This document discusses various screening models used to test potential antidepressant drugs. It begins by defining depression and its symptoms. It then discusses several theories of depression's mechanisms. Various in vivo animal models are described for screening antidepressants, including the forced swim test, tail suspension test, and learned helplessness test. The document provides details on procedures and evaluations for these and other models, such as amphetamine potentiation and NE toxicity tests in mice. The goal of screening models is to see how potential drugs correlate with known antidepressants' clinical efficacy and pharmacological effects.

1. ANTIDEPRESSANTS
SCREENING MODELS
Mohd Riyaz Beg| M. Pharm (Medicinal Natural Products), Institute
of Chemical Technology, Mumbai

2. CONTENTS TO DISCUSS
1. What is Depression?
2. Symptoms of Depression.
3. Possible mechanisms behind
Depression.
4. Drugs used for treatment of
Depression.
5. Why do we require screening models?
6. Different kinds of Screening models
7. Different in-vivo Screening models
8. Discussion on various screening
models.

3. WHAT IS DEPRESSION
By APA: Depression (major depressive disorder) is a common and serious
medical illness that negatively affects how you feel, the way you think and
how you act.
Depression is the most common of the affective disorders (defied as
disorders of mood); it may range from a very mild condition, bordering on
normality, to severe (psychotic) depression accompanied by hallucinations
and delusions.
It is a mental disorder, in which the patient is characterized by pervasive and
low mood with low self-esteem and the patient may loss interest in daily
social life and activities, sad mood, tiredness, guilt, sense of worthlessness,
lack of concentration and suicidal thoughts.
Worldwide, depression is a major cause of disability and premature death. In
addition to the significant suicide risk, depressed individuals are more likely
to die from other causes, such as heart disease or cancer. Depression is a
heterogeneous disorder, with patients presenting with one or more core
symptoms, and depression is often associated with other psychiatric

4. WORLDWIDE STATISTICS OF
DEPRESSION

5. SYMPTOMS OF DEPRESSION
The symptoms of depression include emotional and biological
components.
Emotional symptoms include:
• low mood, excessive rumination of negative thought, misery,
apathy and pessimism
• low self-esteem: feelings of guilt, inadequacy and ugliness
• indecisiveness, loss of motivation
• anhedonia, loss of reward.
Biological symptoms include:
• retardation of thought and action
• loss of libido
• sleep disturbance and loss of appetite

6. SYMPTOMS OF DEPRESSION

7. TYPE OF DEPRESSION
There are two distinct types of depressive syndrome, namely unipolar
depression, in which the mood changes are always in the same
direction, and bipolar disorder, in which depression alternates with
mania.
1. Unipolar depression is commonly (about 75% of cases) non-
familial, clearly associated with stressful life events, and usually
accompanied by symptoms of anxiety and agitation; this type is
sometimes termed reactive depression. Other cases (about 25%,
sometimes termed endogenous depression) show a familial pattern,
unrelated to obvious external stresses, and with a somewhat different
symptomatology.
2. Bipolar disorder, which usually appears in early adult life, is less
common and results in oscillating depression and mania over a
period of a few weeks. It can be difficult to differentiate between mild
bipolar disorder and unipolar depression. Also, bipolar manic

8. CAUSES OF DEPRESSION
However, the exact cause is still not known. It can be caused by any
change in neurotransmitter levels such as changes in Norepinephrine,
Serotonin, Dopamine etc. and it can also cause by any physical or
environmental factor.
Genetics of a person also cause depression as some have a family
history of depression they are more prone to have depression.

9. SOME OTHER CAUSES

10. DIFFERENT THEORIES OF
DEPRESSION
1. THE MONOAMINE THEORY
2. NEUROENDOCRINE MECHANISMS
3. TROPHIC EFFECTS AND
NEUROPLASTICITY

11. GENERAL OVERVIEW ON VARIOUS
THEORIES
Simplified diagram showing mechanisms believed to be
involved in the pathophysiology of depression. The main PR
depressive pathways involve the hypothalamic–pituitary–
adrenal axis, which is activated by stress and in turn enhances
the excitotoxic action of glutamate, mediated by NMDA
receptors, and switches on the expression of genes that
promote neural apoptosis in the hippocampus and prefrontal
cortex. The antidepressive pathways involve the monoamines
noradrenaline (NA) and 5-hydroxytryptamine (5-HT), which
act on G protein coupled receptors, and the brain-derived
neurotrophic factor (BDNF), which acts on a kinase-linked

12. THE MONOAMINE THEORY
The monoamine theory of depression, fist proposed by Schildkraut in
1965, states that depression is caused by a functional deficit of the
monoamine transmitters, noradrenaline and 5-hydroxytryptamine (5-
HT) at certain sites in the brain, while mania results from a functional
excess

13. NEUROENDOCRINE MECHANISMS
Raised CRH levels are associated with stress and, in many cases,
depression is preceded by periods of chronic stress. The plasma
cortisol concentration is usually high in depressed patients.
Hypothalamic neurons controlling pituitary function receive
noradrenergic and 5-HT inputs, which control the discharge of these
cells. Hypothalamic cells release corticotrophin-releasing hormone
(CRH), which stimulates pituitary cells to secrete adrenocorticotrophic
hormone (ACTH), leading in turn to cortisol secretion.
CRH concentrations in the brain and cerebrospinal fluid of depressed
patients are increased. Therefore CRH hyperfunction, as well as
monoamine hypofunction, may be associated with depression.

14. TROPHIC EFFECTS AND
NEUROPLASTICITY
Depressive behaviour is often associated with a reduction in brain-derived
neurotrophic factor (BDNF) expression and treatment with antidepressants
elevates BDNF levels.
Lowered levels of BDNF or malfunction of its receptor, TrkB, plays a
significant role in the pathology of Depression.
Antidepressant treatment may reduce glutamate release and depress NMDA
receptor function. Changes in glutamatergic neurotransmission may also be
involved in depression.

15. ANTIDEPRESSANT DRUGS
Antidepressant drugs fall into the following categories
I) Inhibitors of monoamine uptake:
1. Selective serotonin reuptake inhibitors (SSRIs) E.g. Fluoxetine, Fluvoxamine, Paroxetine, Sertraline, Citalopram,
Escitalopram, Dapoxetine
2. Serotonin and noradrenaline reuptake inhibitors (SNRIs) E.g. Venlafaxine, Duloxetine
3. Tricyclic antidepressants (TCAs):
A. NA + 5-HT reuptake inhibitors: E.g. Imipramine, Amitriptyline, Trimipramine, Doxepin, Dothiepin,
Clomipramine
B. Predominantly NA reuptake inhibitors: E.g. Desipramine, Nortriptyline, Amoxapine, Reboxetine
II) Noradrenaline reuptake inhibitors (e.g. bupropion, reboxetine, atomoxetine).
The herbal preparation St John’s wort, whose main active ingredient is hyperforin: it has similar clinical efficacy to
most of the prescribed antidepressants. It is a weak monoamine uptake inhibitor but also has other actions
III) Monoamine receptor antagonists
Drugs such as mirtazapine, trazodone, mianserin are non-selective and inhibit a range of amine receptors
including α2 adrenoceptors and 5-HT2 receptors. They may also have weak effects on monoamine uptake.
IV) Monoamine oxidase inhibitors (MAOIs)
• Irreversible, non-competitive inhibitors (e.g. phenelzine, tranylcypromine), which are nonselective with respect
to the MAO-A and -B subtypes.
• Reversible, MAO-A-selective inhibitors (e.g. moclobemide).

16. SCREENING MODELS
Principles:
To see the relationship between the Clinical efficacy of known
antidepressants and their effects on various pharmacological test
models.
Motor activity of these tests also allow assessment of the specificity
of antidepressant activity by establishing a ratio between the
antidepressant dose and sedative or stimulant dose.

17. CLASSIFICATION OF SCREENING
MODELS
Antidepressant screening models
In-vitro
models
Inhibition
of NE
uptake in
rat brain
Inhibition
of
Dopamine
uptake in
rat striatal
In-vivo Models
Forced
swim test
Tail
suspension
method
Learned
helpless
ness in
rats
Muricide
behavio
r in rats
Amphet
amine
potentia
tion test
Cataleps
y
antagon
ism in
chicken
Potentia
tion of
NE
toxicity
in mice
Reserpi
ne
induced
hypothe
rmia.
Yohimbi
ne
toxicity
enhance
ment

18. VARIOUS IN-VIVO ANIMAL
MODELS FOR
ANTIDEPRESSANTS
SCREENING
Detail discussion

19. FORCED SWIM TEST
Principle
It was suggested by Porsolt et al.
• When rat or mice are subjected to force swim in a limited space with no way to escape
then a characteristic immobility develops in them after some time of forced swimming.
• The anti-depressants drugs decrease the duration of immobility. It is most widely used
method for screening of acute antidepressants.
Procedure
• Adult rats are allowed to swim in a cylinder with no escape filled with water at 25 ºC.
• When the rats are forced to swim in water initially is was hyperactive but approx.. 5
minutes later the activity slow down and the phase of immobility starts.
• After 15 minutes the rats were removed and allowed to dry. The duration of immobility
was measured.
• The same activity was done for standard and test groups and drug was administered 1
hour earlier when the test starts.

20. CONTINUED…
Evaluation
• The duration of immobility was measured for test, control and
standard groups treated with various drugs.
• The antidepressants drugs decrease the duration of immobility.

21. TAIL SUSPENSION TEST
Principle
• When a rat is suspended by its tail, the immobility is displayed because of
inescapable stress.
• It reflects behavioural despair. The antidepressants drugs decrease the
immobility in a tail suspended rat.
Procedure
• Three group of rats are divided and proper food and water are given.
• Control, test and standard groups are divided and are subjected to respective
drugs via IP route.
• The rats are suspended upside down through its tail. At the start of test, the
rat try to escape but was unable and become immobile after some time.
• The readings were taken for 6 minutes by use of a camera and computer and
the time for activity and immobility was recorded and compared with test and
standard groups.

22. CONTINUED…
Evaluation
• The duration of immobility of standard and test was compared with
control groups and the decrease in duration of immobility was
calculated.
• For different drugs, ED50 was calculated.

23. LEARNED HELPLESSNESS TEST
Principle
• The rats are exposed to an unavoidable electrical shock for some time and after
that, the animal was unable to escape in a situation where escape is possible.
• An electrical grid box was constructed and rat was placed in it and shock was give
(0.7mA) for 1 hour every 10 seconds per minute interval. A 30 to 80% learned
helplessness behaviour was calculated. Antidepressants decrease the escape
failure.
Procedure
• Sprague Dawley rats of weight 250-300gm are used for this test. An electrical
grid box was constructed and rat was placed in it and shock was give (0.7mA) for 1
hour every 10 seconds per minute interval. A platform was not given during the
training of rats.
• After the training, a platform was inserted into the grid and the drugs are given
and a shock of 0.4mA was given for 10 seconds if the rat did not escape to
platform.
• If any escape response occur, the animal was allowed to the platform for 10
seconds and then it was returned to the grid. Readings were taken for 10 responses

24. CONTINUED…

25. CONTINUED…
Evaluation
• The test and standard groups reduce the learned helplessness in
comparison with the control group.
• The number of escape increases and the drug was considered
effective antidepressants.

26. MURICIDE BEHAVIOR IN RATS
Principle
• Some rats have behaviour of mouse killing when they were isolated for some time. This is
called as Muricide behaviour in rats.
• Pre-treatment with Pilocarpine can induce Muricide behaviour. The rats, which consistently
kill mouse for 5 minutes, are used. The drugs, which cause inhibition in Muricide behaviour,
have anti-depressant action.
Procedure
• Male Sprague Dawley rats of 300-350gm are isolated for some weeks.
• Mouse was placed in the rat’s cage and 10-30% of rats kills the mouse by biting.
• The rats, which kill the mouse consistently for approx. 5 minutes, are used for test.
• Test and standard drug are injected respectively via intra peritoneal route before test.
• Mice was placed in the cage for about 30, 60 and 120 minutes and ED50 value was
calculated.
Evaluation
• If the rats fails to kill the mouse in 5 minutes then the drug is considered to inhibit in
Muricide behaviour.

27. SOMETHING EXTRA…
Female rats of Holtzman strain exhibit compulsive mouse killing
behaviour irrespective of their satiety status.
Nonmuricidal rats can be rendered muricidal by pre-treatment with
pilocarpine(2.5-5mg/kg, ip).

28. CATALEPSY ANTAGONISM IN
CHICKENPrinciple
• This test is a method of screening of antidepressants and is done on Leghorn chickens.
Procedure
• For this test, we select adult Leghorn Chickens and divide it into test, control and standard
groups.
• The chickens were pretested for cataleptic behaviour.
• The chickens were grasped and turned back and hold for 1-2 minutes.
• Due to this cataleptic numbness occurs in them.
• Hand was removed carefully and the animal remains in numbness. • By clapping hand
around its head, the chicken jumps and run away.
• The test and standard drugs were given and the above test was performed for 4 time every
30 minutes of 2 hours period.
Evaluation
• If the occurrence of cataleptic behaviour is less than the control group, the test is positive.
• For 2 hours period the cataleptic behaviour was interrupted

29. AMPHETAMINE POTENTIATION
TESTPrinciple
• This test is done to evaluate the exploratory and locomotor activity of rats.
• Amphetamine when given to rats which are previously treated with antidepressants, the
amphetamine effects were potentiated and are evaluated as locomotor activity.
Procedure
• Male Wistar rats of weight 200-300 g were taken for control, standard and test groups.
• They were kept at room temperature with 12-hour light and 12-hour dark cycle and were fed
with food and water.
• For a time of two weeks, the rats receive test and standard drugs respectively.
• When they receive last dose of drug 90 minutes later D-amphetamine (10mg/kg body weight)
is injected through i.p. route.
• After 30 minutes, they were placed back and their locomotor activity was recorded and
compared with control group.
Evaluation
• Evaluation is done on the basis of their locomotor activity.
• Locomotor activity of control group is lowest while that receiving test and standard have more

30. HYPERMOTILITY IN OLFACTORY
BULBECTOMIZED RATS
1. Bilateral olfactory bulbectomy in the rat is associated with changes
in exploratory behaviour that are reversed by chronic, but not
acute treatment with antidepressant drugs.
2. Male Sprague Dawley rats.
3. The animals are allowed to recover for 14 days after surgery.
4. The animals are treated s.c. with the test drug / standard / vehicle
once daily for 14 days.
5. The behaviour of the animals is tested from the 12th day onwards.
6. The rats are placed singly in the centre of an open field apparatus

31. TESTS BASED ON MECHANISM OF
ACTION

32. POTENTIATION OF NE TOXICITY IN
MICEPrinciple
• When we give antidepressants to an animal, it blocks biogenic amines and potentiate the
toxicity produced by Norepinephrine.
Procedure
• Male NMRI mice of weight 22-25g were chosen randomly and divided into the groups:
control, standard and test.
• The test group were treated with test drug, standard group were treated with standard
drug, and control was treated with vehicle all given orally.
• After 1 hour, a sub lethal dose of 3mg/kg body weight of NE is given via sub-cutaneous
route.
• They were kept under supervision for 48 hours.
• The mortality rate was calculated.
Evaluation
• For next 48 hours after the sub lethal dose of NE was given the mortality rate of mice
was assessed.
• The dose causing death of at least 50% of treated animal was calculated for all the
respected groups. ED50 value can also be calculated.

33. RESERPINE INDUCED
HYPOTHERMIAPrinciple
• The main biogenic amine are Noradrenaline, 5HT and Dopamine.
• When depletion of these occur it induce hypothermia in mice.
• Reserpine is given to induce Hypothermia and antidepressant antagonize its action.
Procedure
• Male NMRI mice of weight 20-25g were selected and grouped as test, control and standard
groups.
• A dose of 2mg/kg body weight of Reserpine was given to selected animals.
• Approx.18 hours later the animals were placed into their cages.
• With the help of an electric thermometer recorded the initial rectal temperature.
• Test, standard and vehicle compound were given to respective groups via IP route.
• The rectal temperature was recorded every 60 minutes for 7 hours.
• The difference in respective temperature is calculated.

34. CONTINUED…
Evaluation
• Evaluation is done on the basis of difference in recorded
rectal temperature.
• The maximum difference in recorded temperature is
calculated.
• Amphetamine and chlorpromazine can also induce
hypothermia

35. YOHIMBINE TOXICITY
ENHANCEMENT IN MICE
Principle
• Yohimbine is a selective alpha 2 blocker. It prevents binding of NE to alpha 2
receptors. The reuptake of NE to nerve terminals is blocked by
antidepressants.
• Yohimbine when given with antidepressants cause toxicity of NE and finally
death of animal.
Procedure
• Male NMRI mice of weight 22-25g were taken and grouped as control, test
and standard groups.
• Each mice was treated with standard, test and vehicle compound respectively
according to their groups.
• 30 minutes later, a sub lethal dose of 25mg/kg body weight of Yohimbine
was injected to each mice via sub cutaneous route.
• They were kept under supervision for 24 hours.

36. CONTINUED…
Evaluation
• After the dosing of Yohimbine the mortality of mice were assessed
for 1, 2, 3, 4, 5 and 24 hours.
• The mortality of test, standard and control groups were compared
after 24 hours. ED 50 value is calculated

37. IN VITRO ASSAYS

38. IN-VITRO ANTIDEPRESSANT
MODELS
Assays based on inhibition of amine uptake
Assays based on binding to the receptors
Though complicated but these are precise and accurate

39. LIST OF SOME MODELS
1. Inhibition of [3H] norepinephrine uptake in rat brain synaptosomes.
2. Inhibition of [3H] dopamine uptake in rat striatal synaptosomes.
3. Inhibition of [3H] serotonin uptake in synaptosomes.
4. Binding to monoamine transporters.
5. Measurement of β adrenoceptors stimulated adenylate cyclase.
6. [3H] yohimbine binding to α2 adrenoceptors in rat cerebral cortex.

40. INHIBITION OF NE UPTAKE IN RAT
BRAINPrinciple
• The reuptake of NE is an important physiological process.
• It is important for removing NE in synaptic cleft.
• Antidepressants inhibits the reuptake of NE.
• Hypothalamus is used for this as it has shown great uptake of NE.
Procedure
Tissue preparation
• Male Wistar rats were taken in groups.
• They were decapitated and their brain are removed rapidly.
• The hypothalamus is weighed and its preparation is made.
• Homogenization of this is done by using ice-cold sucrose solution 0.32M 9 volumes with the
help of POTTER- ELVEJHEM HOMOGENIZER.
• Centrifugation is done at 1000g at 0-4 ºC for 10 minutes and supernatant is decanted which is
used for experiment.

41. CONTINUED…
Assay • 800µl 62.5 Nm NE and 200µl of tissue suspension were mixed and incubated in
KREBS-HENSELEIT BICARBONATE BUFFER at temperature 37ºC with 20µl test or standard
drug or vehicle in control.
• Incubation of each assay tube is done at 0ºC ice bath with 20µl vehicle at 95% O2 and 5%
CO2.
• Centrifugation is done for 10 minutes at 4000g.
• After that, firstly, the supernatant is aspirated and then pellets were dissolved in
solubilizer.
• This was shaken and then decanted into SCINTILLATION VIALS.
• This was counted in 10 ml liquid scintillation counting cocktail.
• The difference between cmp at 37ºC and 0ºC is the active uptake.
Evaluation
• The mean of at least three determination is calculated and this is the percent inhibition at
each drug concentration.
• From log probit analysis IC50 values can be determined.
• DESIPRAMINE is a standard drug with IC50 value of 20nM.

42. INHIBITION OF DOPAMINE UPTAKE IN
RAT STRIATALPrinciple
• Dopamine has sodium dependent transport of high affinity in various tissue
preparation.
• Dopamine is found in high amount in Striata.
• Antidepressants and cocaine inhibit the uptake of dopamine.
Procedure
Tissue preparation
• Male Wistar rats were taken in groups.
• They were decapitated and their brain are removed rapidly.
• The Corpora Striata is weighed and its preparation is made.
• Homogenization of this is done by using ice-cold sucrose solution 0.32M 9 volumes
with the help of POTTER- ELVEJHEM HOMOGENIZER.
• Centrifugation is done at 1000g at 0-4 ºC for 10 minutes and supernatant is decanted
which is used for experiment.

43. CONTINUED…Assay
• 900µl 55.5 Nm Dopamine and 100µl of tissue suspension were mixed and incubated in
KREBS-HENSELEIT BICARBONATE BUFFER at temperature 37ºC with 20µl test or standard
drug or vehicle in control.
• Incubation of each assay tube is done at 0ºC ice bath with 20µl vehicle at 95%O2 and 5%
CO2.
• Centrifugation is done for 10 minutes at 4000g.
• After that, firstly, the supernatant is aspirated and then pellets were dissolved in 1 ml
solubilizer which is 1:4 ratio of Triton X-100 + 50% ethanol.
• This was shaken and then decanted into SCINTILLATION VIALS.
• This was counted in 10 ml liquid scintillation counting cocktail.
• The difference between cmp at 37ºC and 0ºC is the active uptake.
Evaluation
• The mean of at least three determination is calculated and this is the percent inhibition
at each drug concentration.
• From log probit analysis IC50 values can be determined.

44. BASIC PROCEDURE OF RECEPTOR
BINDING ASSAY
Isolate the cells with receptors
Add radioactive ligand for that receptor in presence and absence of
test drugs
Count the receptor ligand binding by liquid scintillography.

45. CONCLUSION
After getting insight of all the generally used models we can conclude
that Depression is a heterogenous ailment and having very complex
etiology and mechanism. Therefore, neither of the model set
satisfactorily for all types of depression and not reproduce the human
major depressive illness.
These model may show good predictive validity but poor in construct
validity.
Also these models don’t have a clear face validity.

46. THANK YOU!
Presented by: Mohd Riyaz
Beg | M. Pharm Scholar,
Institute of Chemical
Technology, Mumbai
mohdriyazbeg@chemist.co
m
https://mirzariyazbeig.blog
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