STUDY OF THE GENETIC &

MOLECULAR BASIS OF
BEHAVIOR
& LEARNING USING MODEL
ORGANISMS
 PROJECT WORK DONE AT :

The Brain Research & Cognitive Sciences

Laboratory,
Department of Life Sciences,
SOPHIA COLLEGE FOR WOMEN,MUMBAI (INDIA)
Under the guidance of :
Dr. M. C. Arunan
 
Mentor, Brain Research & Cognitive Sciences Laboratory,
&
Head, Department of Life Sciences.
 
1
ST
PART OF MY PROJECT
WORK………..
 • Learning is the act of acquisition of knowledge and
the processing of that knowledge into memory
• Behavior, or to be more precise, modified behavior is
the external manifestation of this acquired
knowledge. Behavior reflects nervous system activity
and is dependent on multiple factors including
external stimuli, past experience, neuronal structure
and changes in the internal milieu of the animal.
• Therefore, behavioral assays offer the
researcher simple, sensitive and powerful
tools to interrogate neuronal function.
 WHY DID WE CHOSE THESE MODEL ORGANISMS,……NOT
HUMAN !!??

☻The key to understanding the mystery of learning and memory

is in identifying suitable experimental model systems, which are
simple enough to handle, and at the same time sufficiently rich to
address sophisticated questions.
☻ So human brain , with the 100,000 million nerve cells, its 3
kilometers of nerve fibers and its astronomical number of
precisely organized connections,
connections is far from being a feasible
candidate for these type of assays !!
☻On the other hand, the East African Land Snail, Achatina fulica,
has fewer than 100,000 cells , neatly packaged into about half a
dozen ganglia.
ganglia The real attraction is that these cells are huge in
comparison to the human or other mammalian nerve cells: more
than 20 times larger!
☻ The most important point is that, mollusks and mammals share
a close homology in regard to the molecular mechanisms
underlying behavior and learning.
 ERIC KANDEL
• It is impossible to
understand the behavior of
snails in the light of
neurobiology, without
mentioning about the works
of Dr.Eric Richard Kandel
(born November 7, 1929),
co-recipient of the 2000
Nobel Prize in Physiology or
Medicine for his research on
the physiological basis of
memory storage in neurons,
along with Arvid Carlsson
and Paul Greengard.
 OBJECTIVES:
The main objectives of our experiment were to:

► Study the genetic and molecular basis of learning

and memory, using snails (Achatina fulica) as the
model for certain behavioral assays.
► Also to study the basis of long–term and short-term
memory storage in these animals, and thereby, have a
clue of how these processes occur in mammals -
because, as mentioned earlier, mollusks and mammals
share a close homology in regard to the molecular
mechanisms underlying behavior and learning.

 
 RESULTS
• We carried out our experiments with the olfactory behavior of the
snails and decided to modify these particular behavior .
• The Olfactory behavior measuring instrument consists of a
circle of diameter 15 cm drawn on a white sheet of paper and a
clean glass plate placed over it .
• At the opposite poles of this circular area stimulus and control were
presented in the form of filter paper wetted with distilled water
and odorant solution respectively. However, before performing
any assay, it is necessary to do the control assay, keeping
distilled water on both the sides.
• For this purpose, we had to find out an olfactory attractant for the
snails so that a reproducible O.R.I reading could be obtained
using that odorant.
O.R.I (Olfactory Response Index) = (S – C) / N , where,
S is the number of animals that moved towards the stimulus
zone, C is the number towards the control zone and N the total
number of trials.
THE EXPERIMENTAL SET-UP FOR OLFACTORY ASSAYS IN
LAND-SNAILS
 

2 cm

15 cm

The snail is
placed here

2 cm
 THE TRAINING SESSION:
۞An important phase of our experiment was the training session of
the snails, so as to modify the behavior of these animals by
associating a good stimulus with a bad one, e.g, a good smell with
a bad taste.
۞ After trying with different concentrations of cucumber and
cabbage, we find out an olfactory attractant for the snails so that a
reproducible O.R.I reading could be obtained using it - 2.5 %
concentration of Cucumber extract – it was found to be a
consistent attractant for the snails.
۞ We worked with two sets of animals (each set comprising a
young - 5 whorled and an adult- 6 whorled) – one was treated
as the control, or untrained (Set 1) and the other as the
trained (Set 2). 2)
۞ The animals of the trained set were trained in the following
fashion: they were allowed to go towards the attractant (2.5%
cucumber extract) and taste them ; just when they started tasting,
a few drops of Quinidine Sulphate (a chemical with bitter taste)
were added to the extract - after treating them for 5 minutes in
this fashion, they apparently started avoiding the cucumber
 OBSERVATIONS
DATE ASSAY O.R.I
PRE-TRAINING SESSION
D-H2O v/s D-H2O 0
24/07/2008
D H2O v/s Cucumber Extract (2.5%) 0.56

TRAINING SESSION
28/07/2008 D-H2O v/s D-H2O (Set 1) 0

D-H2O v/s D-H2O (Set 2) -0.33

UNTRAINED SET D H2O v/s Cucumber Extract (2.5%) 0.143

(Set 1)
TRAINED SET D H2O v/s Cucumber Extract (2.5%) -0.2
(Set 2)
 OBSERVATIONS……continued
DATE ASSAY O.R.I

POST-TRAINING SESSION

29/07/2008

D H2O v/s Cucumber Extract 0.43

UNTRAINED SET (2.5%)
D H2O v/s Cucumber Extract 0.2
(2.5%)
TRAINED SET
 DISCUSSIONS AND
CONCLUSIONS
◙ In our experiment, the reduced olfactory response for
Cucumber shown by the trained animals as compared to the
untrained animals suggests that the snails in the former group
had associated cucumber odour with the aversive Quinidine
sulphate exposure.
◙ From the results and observations given above, it is evident
that the behaviors of the trained snails towards their attractant
(2.5% cucumber extract) were modified to a great extent, viz.,
from 0.56 during the pre-training session, to 0.2 during the post-
training period (taking consideration the no. of times the snails
moved within the chord); or, alternatively from 0.5 during the
pre-training session, to 0.2 (taking consideration the no. of times
the snails moved towards either hemisphere of the circle)
after they had been trained with the Quinidine sulphate. This
important piece of information can prove handy in a number of
gene-level and protein- level expression studies in the brain of
the trained snails.
◙ Once the behavioral modification is achieved using this
paradigm, one may look at the structural and molecular changes
1› A Southern Blotting assay can be carried out to test if a
gene has been differentially expressed in the trained animals,
during the post-training session, which is responsible for its
modified behavior.
2› In a similar fashion, a Northern Blotting assay can also be
carried out to test if there has been a difference in the RNA level
due to the training.
3› However, it is the most important task to study the
proteomics of the brain of the trained snails, either by
Western Blotting, ELISA or Immunohistochemical staining
studies – to study if there has been a differential protein
expression, either in the form of over- or under- expression of a
specific protein, or in the form of post-translational modification
of any proteins.
۞ Any new findings revealed through such studies will throw light
on the genetic and molecular mechanisms underlying the
processes of learning and memory-storage in humans also.
۞ As a matter of fact, we could not carry out any such studies
with the trained set of mollusks due to an acute shortage of
time, but it is a real challenge for us to find out the basis of all
2nd PART OF MY PROJECT
WORK………..
Rationale for studying invertebrate behavior:
 Invertebrates offer an excellent system to study behavior
because of two reasons:
◘ Several of the molecular cues governing developmentally
controlled mechanisms are shared between vertebrates and
invertebrates, and,
◘ Due to the sheer simplicity of their body organization
compared to vertebrates, their cells can be easily identified,
isolated and subjected to experimental manipulations.
◘ The earthworms provide an excellent cost-effective system for
culturing in laboratory conditions.
 OBJECTIVES

The main objectives of our experiment are,

► To develop an assay set-up to study the Gustatory Behavior
of the earthworms.
► To find out their dose-dependant behavior towards different
concentrations of the speculated gustatory stimulants.
► To investigate the role of these chemosensory modalities in the
learning and memory formation of earthworms.
 
 
 REARING OF EARTHWORMS

Vermiculture Cup culture

• The rearing of earthworms in soil culture (their natural habitat) is

known as Vermiculture.
• However, for assay purposes, 1-2 earthworms were taken in a
plastic cup having a moist filter paper at the base so that the
earthworm never falls short of moisture.
• Besides, 4-5 tiny holes were made at the bottom of the cup,
which is kept on a moist tray – so that the water can move up the
cup by capillary action.
 THE EXPERIMENTAL SET-UP FOR
GUSTATORY ASSAY
◘ On a clean glass plate (size 20.5 x 20.5 cms),
cms two strips of filter
paper (15 x 7 cms)
cms are placed, 1cm apart from each other. For each
of the assays, a control assay is necessary, where both the filter
papers are soaked in distilled water.
◘ Now, the earthworm is placed at the middle of the glass plate,
where the filter papers are separated by the prescribed distance.

FILTER PAPERS

WIDTH 1 cm

The earthworm
is placed here
 THE EXPERIMENT –
continued……
◘ After performing the control assay, the stimulus assay is
performed where one of the strips of paper is soaked with
distilled water and the other with the chemical to which the
worm is supposed to move. To prevent biasness of the worm
towards any particular direction to interfere with our readings,
the filter papers are always placed in alternate directions for
each new reading.
◘ The readings are then noted down, and the Gustatory
Response Index (G.R.I) = S-C/S+C of the worms is calculated,
where, S → No. of times when the worm moved towards
the chemical, and, C → No. of times when the worm
moved towards distilled water.
The value of G.R.I ranges from +1 to -1.
If G.R.I is +1, or nearing +1, then the stimulus is called an
Attractant.
If G.R.I is 0, or nearing 0, then the organism is indifferent
towards the stimulus (this is the case for control assay also).
 OBSERVATIONS – Page 1
DATE ASSAY G.R.I
25/6/200 distilled-H₂O v/s distilled-H₂O 0.133
8
26/6/200 distilled-H₂O v/s distilled-H₂O 0.04
8 d-H₂O v/s 200mM NaCl -0.8

distilled-H₂O v/s distilled - H₂O -0.067

27/6/08 d-H₂O v/s 200mM Dextrose 0.067
30/6/08 distilled-H₂O v/s distilled - H₂O -0.067
d-H₂O v/s 200mM Dextrose -0.067

2/7/2008 distilled-H₂O v/s distilled - H₂O 0.133

d-H₂O v/s 50mM Dextrose 0

3/7/2008 distilled-H₂O v/s distilled - H₂O 0

d-H₂O v/s 4% Soil solution 0
 OBSERVATIONS – Page 2
DATE ASSAY G.R.I

4/7/2008 distilled-H₂O v/s distilled - H₂O 0.067

d-H₂O v/s 1:1000 Benzaldehyde -0.733

7/7/2008 distilled-H₂O v/s distilled - H₂O 0

d-H₂O v/s 200mM Dextrose 0

As is evident from our recordings, we couldn’t find out any satisfactory attractant
for Earthworm; however we obtained some repellents (200mM NaCl and 1:1000
Benzaldehyde).
However, at this point, certain questions are bound to arise regarding the set-up!
They are being discussed below.
 DISCUSSIONS AND
CONCLUSIONS
◙ It is a well established fact that a moist environment and a minimal
food resource are good enough for an earthworm to survive – thus, a
moist filter paper is a good attractant itself. So if the animal finds one
moist filter paper at one side, it may not bother at all, about the
opposite filter paper!
◙ Moreover, if it goes to the stimulant side,- can it be conclusively said
that the animal is attracted towards stimulant only? As the solutions are
made in water, it may be attracted towards the solvent water only. So,
the same experimental set-up may not be applicable for
detecting both attractant and repellent; however, it is definitely a
feasible set-up for detecting only repellants for the organism.
◙ Alternately, we missed out in identifying a strong attractant stimulus.
This is possible because not many studies have taken place in the
olfactory and gustatory behavior of earthworms
However, the discrepancy in the set-up can be accounted for, by
introducing physical stimulants.

◙ This newly proposed set-up can be described as given below:

 DISCUSSIONS AND
CONCLUSIONS –
continued………………………
◙ A mild repellent for the organism is detected and the
Gustatory Assay is started with that chemical.
◙ Then we apply an electric shock to the animal once it tries
to come towards this repellent.
◙ Now it is expected that the organism will be much more
aversive towards the chemical,-in short, the chemical turns into
a strong repellent from a mild one.
◙ Thus a behavioral modification can be said to take place in
this organism – now we can study the basis of this behavioral
change, in the molecular and genetic level, as is the objective of
our work
As a matter of fact, we had a very limited time-period to carry out our plans, so we
could not give a concrete shape to the set-up proposed above. However, provided
with a prolonged tenure in future, we can certainly work out this experiment and
study the role of chemosensory modalities in the learning and memory
formation of earthworms.
 ACKNOWLEGDEMENTS
☼ I would like to express my utmost gratitude to my mentor Dr.
M.C.Arunan, Head, The Department of Life Sciences, Sophia
College, whose guidance and untiring efforts were the main driving-
force behind the accomplishment of my project work. I was more than
benefited by his invaluable suggestions regarding each and every
detail in this work, which I dare not to claim to be exclusively mine.
 
☼ I sincerely express my gratitude to my teachers in Calcutta
University, especially to my Program Coordinator Prof. S. Jha and to
Dr. K. Nandagopal, for their kind assistance, during each and every
step, of applying for my project.
 
☼ I shouldn’t let go of this opportunity to thank the rest of our
teaching faculty at The Department of Life Sciences, Sophia College -
Dr. M. Rajadhakshya, Dr.H.Subramaniam, Dr. H. Ramchandran,
and Dr.Y.Khan, and the research fellows – Ms. Jennifer Italia and
Mr. Mustafa Motiwala – who never seemed short of exuberance to
have a discussion about my project and the questions arising out as a
consequence opened up new avenues for me!
☼ It would be the gravest mistake on my part if I overlook the
assistance rendered to me by the non-teaching staff of the institution,
☼ I consider this the best opportunity to thank the person, without whom
this project couldn’t have seen the light of daytime, i.e., Dr. (Sr.)
A.Verghese, Principal, Sophia College, and also The University of
Mumbai, for providing us residential accommodation so close to our
institute. 
☼ I am also highly indebted to Mr.Kshaunis Misra, a batch mate from
Calcutta University and the sole companion throughout my project-
work; and the rest of my friends in our Department of Genetics. It is
needless to say that this project would not have taken its present shape
without the support of my family.
 

☼ Last but not the least; I thank all my friends at The Department of
Life Sciences, Sophia College, especially, Mr.Viraj Torsekar and
Ms.Ramya Ranganathan, who assisted me with every minute detail of
the techniques, involved in my work – and of course, all the model