REPORT OF LABORATORY

COURSE ON NEUROGENETICS
M.Sc. Neural and Cognitive Sciences
2018-2020
Semester III

Submitted By: Nitisha Singh

Roll No: 17MNMS06
Center for Neural and Cognitive Sciences
University of Hyderabad
Teacher: Dr. Sudipta Saraswati

Submitted on: 06-12-2019

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Preface

This document is the report of what we as students did during our

laboratory course on Neurogenetics taught by Dr. Sudipta Saraswati, as
a part of our M.Sc. course in Neural and Cognitive Sciences, at the
Center for Neural and Cognitive Sciences, University of Hyderabad.
This laboratory course stretched for a month during which we have
participated in laboratory based experiment and demos, as presented in
the following ways:
PART II has been collectively written by Akarsh Sriramoju, Bandita
Choudhary, Kusum Thuwal, Nitisha Singh, Shailaja Pathak,
Vikrant Menia, Vivek Dasoju.
PART I and III has been written by Nitisha Singh

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Acknowledgement

I would like to thank Sayantan Dutta, Research Scholar, TIFR-

Hyderabad, for his precious time to demonstrate adult drosophila Brain,
larval neuromuscular junction and imaginal disc dissection, along with
the introduction to Fiji software. I would also like to thank Arvind,
Research Scholar, TIFR-Hyderabad, for his help in confocal imaging;
D. Chandrika for demonstrating Foldscopes and Anujay, Post Doctoral
fellow, TIFR-Hyderabad for demonstrating a behavioral experiment.
I would sincerely like to thank Dr. Manish Jaiswal for giving us an
opportunity to use the confocal microscope at TIFR-Center for
Integrated Sciences, Hyderabad.
I would also like to thank all team members for their collaboration in
Part I of this report.

Name: Nitisha Singh

Roll No: 17MNMS06
M.Sc. Neural and Cognitive Sciences
Center for Neural and Cognitive Sciences
University of Hyderabad
Date: 06-12-2019

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Contents
PART I
Section 1: Introduction
PART II
Section 2: Morphological features of drosophila melanogaster
Experiment 1: Observation of Life cycle of drosophila melanogaster

Experiment 2: Identification of males and females of drosophila melanogaster

Experiment 3: Observation of different body parts of drosophila melanogaster

Section 3: Anatomical experiments

Experiment 1: Anatomy of late 3rd instar larva

Experiment 2: Larval neuromuscular junction dissection

Experiment 3: Larval brain dissection

Section 4: Classical Genetics experiments

Experiment 1: Identification of mutants using morphological/behavioral
phenotypes

Experiment 2: Virgin collection (pupa and adult)

Experiment 3: Setting up of a genetic cross.

Experiment 4: Complementation test

Section 5: Observations using Foldscopes

Section 6: Immunohistochemistry of developing neurons of drosophila
melanogaster larva.
Section 7: Confocal imaging

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PART III
Section 8: Analysis of drosophila melanogaster behavior.
Experiment 1: Alcohol tolerance in flies

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 PART I

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Section 1: Introduction
1. What is a Model system?

A model organism is a non-human species which is used for studying

specific features, traits, disease or phenomena. The model organism is
chosen such that its biology is well known and assessable for conducting
studies and experiments in the laboratory [1, 2]

2. Why do we need a model in neuroscience?

A model organism is needed to conduct studies which cannot be conducted

on humans ethically. Model organisms which are used have similar genome
to that of the genome of the humans and hence, studies conducted on a
model organism can be linked to the human studies.
Also, using a model organism is handy as they breed in large numbers and
have a short generation time as compared to humans, which is advantageous
for conducting trial studies [2]

3. What models are used?

Some of the model organisms which are widely used are:

 Yeast (Saccharomyces cerevisiae)

 Fruit fly (Drosophila melanogaster)
 Nematode worm (Caenorhabditis elegans)
 Western clawed frog (Xenopus tropicalis)
 Mouse (Mus musculus)
 Zebrafish (Danio rerio)[2]

4. Who first introduced Drosophila melanogaster as a model system?

Thomas Hunt Morgan was the first one to use drosophila to prove the
chromosomal theory of inheritance for which he received a Nobel Prize. He
along with his colleagues defined many principles of genetics, including the

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 effects of X-rays on mutation rates, for which Hermann Muller also won the
Nobel Prize. Similarly, more advancement in genetic tools helped address
more complex problems in biology. Many scientists like Seymour Benzer
who used to work on topology of genes using bacteriophage, turned
to Drosophila to study the influence of genes on behavior. His great work in
the field of Neurogenetics contributed to how genes maintain higher order
brain functions.
Ever since then, Drosophila melanogaster has been widely used in
Neurogenetics and many fields of biology.

5. What are the strengths of using Drosophila melanogaster as a

model organism?

Drosophila melanogaster, commonly called a ‘fruit fly’ belongs to the

family Drosophilidae.

Figure 1: Drosophila Melanogaster

Image source: https://www.eurekalert.org/multimedia/pub/91875.php?from=296349

It is one of the most widely used model organism till date and is used
because of the following reasons:
 Ease in culturing as it requires less space, equipments and expenses.
 It has a relatively shorter generation time therefore; several generations can
be studied within a few days.
 Its life cycle and development is well understood
 An adult female fly can lay up to 100 eggs per day
 Its complete genome has been sequenced
 Male and female adult flies can be easily distinguished
 Collection of virgin females is easy.
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  It has only four pairs of chromosomes in which three are autosomes and one
pair of sex chromosomes
 Presence of giant chromosomes called ‘Polytene chromosomes’ in the
salivary gland of the larva is an advantage to study transcription. [4]

6. What are the weaknesses of using Drosophila melanogaster as a

model organism?

The only disadvantage of using Drosophila melanogaster is that it canned be

used as a model for complex diseases like multiple sclerosis, brain infarcts
and brain hemorrhage because it lacks blood cells and blood vessels [5]

7. What are the most important achievements of Drosophila

Neurogenetics?

Seymour Benzer, known as the Father of Neurogenetics, successfully solved

the mystery of circadian rhythms and found out all the genes which are
involved at the molecular level.
With the new advances in genetics such as recombinant DNA technology
and reverse genetics allowed us to find out genes responsible for
Alzheimer's disease, Parkinson's disease and epilepsy.[6]

8. Concluding remarks:

Drosophila melanogaster has been widely used in genetic studies because

of number for advantages listed above. It has been useful in the discovery of
many important genes and their functions which are crucial for the survival
of the organism. Drosophila melanogaster is one of the best model
organisms in Neurogenetics.

References:

1. (n.d.). Retrieved from https://www.nature.com/scitable/definition/model-organism-

model-genetic-organism-139/.
2. What are model organisms? (2017, March 3). Retrieved from
https://www.yourgenome.org/facts/what-are-model-organisms.

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 3. Tolwinski N. S. (2017). Introduction: Drosophila-A Model System for Developmental
Biology. Journal of developmental biology, 5(3), 9. doi:10.3390/jdb5030009
4. Drosophila melanogaster. (2019, November 20). Retrieved from
https://en.wikipedia.org/wiki/Drosophila_melanogaster.
5. Jeibmann, A., & Paulus, W. (2009). Drosophila melanogaster as a model organism of
brain diseases. International journal of molecular sciences, 10(2), 407–440.
doi:10.3390/ijms10020407
6. Wikipedia contributors. (2019, September 24). Neurogenetics. In Wikipedia, the Free
Encyclopedia. Retrieved 19:20, December 4, 2019,
from https://en.wikipedia.org/w/index.php?title=Neurogenetics&oldid=917550693

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 PART II

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Section 2: Morphological features of drosophila melanogaster
Experiment 1:: Observation of Life cycle of drosophila melanogaster
A drosophila takes about 10 days to grow into an adult fly from the fertilized egg. The optimum
temperature for development of drosophila is 25°C. The drosophila life cycle comprises of the
following development stages:

1. Embryogenesis: This stage occurs after 24 hours of fertilization of the oocyte by the male
drosophila sperm. During this stage, rapid DNA replication
eplication and nuclear division occurs.
occurs
2. Larval stage: There are three larval stages (3 instars) which collectively stretch for almost
4 days.
3. Pupal stage: This stage refers to the encapsulation of the 3rd instar larva into a capsule
called pupa. This stage lasts for 4 days. The initial stage of pupa is white in color and is
called ‘white pupa’ or ‘pre
‘pre-pupa’.
pupa’. The later stage of pupa appears dark or black
blac in color
and is called ‘black pupa’.
4. Adult life: Adult
ult fly emerges upon eclosion from the black pupa after 22-3
3 days.
days The
lifespan of an adult fly is 30 days.
The life cycle of drosophila melanogaster is demonstrated below:

Figure 2: Life cycle of Drosophila Melanogaster.

Image source: https://www.cherrybiotech.com/scientific
https://www.cherrybiotech.com/scientific-note/drosophila-life-cycle-and-fly-anatomy

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Experiment 2: Identification of males and females of drosophila melanogaster
For setting up a cross, it is important to differentiate between the adult male and female flies.
The main visible characteristics to differentiate adult male and female flies are as follows:

Table 1: Characteristic differences between adult male and female fly

CHARACTERSTIC FEMALE MALE

Body Size Longer than males Shorter than females

Body shape Pointed abdomen. Rounded abdomen

Each abdominal segment Abdomen with entirely dark

Color
carries a narrow dark band segments (A5 and A6)

External genitals called External genitals called

Genetelia Ovipositor are smaller and Epandrium are larger and
lighter in color darker in color

Bristle-like structures present

Sex comb None
on forelegs

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 Figure 3: Characteristic difference between adult male and female fly
Image source: Drosophila Melanogaster. (n.d.) REtrieved from https://www.sciencedirect.com/topics/immunology-and-
microbiology/drosophila-melanogaster.

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Experiment 3: Observation of different body parts of drosophila melanogaster
A. HEAD- They has a movable head which has sensory organs for vision, olfaction,
gestation, hearing and touch.

1. Antennae: These are a pair of sensory antennae present on the head of the adult fly.
2. Johnston organ: This is a hearing organ of the fly composed of mass of receptor cells to
sense different mechanosensory stimuli.
3. Compound Eye: Compound eyes are the most prominent structures located on head
capsule. Each eye has 750 ommatidia. A darker area at the center of eye is called pseudo
pupil which is present in the wild type and may not be present in the mutants.
4. Proboscis (mouthpart): The mouth part helps in detecting food or detection of toxic
compounds.

5.
Figure 4: Body parts of drosophila melanogaster
Image source: The Biology. (n.d.). Retrieved from https://morphologicallydisturbed.weebly.com/the-biology.html.

B. THORAX- It consists of three segments: Prothorax, Mesothorax and Metathorax. All

three segments possess a pair of legs Prothoracic legs, Mesothoracic legs, Metathoracic
legs respectively. In males only, the Prothoracic legs have sex combs. The Mesothorax
supports the entire flight system including wings and internal musculature that drives

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 flight. The halteres on the Metathorax are the morphological equivalent of the
metathoracic wings. Halteres help in balance during flight.

C. ABDOMEN- Abdomen is segmented, and female abdomen is comparatively larger than

males and a dark patch is present at the end of abdomen in males and no dark patch in
females. Female abdomen is pointed at end while male abdomen is round. The genitals
are present on the abdomen. .

Reference:

1. Drosophila life cycle and fly anatomy. (2019, July 24). Retrieved from
https://www.cherrybiotech.com/scientific-note/drosophila-life-cycle-and-fly-anatomy
2. The Fly Manual A guide to working with Drosophila. University of Leicester
https://www2.le.ac.uk/departments/genetics/research/staff-research-interests/tauber-
research-lab/livegene/fly-manual-1

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Section 3: Anatomical experiment

Experiment 1: Anatomy of 3 instars larvae of drosophila melanogaster.

Instar is the name given to the developmental stage of drosophila .If we put a stock of
larvae in a culture dish; we can easily distinguish between the three larval stage. The first
and the second instars are found nearer to the medium and the late thirds instar will be
seen climbing up the vile and preparing to pupate.

The larvae looks transparent under the light and under proper lighting we can easily
identify the strands of trachea (tough, white, branched, string like structures), the salivary
glands, the fat bodies, and the many pairs of imaginal discs hanging to the tracheae.

Figure 5: Schematic diagram of a third instar larva.

Fat body: A whitish sheet of fat cells running across the length of the body. It is a site fat
storage in drosophila.

Trachea: It is tube-like invaginations of the body wall which serves as an air channel for
the exchange of respiratory gases. They open to the outside at the spiracles.

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Salivary glands: They are soft looking and large, and their chromosomes are polytene (as
are most of the differentiated cells of the larva), making them suitable structures for
creating chromosome squashes.

Imaginal discs: In the fly, these are packets of folded epithelium near the brain region of
the Drosophila that later on differentiate into many structures of the adult such as the
wings, legs, antennae, eyes, and proboscis.

Labial discs: It will form the proboscis of the adult fly.

Haltere disc: In the fly, the haltere disc will later form halters with are used for balance.
These are club-shaped organ posterior to the wings. These are homologous to the hind
wings of non-dipterans insects.

Genital disc: they are the only unpaired imaginal disc .This disc later forms the genital
ducts, accessory glands and external genitalia of the adult fly.

Figure 6: Schematic diagram of imaginal discs from a third instar larva.

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Experiment 2: Larval neuromuscular junction dissection
Introduction:

The Drosophila neuromuscular junction (NMJ) has been an established model system for
studying the synaptic development and plasticity. The widespread use of the Drosophila
motor system is due easy accessibility. Here our aim is to dissect Drosophila 3 instar
larvae for study of the NMJ by removing all internal organs while leaving the body wall
intact.

Protocol:

1. Put a drop of cold PBS on the dissection plate. This will keep the animal from drying
and stun the animal making it easier to work with.

2. Using the long forceps, select a wandering third instar larva and place it on the
dissecting plate.

3. By using short forceps hold the minutien pin. Place the pin near the head anterior part
of the larva and then stretch the larva and put the second pin in the posterior end. This
will give us maximum space of exposed body for cutting

4. Using spring scissors make vertical incision just from anterior to the posterior pin on
the dorsal side of the larva.

Open the incision by using the forceps, the finished result should be an I-shaped filet.
After that we pin the left and the right flaps clockwise by stretching the body walls.

5. Next we remove the organs by pouring some additional PBS on the larva. This will
enable the organs to float up out of the body allowing you to remove them much more
easily. We remove the tracheal system and the rest of the gut

6. During step 3 you made a left flap and right flap in the body wall. Pin the flaps in a
clockwise order making sure to stretch the body wall both horizontally and vertically

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Experiment 3: Larval brain dissection

Protocol:

First we collect late third instar larvae from the stock. The 3 rd instar larvae will be seen
crawling up the medium, away from the food.

We then place the larva in the dissecting plate and pour some cold PBS to anesthetize the
organism.

We cut the posterior gut part of the larva.

Using two pairs of fine forceps we start the dissection. With one, we hold on to the
posterior open end of the larva. We hold it firmly and with the second forceps, we push
the anterior head region inside out like a sleeve. We will be able to see the inner part of
the larva in the correct anterior-posterior manner.

Reference :

 Drosophila melanogaster. J. Embryol. Exp. Morph. 66: 57–80.

 Campos-Ortega, J.A. and V. Hartenstein. 1985. The Embryonic Development of
Drosophila Bainbridge, S. P. and M. Bownes. 1981. Staging the metamorphosis of
melanogaster. Springer-Verlag, Berlin.
 Jonathan R. Brent, Kristen M. Werner, Brian D. McCabe. Drosophila Larval NMJ
Dissection. DOI: 10.3791/1107.

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Section 4: Classical Genetics experiments
Experiment 1: Identification of mutants using morphological/behavioral phenotypes.

Requirements: Flies and microscope.

Mutant analysis is a crucial part in Neurogenetics research especially in forward genetics

approach. Mutant analysis is done for a specific phenomenon of interest like learning and
memory etc. Following are some morphological mutants:

Figure 7: Image of Bar mutant (bottom) and Wilt type fly (top)

Description: Above image is of Bar mutant which is caused by a dominant mutation. The
mutant shows a kidney shaped eyes as compared to the wild type eyes which are round. These
mutants are useful in studying the developmental aspects of photoreceptors.

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 Figure 8: Image shows a white eyed mutant along with Curly gene mutant and red eyed wild-type fly.

Description:
Above image is of a mutant totally lacking any photoreceptor which gives it a white eye as
compared to the wild type fly. Along with that white eye mutant fly also exhibits curled wings as
compared to the wild type, this phenotype is caused due to a mutation in Curly gene present on
the chromosome 2.

Figure 9: Image of Serrate gene mutant and wild type fly.

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Description: above image shows another wing mutant where the mutant (bellow) shows
abnormally shaped wings as compared to the wild type fly (above).

Experiment 2: Virgin collection (pupa and adult).

Virgin collection is important step before setting up any experimental cross as this will ensure
the absence of any contamination. Female flies are capable of mating as early as they emerge
from the pupa and are polyandrous (capable of mating with several males). Once mated the
female can retain the sperm in its body and use it later for few days hence it gets even more
crucial to collect virgins as soon as they emerge from the Pupal stage.
There are two ways of collecting virgin females one is during the Pupal stage and one is at the
adult stage. For adult virgin females one has to visit the lab at timings when the most number of
pupae eclose in order to collect the females from the vials. Another way is to start at the Pupal
stage itself by looking for female pupae. One can differentiate the female from the male pupae
by looking at its legs under the microscope.

Experiment 3: Setting up a genetic cross.

Genetic crosses are cross breeding deliberately done between two appropriate sexes within a
species. Genetic crosses are done for several reasons like creating mutant lines, complementation
test among others. Genetic cross can be contaminated by females who have mated already and
the produced progeny and traits cannot be relied on for phenotype of interest.
An “X” is used to indicate that two individuals have been mated together. The parents are
designated as P (for parental) and the offspring as F (for filial). For several generations involved
subscripts are added to the letters.

If the F1 are crossed together they become P2 and their progeny F2. A cross between members
of the F1 and members of the P1 is a backcross. A cross between members of the F1 and the true
breeding recessive P1 is a test cross.

Experiment 4: Complementation test.

Complementation test is carried out to find out if the observed phenotype is caused due to one
gene or multiple genes. To carry out the test one needs to set up a cross between a wild type fly
and the mutant fly and observe the phenotype produced in progeny. If the progeny exhibits wild
type phenotype, it would mean that the mutation can be caused by two different genes and due to
which the mutation is being compensated with the help of the wild type copy in the parents. This
compensation will not occur in mutations which occur due to a single gene.

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References:
1. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular
biology of the cell. New York: Garland Science.

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 Section-5: Observations using Foldscopes

Story of Microscopy and Foldscopes

Foldscopes:

Microscopes are a remarkable tool in science, which bridges the familiar visual macro-world
macro and the
underlying unseen micro-world.
world. The microscope is invented by a D Dutch
utch researcher called Antoine van
Leeuwenhoek (1632 – 1723), his microscope is consisting of a single high-quality lens and very short
focal length, through these he discovered for the first in 1674 the protozoa and later on the bacteria (he
isolated these miniature organisms from rivers, ponds, and human intestine & mouth). At that time his
research was recognized by royal society and made him distinguish royal fellow. Thus, his microscopic
observations paved a way for the science of bacteriology and protozoology.

Figure 10: Replica of Van Lee

Leeuwenhoek Microscope (Source: Wikipedia)

Many researchers have contributed

ributed to developing the reliable, maximum magnifying microscopes
th
after 17 century, popular among those are

1. Compound microscope developed by Galileo Galilei with a convex and concave lens, this
works with the help of visible light and the two lenses aree in compound arrangement. Even
today we use different variations of the compound microscope (Magnifying range between 4x
to 100x)

2. Electron microscope developed by ErnstRuska,, which works on the principle of subjecting

samples to a beam of electrons rathe
rather than light (the beam of photons) in the compound
microscope. Here wavelength of electrons is much smaller than visible light, so the resolution
power of the electron microscope is much better than a light microscope. Ernst Ruska in 1986
won the Nobel Prize in Physics for his study in the electron microscopy.

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3. Confocal imaging or confocal microscope is a special kind because it uses a laser rather than
the illumination (light source), this principle is patented by Marvin Minsky in 1957. The
confocal microscope illuminates the point by point fashion, and laser light short in wavelength
(eg: blue) is used to excite the sample.

4. Super-resolution Imaging or Fluorescence Microscope is developed by Stefan Hall in the

year 1993 to increase the resolution power so that high-resolution optical methods can be
utilized to capture images of high definition. A fluorescence stain is used with the sample and
it is subjected to illumination with a particular wavelength of light, then stain on the sample
absorbs it and emits another wavelength, and the image is reconstructed by the filter from this
emitted light. This imagining technique paved a path to look deep into the nanoworld for the
inaugural time. Thus, he along with others awarded a Nobel Prize in chemistry in the year
2014

Despite all of these remarkable inventions in the microscopy technology, the affordable and
portable microscopy has not been realized till the 20th century, but in 2010 at Stanford University
Professor Manu Prakash came up with an idea of an origami-based foldable microscope. This
scope’s working principle is inspired by Antonie van Leeuwenhoek microscope model, which has
small spherical lens held closer to eyes to visualize the microworld.

Figure 11: Foldscopes (source: Google images)

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Foldscopes:

In the 21st century, cost-effective and scalable manufacturing is the key to frugal innovation;
these foldable scopes can be a great resource for educational purposes and personal scientific
endeavors. Here the inventors have combined the optical microscopy principle with origami
(that can be assembled into the microscope within 10mins). Though it costs less than a dollar,
it can magnify the sample from 140x to 2000x. This microscope is application-specific, which
gives less functionality by reducing the cost, unlike the general-purpose microscopes.

Figure 12: Schematic diagram of disassembled elements of fold scope( Source: James, James, Manu Prakash “Foldscopes:
Origami-Based Paper Microscope” 2014)

Working principle :

A fold scope is operated by mounting the sample on a slide and inserting this slide into the
gap between lens and eyepiece column. The sample is viewed by holding the scope with
the hands and placing it near to our eyes. Now we can use an LED or a natural light to
illuminate the sample by subjecting the specimen to the light source. The two basic features
of a microscope to get an image are panning and focusing. Here panning (getting the
required sample under focus by blurring the background) is achieved by using our thumbs
over the yellow color element (in the above figure) moving in unison. The focusing or
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 zooming is achieved by using the same thumbs to push together or pull apart for getting -z
and +z direction movement. Unlike the traditional scope (where the sample is moved), here
sample is kept at fixed location and optics and illumination stages can be moved to get the
image.

Observations utilizing fold scope:

1) This is the divot plant (this structure is known by Reticulate venation: veins form like an
interconnected web-like network) taken from UOH campus, and we applied nail polish to
the leaf for visualizing it under light, because of opaque nature of a leaf

2) The brain of drosophila larvae taken with the help of staining: Hematoxylin &Ecosin

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3) Wing disc of drosophila larva with the help of staining: Hematoxylin &Ecosin

4) Head of adult Drosophila

5) Legs of adult Drosophila

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6) Wings of adult Drosophila

7) Chemosensory organs of adult Drosophila

Conclusion:
This Foldscope is very cost-effective and can be utilized for experimentation and
demonstration purposes in schools, which can increase the intrinsic motivation of students
to learn new things due to its hands-on exposure, and also an affordable device for
children’s (who have never experienced the micro-world out there). This is also a feasible
device for field-based investigations for researchers. In future we may have general-
purpose origami-based foldscopes for all kinds of investigations.

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References:

1) James S. Cybulski1, James Clements2, Manu Prakash “Foldscope: Origami-Based Paper

Microscope” 2014

2) www.sciencelearn.org.nz/resources/1692-history-of-microscopy-timeline

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Section-6: Immunohistochemistry (IHC) of Neuromuscular junction (NMJ) of
Drosophila melanogaster larvae

Introduction:
Immunohistochemistry combines immunological, histological and biochemical
techniques for the detection of a specific antigen in the cell with labelled antibodies,
based on antigen-antibody interaction. The principle has existed since the 1930s, but the
first IHC study was reported in 1942 by Albert Coons where he identified pneumococci
using a direct fluorescent method. Since then, advancements in tissue fixation methods,
protein conjugation and microscopy have led to increased use of IHC in scientific
research.

Terminologies:

Antigen (Ag): An antigen is any substance or molecule which is capable of stimulating

an immune response. Each antigen is specific and differs in terms of their epitopes.

Antibody (Ab): An antibody is a Y-shaped protein, which is produced by B-cell in

response to exposure to antigen. Antibody contains a paratope to which specific antigen
(epitopes) binds.

Primary antibody: one which directly binds to the antigen. The epitope of the antigen is
recognised by the variable region of the antibody.

Secondary antibody: one which binds to the primary antibody and not with the antigen
directly. Secondary bodies bind to the constant region of the primary antibody.

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Principle of IHC:

1. The primary antibody binds to the epitopes of the antigen expressed in a cell
within tissue with high specificity.
2. After this binding, many secondary antibodies bind to the primary antibody due to
the presence of a constant region of primary antibody.
3. The secondary antibody is coupled with a fluorophore (immunofluorescence),
which produces colour when excited with a light of a particular wavelength.

Figure 13: Direct labeled First Antibody Vs Indirect Secondary Antibody Labeled.

Basics of IHC Experiment

Figure 14: Basics of an IHC eexperiment (Source: IHC Handbook by Novus)

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Protocol:

1. Larval Dissection:

● Pour PBS buffer in the dissecting plate, it will keep the larvae from drying and
slow the activity of larvae.
● Using forceps, collect a third instar larva, which can be found wandering up the
sides of the bottle or vial and keep it in the PBS buffer.
● Keep the dissecting plate under the microscope to start the dissection.
● Using short forceps grasps the minutein pin. Place one pin on the anterior part
(mouth hook) and another pin on the posterior part, it stretches the larvae
lengthwise.
● Using scissors make a verticle incision on the dorsal side of larva from anterior to
posterior pin.
● Open the incision using forceps, stretch the larva and pinned it into I shape filet by
making a right and left flap in the body wall.
● Remove the organs. First, remove the tracheal system and then grab the rest of the
organs.

Figure 15: Fillet preparation (source:Budnik et al. 2006)

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 2. Fixation:

● Fixation of the tissue helps to maintain tissue structure and retain antigenicity. Fix
the tissue in 4% paraformaldehyde in PBS for 20 minutes, it mummified the tissue
at a particular position. Paraformaldehyde form crosslink between protein.
● Wash 1x with PBS with 0.2% Triton-X(PBST), PBST act as a detergent.
● Block in 5% Normal Goat Serum (NGS) in PBST for 1 hour.
● Incubate in primary antibody (diluted in PBST) overnight at 4°C.
● Save antibody, wash 3x with PBST each wash lasting 10min.
● Incubate secondary antibody (diluted in PBST) for 2 hours.
● Wash 3x with PBST, each wash for 10min.
● Mount appropriately and observe under a confocal microscope.

Motor neuron

Muscle fibre

Figure 16: Neuromuscular Junction, under a compound microscope. (Source: image was

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References:

1. Wang, S., Yoo, S., Kim, H. y., Wang, M., Zheng, C., Parkhouse, W., Krieger, C.,
Harden, N. Detection of In Situ Protein-protein Complexes at the Drosophila
Larval Neuromuscular Junction Using Proximity Ligation Assay. J. Vis. Exp. (95),
e52139, doi:10.3791/52139 (2015).
2. Brent, Jonathan R et al. “Drosophila larval NMJ dissection.” Journal of visualized
experiments :JoVE ,24 1107. 4 Feb. 2009, doi:10.3791/1107
3. Parton, R. M., Valles, A. M., Dobbie, I. M., & Davis, I. (2010). Drosophila Larval
Fillet Preparation and Imaging of Neurons. Cold Spring Harbor Protocols,
2010(4), pdb.prot5405–pdb.prot5405. doi:10.1101/pdb.prot5405
4. Immunohistochemistry (IHC) Handbook (Novus biologicals)
5. Immunohistochemistry Principles, uses and methods (Expedeon)

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SECTION 7: CONFOCAL MICROSCOPY
EVOLUTION OF CONFOCAL MICROSCOPY

The confocal microscope was invented by Marvin Minsky, who produced a working microscope
in 1955. The purpose of developing the confocal approach was to image biological events as they
occur in living tissue (in vivo), and Minsky was having the intention of imaging neural networks in
unstained preparations of living brains. The principle of confocal imaging advanced by Minsky,
and patented in 1957, is employed in all modern confocal microscopes. Minsky used a pinhole
placed in front of a zirconium arc source as a point source of light.

WORKING PRINCIPLE OF CONFOCAL MICROSCOPY

The point of light was focused by an objective lens at the desired focal plane in the specimen, and
light that passed through it was focused by a second objective lens at a second pinhole having the
same focus as the first pinhole (the two were confocal). Any light that passed the second pinhole
struck a low-noise photomultiplier, which generated a signal that was related to the brightness of
the light from the specimen. The second pinhole prevented light originating from above or below
the plane of focus in the specimen from reaching the photomultiplier. The use of spatial filtering to
eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus, is the key
to the confocal approach.

Figure 17: Principle Light pathways of Confocal microscopy

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 The image in the red and green are of two separate proteins and the third image is that
of both the proteins mentioned before merged together to show co-localization.

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 PART III

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Section 8: Analysis of drosophila melanogaster behavior.
Experiment 1: Alcohol Tolerance in flies

Aim of this experiment is to quantify the alcohol tolerance/addiction behavior of the adult
flies. From genetic analysis, we are familiar with the fact that there is almost 60%
similarity between the human and drosophila genome, therefore drosophila is the best
model to study the alcohol tolerance. Similar tolerance behavior which is seen in Humans
and rodents is also observed in drosophila. To investigate about alcohol tolerance, we can
track the postural control mechanism of flies during intoxication.

Day 1:
Four empty vials were taken and adequate numbers of flies were transferred from the
incubated vials. The vials were closed with cotton plugs and labeled according to the
treatment they will be given. The four treatment conditions will be given are:

1. Air (control)
2. Water (control)
3. 50% ethanol
4. 70% ethanol

The solutions were pipette on the cotton plug and the motor behavior of the flies was
observed.

The experiment will be analyzed into two phases. In Phase 1, we will analyze the falling
rate of flies in all the four treatment conditions. Falling depends on the postural control
mechanism of the flies. In Phase2, flies will be transferred into new empty vials and we
analyze the recovery time of the flies from the sedation state.

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 Phase one: (Falling phase) Graph -1

Falling phase
110.00
Percentage of flies on the ground

100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56

time

70% ethanol 50% ethanol Air Water

Graph -2

Percentage of flies falling in a single time instant

140.00
Percentage of flies on the ground

120.00
100.00
80.00
60.00
40.00
20.00
0.00
0 5 10 15 20 25 30 35

Time

50% ethanol 70% ethanol

Inference from the graphs

Here from the above graph-1 we can calculate the at what time instant 50 % of flies were
falling down in each treatment mainly in 50% ethanol and 70% ethanol.

1) For 50% ethanol vial the time at which 50 percentage of flies fell down is 30 th min
2) For 70% ethanol vial the time at which 50 percentage of flies fell down is 7 th min 30 s

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From this we can infer that the amount of alcohol has an inverse proportional relation to
the time at which the fly loses its motor control function. This means that higher the
concentration of alcohol, less time is taken for the fly to lose its motor function.

From the graph -2 we can calculate at what time instant maximum number of falls has been
registered:

1) 50% ethanol vial maximum falls registered at 31st min

2) 70% ethanol vial maximum falls registered at 8th min

In the second phase, we will calculate the recovery time taken by the flies from which we
estimate the sensitivity of files to different percentages of concentrations

Phase two: Recovery phase

Recovery phase
110.00
100.00
Percentage of flies on the wall

90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190

Time
Air water 50% ethanol 70% ethanol

Here from this above graph we can infer that:

1) 50% ethanol case, 50% of flies recovered by the 95th min

2) 70% ethanol case, 50% of flies recovered by the 41st min

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 Day 2:
Procedure followed on next day was same as day 1, but we have used another 2 vials
additionally. We have transferred the flies from incubating vials to new vials and named it
according to our treatments:

1. Air (control)
2. Water (control)
3. 50% ethanol (naïve)
4. 50% ethanol (experienced)
5. 70% ethanol (naïve)
6. 70% ethanol (experienced)

 In the phase one (falling phase) we should look for the motor coordination or postural
control mechanism (falling rate of flies) in all the treatment conditions.
Graph1

Falling phase
110.00
Percentage of files on the ground

100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0 2 4 6 8 10 12 14 16 18 20

Time

Air water N 50% E 50% N 70% E 70%

Graph -2

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 Percentage of files falling in single time instant
110.00
100.00
Percentage of flies falling

90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time

N 50% E 50% N 70% E 70%

Inference from the above graphs

From the above graph 1 we can infer that

1) In the 50% ethanol naïve vial the 50% flies fell down at 7 th min
2) In the 50% ethanol experienced vial, the 50% flies fell down at 9 th min
3) In the 70% ethanol naïve vial, the 50% flies fell down at 6 th min
4) In the 70% ethanol experienced vial, the 50% flies fell down at 8 th min 30s

This data clearly shows that experienced flies are tolerant to the alcohol in the 50% and 70
% case, this means they take much longer time than naïve flies. Therefore, we can say that
repeated exposure to alcohol can increase the tolerance level of flies.

From the graph 2 we can calculate at what time instant maximum falls were registered.
1) In the 50% ethanol naïve, the time instant is 18th min
2) In the 50% ethanol experienced, the time instant is 7 th min
3) In the 70% ethanol naïve, the time instant is 4 th min
4) In the 70% ethanol experienced, the time instant is 9 th min

 In the second Phase, we calculate the recovery time for all the cases, this gives us the
information about the sensitivity of flies to different percentage of concentration.

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 Recovery phase
110
100
Percentage of files on the wall

90
80
70
60
50
40
30
20
10
0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96

Time

Air water n50% E50% N70% E50%

From the above graph we can infer that

1) In the 50% ethanol naïve, the 50 percent of flies recovered by time instant: 12 th min
2) In the 50% ethanol experienced, the 50 percent of flies recovered by time instant: 36 th min
3) In the 70% ethanol naïve, the 50 percent of flies recovered by time instant: 68 th min
4) In the 70% ethanol experienced, the 50 percent of flies recovered by time instant: 67 thmin

Conclusion:
In the day 1, we have seen that flies postural control mechanism depends on the
concentration of alcohol, and recovery time signifies about the sensitivity of flies towards
different concentrations. In the day 2, we noticed that experienced flies are more tolerant to
the alcohol than the naïve flies (this we concluded based on the falling time in both the
cases), this signifies that repeated exposure of flies to alcohol can make them tolerant, that
mean addicted to alcohol

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