By,

Dr. Manoj Upadhya,

SKBCOP, Kamptee
Extremely common psychiatric condition
o Intense feeling of sadness
o Hopelessness
o Despair
o Inability to experience pleasure in usual activities
SITUTATIONS
*Loss
*Isolation
Conflict
*Stress
THOUGHTS
* Negative thinking habits
*Harsh self-criticism
*Unfair & unrealistic thoughts
EMOTIONS
* Discouragement / Anxiety
* Sadness
* Irritability/Anger
* Numbness
ACTIONS
* Social withdrawal
*Reduced activity level
*Poor self care
PHYSICAL STATE
*Altered Sleep
* Low energy/fatigue
*Agitation
*Changes in brain chemistry

Low Mood
&
Depression
Dopamine
Pleasure,
Reward,
Motivation/Drive
Norepinephrine
Alertness,
Concentration,
Energy.
Serotonin
Obsession &
Compulsions,
Memory.
Mood,
Cognitive
Function
Anxiety,
Impulse,
Irritability
Appetite,
Sex,
Aggression
Attention
o Tricyclic /polycyclic antidepressants
Imipramine, Desipramine, Amitriptyline etc.

o Selective serotonin reuptake inhibitors (SSRI)
Fluoxetine, Venlafaxine, Sertraline etc.

o Monoamine Oxidase Inhibitors (MAO inhibitors)
Isocarboxazid, Phenelzine, Tranylcypromine etc.
Based on reuptake inhibition concept
Procedure:
Tissue Preparation
W. Rats 200-250 g
Brain is removed rapidly
Hypothalamic region is prepared, weighed and homogenized in 1:9 ice cold 0.32 M sucrose
solution
Supernatant decanted and used for assay
(Decapitation)
(Centrifuge at 1000g, 0-4
O
C for 10 min)
Assay procedure
200l tissue suspension + 800l 62.5nM [
3
H]-norepinephrine in Krebs-Hanseleit bicarbonate
buffer and 20l of appropriate drug concentration or vehicle
(incubate at 37
O
C under 95% O
2
:5%CO
2
for 5
min, parallel reaction at 0
O
C)

Immediately centrifuge at 4000g for 10min.
Supernatant is aspirated and the pallets are dissolved adding 1ml solubilizer
(Triton X-100 + 50 % ethanol, 1:4)
Decanted into scintillation vials, and counted in 10ml of liquid scintillation cocktail
Evaluation
Corpora striata
Hypothalamus or whole brain minus cerebellum
Procedure:
Expression of Human transporters
Human serotonin transporter cDNA ligated into the expression vector pRc/CMV
Or
Human dopamine transporter cDNA ligated into the expression vector pcDNA3
Transfected to HEK293 (human embryonic kidney) cells by Ca
2+
method
Cell lines are grown and harvested
The growth medium is removed by aspiration
Cell lines are washed with modified Pucks D1 solution
Cell lines are incubated with D1 solution for 5 min
(Centrifuge at 110g for 5 min)
Pellets are suspended in respective binding assay buffers
Mixture is centrifuged at 35600g for 10 min at 4
O
C
Supernatant decanted and the final pellets are suspended in respective buffers and stored
at -80
O
C until assay
Radioligand binding assay
[
3
H]imipramine binding to human serotonin transporter
Total protein conc. is determined by the Lowry assay using bovine serum albumin as standard
[
3
H]nisoxetine binding to human norepinephrine transporter
[
3
H]WIN35428 binding to human dopamine transporter
Methods similar as above
Methods similar as above
o p-Chloramphetamine (p-CA) causes selective toxicity to serotonin neurons.
o At 10mg/kg i.p. p-CA causes long term decrease in 5-HT, [
3
H]-5-HT uptake and tryptophan
hydroxylase.
o antagonism of the long-term p-CA induced reduction of synaptosomal
3
H-5-HT in-vitro
uptake is a highly useful index of compounds ability to inhibit 5-HT re-uptake in-vivo
Procedure:
Drug treatment
Group of 8 male W.Rats (150-200g) molar equivalent doses of test drugs are
injected after 30 min , 4rats from each group are injected with saline or with p-CA (10mg/kg;
i.p.). Three days after treatment the rats are sacrificed.
Tissue preperation
Rats are decapitated and brains are rapidly removed


Whole brain minus cerebellum is weighed and homoginized 1:9 vol of ice-cold 0.32M sucrose


The supernatant is decanted and used for uptake experiments
(Centrifuge at 1000g at 0-4
O
C for 10 min)
Assay procedure
200l tissue suspension + 800l Krebs-Hanseleit bicarbonate buffer + [
3
H]-5-HT


Incubate at 37
O
C under 95% O
2
:5%CO
2
for 5min


Supernatant is aspirated and pellets dissolved by adding 1ml of solubilizer (Triton X-100 and
50% ethanol, 1:4 v/v)


Counted in scintillation counting cocktail
(Centrifuge at 4000g at 0-4
O
C for 10 min)
Adult white leghorn chicken


The animal is grasped with both hands (left hand slightly pushes the chicken down and the right
hand supports the animal from the ventral side)


Immediately the chicken is turned on its back and hold for 1min


The chicken remains in cataleptic state for several min up to 1hr
(it may be interrupted by noise or fast movements of the observer)


The animals are pretested in order to be sure about cataleptic activity


After control experiment the animals are treated with test compound or vehicle


The test is performed 4 times every 30 min during 2 h
Evaluation
S.Mice (20-25g) brought to laboratory at least one day before the experiment


Forced to swim inside a vertical plexiglass cylinder
(30cm height with 20cm diameter containing 15cm water at 25
O
C)


After initial 2-3 min activity the phase of immobility or floating increases


After 5-6 min the immobility reaches to plateau


After 15 min in water the animals are taken out and dried in a heated enclosure (32
O
C)


After 24h the total immobility time is recorded during 5 min test for each animal
(test drug or standard are administered 1 h prior to testing)
Evaluation
S.Mice (20-25g) brought to test area at least 1 h before the experiment


Test animal is suspended on the edge of the shelf about 58cm above the table top with adhesive
tape placed approximately 1cm from the tip of the tail.


The duration of immobility recording is for 5min
(considered immobile when they hand passively and completely motionless for at least 1 min)
Animals exposed to inescapable and unavoidable electric shocks in one situation later
fail to escape shock in a different situation when escape is possible
Male SD rats (250 to 300g)


Exposed to electric shock (0.7mA) for 1 h on a
schedule of 10s shock/min.
(Platform not available during training)


During test the platform is attached
Grid floor Platform for escape
0.4mA shock is initiated for 10s if animal not escape
If escape occurs the animal is allowed to rest on platform for 10s and then returned to the grid
floor
10 trials in 20s interval
SD rats (250-300g) are isolated for 6 weeks in individual cages
(Free access to food and water)


After 6 weeks one mouse is introduced into the rats cage


About 10-30 % of rats kill the mouse by biting the animal through the cervical cord


Only rats consistently killing mice within 5 min after presentation are used for the test


Drugs are injected to the rats before the test


Mice are presented 30,60 and 120 min after drug administration
Antidepressants block the re-uptake of biogenic amines into nervous tissue. The toxic
effect of norepinephrine are potentiated.
Mice (20-25g)


Test drugs or standard are administered 1h prior to the administration of sub-lethal dose of
3mg/kg of noradrenaline


The mortality rate is assessed 48h post-dosing


ED50 or dose which causes death of 50% of treated animals is calculated
Apomorphine induces emesis in humans but in rodents it causes compulsive gnawing
instead of vomiting. This is due to dopaminergic stimulation.
Therefore many compounds with psychotropic activity are known to have an
apomorphin-synergistic effect. TCAs also enhance this behavior.
Mice (20-25g)


10mg/kg apomorphine at the same time treated with test drug or vehicle if i.p. or s.c.
(for p.o. activity the drug is administered 30min prior to apomorphine)


Immediately after apomorphine the mice are kept in cages having corrugated paper


The mice start biting the paper causing fine holes or tearing the paper
(Mice are kept for 1h in cage)


This behavior is enhanced by antidepressants
Evaluation
Apomorphine induces hypothermia in mice which can be prevented by
antidepressants
Mice (20-25g)


1 h after oral administration of test or vehicle a dose of 16mg/kg apomorphine in injected s.c.


The rectal temperature of each mouse is measured with electronic thermometer immediately
prior to apomorphine administration and 10, 20 and 30 min later.



Evaluation
Olfactory bulbactomy in rats is associated with changes in exploratory behavior that
are reversed by chronic , but not acute treatment with antidepressants.
W.Rats (200-250g)


Skull is exposed and a burr hole is drilled at points 7mm anterior to bregma and 2mm either side
of midline


The olfactory bulbs are removed by suction and the holes are filled with sterile hemostatic
sponge. (Sham surgery is also carried out)


The wound is closed with due care and application of antibiotic and stitched


Animals are allowed to recover for 14days after surgery


Animals are treated with test drug or standard or vehicle at 09:00 daily for 14 days
The behavior is tested 12
th
day onwards in open-field apparatus


Ambulation (No. of squares crossed), Rearing (forepaws raised from the floor), grooming and
defecation scores are recorded for 3min period observation.
Evaluation