3 SOCIAL DEFEAT PARADIGM

Transgenic nestin-GFP mice were subjected to a chronic social defeat paradigm as described
in earlier reports (Krishnan et al, 2007; Veeraiah et al, 2014, Pathak et al, 2016) with minor
modification such as 5 min of encounter, instead of 10 min, daily. In details, rectangular
cages (35X20X10) with paired steel-wire lids containing hard woodchip bedding were used
for the chronic social defeat (CSDS) procedure. The cage was divided in half by the clear
perforated plexiglas divider, which physically separates the mice following the defeat
sessions. 24 h before starting the first social defeat stress, a CD1 aggressor mouse was placed
on one side of the divided cage (home cage) overnight before the defeat sessions. On the first
day of the defeat, an experimental nestin-GFP mouse was introduced in the home cage
compartment of an unfamiliar aggressive resident CD1 mouse and allowed to interact for 5–
10 min. After 5–10 min of social defeat, the intruder nestin-GFP was transferred across the
perforated divider to the opposite compartment and house within this compartment for the
remaining 24-hour to enable the transmission of visual and olfactory cues. These defeat
sessions were repeated daily for 10 consecutive days with a different aggressor mouse each
time, to minimize inter-aggressor variability. Control mice were also housed on either side of
the similar divided cages and were allowed to interact with each other daily for 5min, upto 10
days. Finally animals were sacrificed and brain tissues collected for stress specific molecular
analysis.

5.4 Behavioral study

5.4.1 Sucrose preference test
Principle
This test is used as in indicator of anhedonia (lack of interest in rewarding stimuli), which is
present in some forms of affective disorder, including depression (Tatyana Strekalova. et al.,
2004). In this task we assess the animal interest in seeking out a sweet rewarding drink
relative to plain drinking water. A bias toward the sweetened drink is typical, failure to do so
is indicative of anhedonia/depression.
Sucrose preference test is carried out in the animal home cage. For this test animals were
presented with two bottles kept side by side in the cage. One bottle contains purified drinking
water while other contains 2% w/v sucrose (SRL- 194957) solution.

Procedure
Habituation period (6 days):
1. For initial two (1st and 2nd) days water bottles were replaced with two approximately
180 ml purified drinking water bottles (Bottle A and Bottle B) in each cages. The position of
the bottles A and B were switched daily so as to avoid a side bias. The fluid consumed from
each bottle was measured daily.
2. For next two (3rd and 4th) days both the water bottles (Bottle A and Bottle B) were
from all cages were replaced with 1% w/v sucrose solution. The position of the bottles A and
B were switched daily so as to avoid a side bias. The fluid consumed from each bottle was
measured daily.
3. For remaining two days (5th and 6th) water bottles were replaced with two bottles, one
containing purified drinking water (Bottle A) and other containing 1% w/v sucrose solution
(Bottle B) in each cages. The position of the bottles A and B were switched daily so as to
avoid a side bias. The fluid consumed from each bottle was measured daily.

5.4.2 Social interaction:

Social interaction test is parameter to evaluate the level of depression in animals, usually
mice socially very interactive and they will immediate give positive response to a new
neighbor, but when they are depressed they will keep themselves in an isolated condition and
does not interact socially this principal in widely being used as tool to detect depression.

5.5 Ethovision:
EthoVision is an automated video tracking, motion analysis and behavior recognition system.
It offers a wide range of video tracking options, extensive analysis of locomotory tracks, and
automatic behavior recognition.
5.6 Brain micro dissection:
Materials required
Surgicals Petriplates
Cotton Eppendorf tubes
Crude alcohol Ice
Fresh blade Liquid nitrogen
Gloves 1X PBS
Marker pen 4% Paraformaldehyde (SIGMA-P6148)

Preparation of 4% Paraformaldehyde (PFA) as fixative

 Initially 400 ml of distilled water was added in a glass beaker and heater at 60 0C using a hot
plate with stirring.
 While stirring, 20 gm of paraformaldehyde powder was added to the heated water. I was
covered with a foil abnd maintained at 60ºC. (Do not heat the solution above 70ºC, PFA will
break down at temperature above 70ºC )
 Now 5 drops of 2 N NaOH (SRL-1949181) was added (1 drop per 100 ml). The solution
cleared within a couple of minutes. (There will be some fine particles that will not go away)
 It was recovered from heat and 50 ml of 10X PBS was added. The pH was adjusted to 7.2
using HCl or NaOH. Final volume was made up to 500 ml. The solution was filtered and
placed on ice and covered with a foil to protect from light.
 The prepared solution can be used immediately or aliquots may be frozen at -20ºC and
thawed as needed.

Preparation of 10X PBS

 A 1lt stock of 10X PBS was prepared by dissolving 80gm NaCl (SRL-1940103), 2gm KCl
(FISCHER SCIENTIFIC-7447-70-7), 14.4gm NA2HPO4(SIGMA-S5136) and 2.4gm
KH2PO4(SRL-1649201) in 800ml of distilled water.
 The pH was adjusted to 7.2 using NaOH or HCL. The final volume was made up to 1lt with
the help of distilled water.

Preparation of 1XPBS
 The 1x PBS solution was made by diluting 10x PBS with Milli-Q water (100ml of 10x PBS
in 1000 ml).

Procedure
 By the method of cervical dislocation, the body of the mouse was elongated on both the
sides, such that its vertebral column breaks.
 Using a scissor the body was decapitated and disposed in designated animal bag that was
later moved into the animal fridge. The blood samples were collected immediately after
decapitation.
 Initially the brain was removed by cutting with scissors the ears and then the top of the
head.
 With the bone cracker, an incision was made down the midline at the base of the skull and
the bones were removed at the base of head.
 Cutting carefully, a cut was made in the middle and then to the sides, removing the bones
along the way.
 Once the full brain was exposed, with a spatula, a cut was made through the olfactory bulb
and around the sides of the skull, under the brain, detaching the optical nerve and removing
all the pia matter.
 After the removal of brain, Hippocampus was dissected and placed in an eppendorf tube and
labeled.
 These tubes were stored in liquid nitrogen and were later placed in an eppendorf box and
stored at –80C.

5.7 Western Blotting

Western blotting identifies with specific antibodies proteins that have been separated from
one another according to their size by gel electrophoresis. The blot is a membrane, almost
always of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the
membrane and application of an electrical current induces the proteins in the gel to move to
the membrane where they adhere. The membrane is then a replica of the gel’s protein pattern,
and is subsequently stained with an antibody.

A. Sample preparation
1. The stored (at -80oC) tissue samples were taken out and about 200 µl of lysis buffer
was added to each of the tissue samples in epitubes.
2. The lysis buffer is prepared as follows
Table 1: Lysis Buffer Composition.
Components For 10 ml

8M Urea 4.8 gm

2 M Thiourea 1.52 gm
 4% CHAPS 0.4 gm

65mM DTT 100.3 mg

The tissue samples were homogenized properly using homogenizer

3. Further the tissue samples were sonicated using sonicater for period of 1 min (Pulse
-10 Sec, Amplitude- 40%).
4. The tubes were centrifuged for 20 min at 12000 rpm at 4°C in a microcentrifuge.
Tubes were removed gently from the centrifuge and placed on ice, supernatant was aspirated
and placed in a fresh tube kept on ice; discarding the pellet.
5. Further 50 µl of lysis buffer was added for necessary dilution of protein.
6. The concentration of proteins were estimated using Bradford Assay

B. Determination of Protein concentration

Bradford Protein estimation
Principle:
The Bradford assay is a protein determination method that involves the binding of Coomassie
Brilliant Blue G-250 dye to proteins (Bradford 1976). The dye exists in three forms: cationic
(red), neutral (green), and anionic (blue) (Compton and Jones 1985). Under acidic conditions,
the dye is predominantly in the doubly protonated red cationic form (Amax = 470 nm).
However, when the dye binds to protein, it is converted to a stable unprotonated blue form
(Amax = 595 nm) (Reisner et al. 1975, Fazekes de St. Groth et al. 1963, Sedmack and
Grossberg 1977). It is this blue protein-dye form that is detected at 595 nm in the assay using
a spectrophotometer or micro plate reader.
Beer's law may be applied for accurate quantitation of protein by selecting an appropriate
ratio of dye volume to sample concentration.

Protocol
1. Preparation of Standard Curve using Bovine Serum Albumine BSA (1mg/10ml)
2. The reagents are pipetted out as follows.
BSA Triplicate Milli-Q Triplicate Bradford Triplicate
(µl) (µl) water (µl) (µl) Reagent (µl) (µl)
 0 0 160 480 40 120

10 30 150 450 40 120

20 60 140 420 40 120

30 90 130 390 40 120

40 120 120 360 40 120

50 150 110 330 40 120

60 180 100 300 40 120
Total Volume = 600 µl
Volume to be added in each well= 200 µl

3. The absorbance value was taken using plate reader.

4. The graph was plotted as concentration (x-axis) vs absorbance value (y-axis) as follows

5. Now protein sample were treated as follows

Protein Triplicate Milli-Q Triplicate Bradford Triplicate
sample (µl) water (µl) (µl) Reagent (µl) (µl)
(µl)
2 6 158 474 40 120
Total Volume = 600 µl
Volume to be added in each well= 200 µl
6. Blank reading was taken by adding the lysis buffer in place of protein sample to the
above reagents.
7. From the standard graph, the concentration of proteins was estimated.
Once the protein concentration was determined, the tubes containing protein can be stored at
-30oC or -80oC for further use.

C. Preparation of samples for loading in to the gel.

1. The stored samples were thawed and suitable volume of loading dye was added. The
loading dye consist of following
2. Table 2: Laemmli buffer 6X, 10ml Composition
 SDS 1.2 g
Bromophenol Blue 6 mg
Glycerol 4.7 ml
Tris pH 6.8 1.2 ml
Milli-Q water 2.1 ml
DTT 0.93 g

(Add all the ingredients and shake it. The DTT should be added at last and ensure that all
ingredients are dissolved. The Perpared buffer was aliquoted and stored at -30 oC. For 5µl of
protein sample; 1µl of 6X Laemmli buffer was used).
3. After adding laemmli buffer the samples were heated at 95°C for 5-7 minutes.
4. The samples were then kept at room temperature and short spinned.

D. Electrophoresis (SDS- PAGE)

The gel consists of stacking and resolving gels. The stacking gel has a very large pore size,
which allows the proteins to move freely and concentrate, or stack, under the effect of electric
field. The Gel was prepared by preparing resolving gel first depending on the molecular
weight of desired protein, after solidification of the resolving gel, stacking gel was added
above it. The composition of resolving and stacking gel are as follows:
Preparation of 30% Acrylamide
1. 29gm of Acrylamide and 1 gm of N, N’ methylene bis acrylamide were Dissolved in
60 ml of Milli-Q water at 37ºC.
2. The pH was adjusted if required and the volume was made up to 100 ml
3. The prepared solution was filtered sterilized and stored in amber colour bottle at 4ºC.

4 .The samples were loaded into the wells and and 5 µl of PageRuler Prestained Protein
Ladder (Thermo Scientific- 26616) was loaded in one of the well. The marker being
separated in the following manner in kDa:

5 . The tank was filled with 1X running buffer and the gel was run at 80 Volts during
stacking and 100 volts during resolving gel.
6.The running buffer was prepared as follows

Table 5. 1X Running buffer composition

 Tris base 3.02 gm
Glycine 18.8 gm
SDS 0.1% 10 ml of 10% SDS
Milli-Q water Up to 1000ml (pH- 8.3)

7. After running an SDA PAGE, gel was equilibrated by placing it in a small container with
transfer buffer for 30 min. Equilibrium helps in removal of electrophoresis buffer salts and
detergents. If salts are not removed, they will increase the conductivity of transfer buffer and
the amount of heat generated during transfer.

Transfer of proteins (Wet Transfer)

1. A piece of blotting/ filter paper was saturated by soaking in a transfer buffer and
placed on a flat chamber.
Table 6: 1X Transfer Buffer Composition
Glycine (39 mM) 2.9 gm
Trisbase (48 mM) 5.8 gm
SDS (0.037%) 0.37 gm
Methanol (20%) 200 ml
Milli-Q water Up to 1000ml (pH- 8.3 or not
required)

2. A pipette or a roller was roller over the surface of the paper to remove all air bubbles.
This is repeated with the second and third piece of blotting/ filter paper and placed directly on
the top of the first piece.
3. Carefully the equilibrated gel in transfer buffer was placed on the top of the blotting/
filter paper.
4. The PVDF membrane was activated by dipping in methanol for 1-2 minutes and
transferred to other container containing 1X transfer buffer. A pre-wetted PVDF membrane
(Merck REF-741260) in 1X transfer buffer was placed on the top of the wetted blotting/ filter
paper, and air bubbles were rolled out.
5. Another piece of soaked blotting/ filter paper was placed on the top of the gel,
carefully removing air bubbles between the gel and blotting/ filter paper. This process was
repeated with the second piece of blotting/ filter paper which was place on the top of the first
piece.
6. The arrangement of the gel, PVF membrane and blotting/ filter paper as follows
7. Carefully prepared sandwich was placed on the wet chamber and the whole unit was
covered safely by closing the lid.
8. The transfer unit was run at 40 Volts for 3.5 hours.

E. Visualization of proteins in gels

This visualization of protein at this stage is useful to determine if proteins have migrated
uniformly and evenly. Use the copper stain if you plan to transfer the separated proteins to a
membrane, as the Coomassie stain is not reversible.

F. Visualization of proteins in gels

1. To check for success of transfer, membrane was washed in TBST. . Dilute the
stock.
2. Ponceau Red 1:10. The stock was made with 2% Ponceau S in 30% trichloroacetic
acid and 30% sulfosalicylic acid.
3. The stock solution of ponceau red prepared was diluted in the ratio of 1:10.
4. The PVDF membrane was incubated on agitator for 5 min.
5. The membrane was washed extensively in water until the water is clear and protein
band were well defined.
6. The membrane was destained completely by repeated washing in TBST or water.
When using a PVDF membrane, the membrane was re-activated with methanol and washed
again in TBST.

G. Target protein detection by immunoblotting

1. Initially the PVDF membrane was blocked with 5% BSA solution for 1 hour at room
temperature.
2. After blocking, the blot was incubated in primary antibody as per dilutions given in
the datasheets overnight (14-16 hours).
3. After incubating the blot, it was washed with 1X TBST for 3 times with 10 min
interval.
4. Then the blot was incubated in secondary antibody for 2 hours at room temperature or
overnight at 4 °C.
5. Then the blot was subjected to three times washings with 1XTBST with 10 min
interval.
6. Finally, the blot was developed in ECL method using Super signal West Dura
Extended Duration Substrate (Thermo scientific- 37071) under chemiluminescent.
7. The substrate was prepared by mixing luminol/enhancer and peroxide solution in the
ratio of 1:1.

5.8 GFP Immunoflorescence

A series of sections were blocked in 10% normal horse serum prepared in 0.3% Triton-X for
2 hrs at room temp, followed by overnight incubation in anti-rabbit GFP (abcam #ab6556,
1:500) at 4 °C. Next day, after washes the sections were incubated in goat anti-rabbit Alexa
fluor 488 (CST #4212S, 1:500) for 1 hours at room temp and finally mounted in Vectashield
medium (Vector labs) with DAPI. GFP positive cells were quantified by stereological cell
counting method.

5.9 BrDU Immunoflorescence

For BrdU immunostaining, the sections were washed with 1xPBS and pre-treated with 50%
formamide in 2X SSC for 2 hrs at 65 °C. After washes, DNA denaturation was carried out by
treatment with 2N HCl for 25 mins at 37 °C. The sections were then blocked in 10% horse
serum for 2 hrs at RT, followed by overnight incubation in anti-Mouse BrdU (Millipore #
NA61, 1:500). Next day, the sections were washed and incubated in Anti mouse Alexa fluor
555 (CST #4409S, 1:500) for 1 hour hours at room temp and finally mounted in Vectashield
medium (Vector labs) with DAPI. BrDU positive cells were quantified by stereological cell
counting method.