubated with primary
antibodies: a rabbit anti-TH antibody (dilution 1:1000) and a mouse anti-β-actin antibody
(dilution 1:8000). This step allowed the specific proteins of interest to be "tagged" for later
detection.
• Secondary Antibody Binding: After incubation with primary antibodies, the membranes
were briefly washed and then exposed to a secondary antibody labeled with IRDye 680,
which can bind to the primary antibodies. This secondary antibody helps in detecting the
presence of the primary antibodies on the membrane.
• Imaging: Immunoreactive bands on the membranes, which correspond to the presence
of TH and β-actin, were visualized using an Odyssey CLx imaging system. This step
captures the signals generated by the antibodies, indicating the quantity of TH protein
present.
• Analysis: Finally, to quantify the amount of TH protein accurately, densitometric analysis
was performed using ImageJ software, a tool for analyzing digital images. This analysis
provides quantitative data regarding the protein levels in the zebrafish brain
samples.(Dongjun 2021)

13. QUANTITATIVE REAL POLYMERASE CHAIN REACTION :
Total RNA was extracted using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μg RNA
was reverse transcribed into cDNA using a cDNA synthesis kit (Roche Diagnostics, Germany). Real‐time
PCR was conducted on the 7500 real‐time PCR system (Applied Biosystems, Foster City, CA, USA) with
the following primers: 18S (forward: 5′‐TCAACACGGGAAACCTCAC‐3′, reverse: 5′‐CGCTCCA
CCAACTAAGAAC‐3′), drd 1a (forward: 5′‐CTAAGGACTCATG ACACC CCC‐3′, reverse:
5′‐CAGTCACACCTCAGGTAGCAT‐3′), drd 1b (forward: 5′‐GACGGTGAACAAACTGCTGA‐3′, reverse:
5′‐CTTACACGTGAATCGG AGC
A ‐3′), drd 2a (forward: 5′‐AGTGCCGTAAACCCAATC‐3′, reverse: 5′‐ GTATCAT TTCCATCCCTTTCTG‐3′), drd
2b (forward: 5′‐GTCTCCATCT CCGTCCTCTC‐3′, reverse: 5′‐TTACCGAACACCACACAGAAG‐3′), drd 2c
(forward: 5′‐ATGCTCCT GACTCTCCTC‐3′, reverse: 5′‐ATCTGCCACCG CCAAG‐3′), drd 3 (forward: 5′‐TT
CAGACCACCACCAACTACC‐3′, re- verse: 5′‐GCTCCGCCGACCACTTC‐3′). The results were normalized to 18S
RNA( Dongjun 2021).
Drd =dopa responsive dystonia (term used to describe specific dystonia disorders that respond to a
medication called levodopa)
cDNA = complementary DNA

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