This document summarizes screening methods for assessing anxiolytic activity. It describes several models used to test anxiety in rodents, including the light-dark test, elevated plus maze test, social interaction test, marble burying test, and Vogel conflict test. Each method exposes animals to an anxiety-provoking stimulus and measures behaviors like time spent in unprotected areas or number of marbles buried. Anxiolytic drugs are expected to reduce anxious behaviors in these tests. The document also provides details on procedures, parameters measured, and interpretation of results for each screening method.

1. Utkarsh Alok
First year ,M.pharm
Department of Pharmacology
RIPS,BERHAMPUR
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SCREENING OF
ANXIOLYTIC ACTIVITY

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Anxiety is an emotional state commonly caused by the perception
of real or perceived danger that threatens the security of an
individual.
 Episodes of mild anxiety are common life experiences and do not
warrant treatment. However, the symptoms of severe, chronic,
debilitating anxiety may be treated with antianxiety drugs
(sometimes called anxiolytic or minor tranquilizers) and/or some
form of behavioral or psychotherapy.
Introduction

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Physiological and Pathological Anxiety
NORMAL
•Jitter
•Stage fright
•Nervousness
•Worrying
Pathological
•Panic attack
•Obsession,compulsion
•Flashbacks,nightmares
•Pathological fears

4. CLINICAL PRESENTATIONCLINICAL PRESENTATION
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 ANXIETY DISORDER I
GAD (Generalized Anxiety Disorder)
PD (Panic Disorder)
Agoraphobia (fear of open or public places)
SAD (Social Anxiety Disorder)
Specific Phobia
 ANXIETY DISORDER II
Obsessive Compulsive Disorder
Post Traumatic Stress
Acute Stress

5. GENERALISED ANXIETY DISORDERGENERALISED ANXIETY DISORDER
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 PHYSIOLOGIC & COGNITIVE SYMPTOMS
Excessive Anxiety
Worries that are difficult to control,poor concentration or mind going
blank
 PHYSICAL SYMPTOMS
 Sleep disturbance
 Irritability
Restlessness
PANIC DISORDERPANIC DISORDER
•Unexpected panic attacks associated with physiological symptoms of
autonomic nervous system.
•Intensive fear
•Phobias
•Avoidance of places and situations where panic attacks occurred.

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 .
SOCIAL ANXIETY DISORDERSOCIAL ANXIETY DISORDER
•Intense, irrational and persistent fear of being negatively evaluated in
at least one social or performance situation.
•Blushing is the principal physical indicator
SPECIFIC DISORDERSPECIFIC DISORDER
Marked and persistent fear of a circumscribed object or situation (e.g.,
insects, heights, or injections).
POST TRAUMATICSTRESSPOST TRAUMATICSTRESS
DISORDER( PTSD)DISORDER( PTSD)
•Develops subsequent to experiencing or witnessing a traumatic event.
•Symptoms (lasting at least 4-6 weeks ) : flash-backs, memories, nightmares

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OBSESSIVE-COMPULSIVE DISORDER (OCD)OBSESSIVE-COMPULSIVE DISORDER (OCD)
• Obsessions (recurrent, intrusive and generally distressing
thoughts, images or feelings).
• Compulsions(repetitive,ritualistic behaviors aimed to alleviate
obsessions

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Modulation of normal and pathologic anxiety states is associated with multiple regions of
the brain and abnormal function in several neurotransmitter systems,which includes:-
norepinephrine (NE)
 γ –aminobutyric acid (GABA)
 serotonin (5-HT)
Neuroanatomic models of fear (i.e., the response to danger) and anxiety (i.e., the feeling
of fear that is disproportionate to the actual threat) include some key brain areas.
Amygdala,(a temporal lobe structure,) role in the assessment of fear stimuli and
learned response to fear.
The locus ceruleus (LC), located in the brain stem, is the primary NE-containing
site in the brain, with widespread projections to areas responsible for implementing fear
responses .
The hippocampus is integral in the consolidation of traumatic memory.
The hypothalamus is the principal area for integrating neuroendocrine and
autonomic responses to a threat.
PATHOPHYSIOLOGY

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CLASSIFICATION
OF ANXIOLYTICS
AND HYPNOTICS
DRUGS

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SCREENING METHODSSCREENING METHODS

11. SCREENING METHODS OF ANXIETY I
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Methods based on unconditioned (spontaneous) response:
Exploratory activity
 elevated plus-maze
 light-dark (two compartment box)
Social behavior
 social interaction
Methods based on conditioned (learned) response
Conflict models
 Vogel punished drinking
SCREENING METHODS OF ANXIETY
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Normal (adaptive) anxiety
Elevated plus-maze
Social interaction
Light-dark

12. SCREENING METHODS OF ANXIETY 2
contd..
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Stress-induced anxiety
Vogel lick conflict
Pathological anxiety
Neurochemically-induced anxiety.
mcpp induced anxiety in rats.

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ANTI-ANXIETY TEST

14. LIGHT-DARK MODEL IN MICE AND RATS
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Purpose and Rationale
 Animals are placed in a two chambered systems, where they can freely move
between a brightly –lit open field and a dark corner.
 After the treatment with an anxiolytic they show more crossings between the
two chambers and more locomotor activity.
The number of crossings between the light and dark sites is recorded.
METHODOLOGY:
The apparatus consists of a dark and a light chamber which are divided by a
photocell equipped zone.
A polypropylene animal cage (44 21 21 cm) is darkened with black spray overˣ ˣ
one-third of its surface.
A partition containing a 13cm(l) ,5 cm (h) opening is used for separating the
dark one-third of the cage.
This case rests on an activity monitor which counts total locomotor activity.

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Another electronic system consisting of
four sets of photocells across the partition
It automatically counts movements
through the partition and records the time
spent in the light and dark compartments.
Experiments are conducted on mice or
rats.
 They are treated 30 min before the test
drugs or vehicle given i.p. placed in the
cage and observed for 10 min.
 Groups of 6-8 animals should be used for
each dose.

16. ELEVATED PLUS MAZE TESTELEVATED PLUS MAZE TEST
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PRINCIPLE: proposed for selective identification of anxiolytic and
anxiogenic drugs. Anxiolytic compounds,by decreasing anxiety, increase the
open arm exploration time; anxiogenic compounds have the opposite
effect.
• Dose response curves are plotted & number of crossings through the
partition between the open and the closed arm are compared with total
activity counts during the 10 min.
• It has been reported that anxiolytics like diazepam and meprobamate
produce a dose dependent facilitatory effect whereas the non anxiolytics are
not effective in this model.
•Two open and two enclosed arms (50 ˣ
20 40cm dimensions).ˣ
•Open roof arrangement
•Two open arms are opposite to each
other

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The rats weighing around 200g are housed in pairs for 10 days prior to testing.
 Six rats are taken for each test group.
 The test drug is administered 30 min prior to experimentation by i.p route.
The rat is then placed in the center of the maze facing one of the enclosed arms.
PROCEDURE:
Parameters Measured During Next
5minutes:
•time spent in the open arms
•entries into the open arms
•time spent in the closed arms
•entries into the closed arms
•total arm entries
Anxiolytic effect indicated by:
•increase in the proportion of time spent in the open arms (time in
open arms/total time in open or closed arms)
•increase in the proportion of entries into the open arms (entries
into open arms/total entries into open or closed arms).

18. SOCIAL INTERACTIONSOCIAL INTERACTION
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Procedure :
Male Sprague-Dawley rats (225–275 g body
weight) are housed in groups of 5 animals.
Apparatus used is Perspex open-topped
box (51 × 51 cm and 20 cm high) with 17 × 17
cm marked areas on the floor.
Two naive rats from separate housing cages are
treated with the test compound orally 1 hr before
the test.
They are placed into the box (with 60 W bright
illumination 17 cm above) and their behavior is
observed over a 10-min period by remote video
recording
Source of anxiety: Presence of an unfamiliar social partner in a brightly lit
environment.

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Parameters measured : exploration, sniffing, rearing, social contacts, sexual
behavior, attack, fighting, biting ,defensive posture, immobility and climbing over
the partner.
Anxiolytic effect:In an unfamiliar and brightly lit environment, the normal
social interaction of rats is suppressed. Anxiolytics counteracts this suppression.
EVALUATION:
The values of treated partners are compared with the data from 6 pairs of
untreated animals using single factor analysis of variance followed by Dunnett’s
t-test.

20. MARBLE BURYINGMARBLE BURYING METHODMETHOD
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Source of anxiety : Presence of
an unfamiliar objects (potential
source of danger)
Parameters measured:duration of
burying of marbles.
Purpose and rationale:
The rats develops a peculiar behavior when
shocked through a stationary prod by burying
the shock source.

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PROCEDURE
Male adult rats (250-400g) are used.
Test performed in a chamber, covered with 5 cm of a commercial bedding
material.
In the centre a shock-prod is inserted through a small hole and is buried in the
bedding material.
Electric current is provided through the wires wrapped around the prod-the
behavior of each rat is monitored for 15 minutes.
The rat is injected with the test drug i.p and placed into the test chamber.
When the rat first touches the prod with its forepaw, it receive a brief electric
shock (1mA) which typically elicits a flinch away from the prod.
Afterwards, the rat moves towards the prod pushing the bedding material
with its forepaws-the duration of burying is recorded.
Anxiolytic effect:Anxiolytics shortens the burying behavior dose-
dependently at low shock intensity (1 mA).

22. VOGEL LICK-CONFLICT (VOGEL PUNISHED
DRINKING)
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Source of anxiety: stressful situation
(48h water deprivation) conflict between
thirst and punishment after drinking
Parameters measured: number of
accepted punishment (electric shock).
Anxiolytic effect: statistically significant
increase in the accepted shocks.
PROCEDURE :
•The apparatus is a clear Plexiglas box (38
× 38 cm) with a black Plexiglas
compartment (10 × 10.5 cm) attached to
one wall and an opening from the large box
to the small compartment, with a stainless-
steel grid floor.
•A water bottle with a metal drinking tube is fitted to the outside of the small
compartment, so that the tube extends into the box at a height 3 cm above the grid.

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•A water bottle with a metal drinking tube is fitted to the outside of the small
compartment, so that the tube extended into the box at a height 3 cm above the grid.
•A drinkometer circuit is connected between the drinking tube and the grid floor of
the apparatus, so that the rat completes the circuit whenever it licks the tube.
•Shock is administered to the feet of the animal by switching the connections to the
drinking tube and grids from the drinkometer to a shocker.
• Thirty min after intraperitoneal injection, the naive adult rat is placed in the
apparatus and allowed to find the drinking tube and to complete 20 licks before shock
(available at the tube for 2 s)is administered.
•The rat controls shock duration by withdrawing from the tube.
•A 3-min timer is automatically started after the termination of the first shock.
During the 3-min period, shocks are delivered following each twentieth lick.
• The number of shocks delivered during the 3-min session is recorded for each
animal.
EVALUATION:
The number of shocks received after treatment is compared with untreated animals.
Benzodiazepines increase dose-dependent the number of shocks.

24. mCPP INDUCED ANXIETY IN RATSmCPP INDUCED ANXIETY IN RATS
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mCPP - metabolite [ 1-(3-chlorphenyl) piperazine] of antidepressant drug
trazodone.
 mCPP induces hypophagia and hypolocomotion , inhibits social interaction in
rats, diminishes exploratory activity of rats in the open field test and in the light-
dark box test etc.
 Antagonism of these symptoms has been used for the screening of anxiolytic
drugs.
Parameters measured : time spent in both side (horizontal, vertical activity)
frequency of motion, number of transition
Anxiolytic effect : statistically significant increase in parameters measured in
the light/dark box or in number of transition

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PROCEDURE:
Male Sprague Dawley rats (200-
250g) are housed in groups of six
exposed to 12 h light/dark cycle
with free access to food and water.
 Test compound or vehicle are
administered orally 1h or i.p 30
min before the locomotion test.
 mCPP is injected i.p. in a dose of
7 mg/kg 20 min before the test.
The animals are placed individually
in an automated locomotor activity
cages and locomotion is recorded
for 10 min.
Anxiolytic effect : Disinhibition of locomotion.

26. IN-VITRO METHODIN-VITRO METHOD

27. Benzodiazepine receptor: [3H]-
flunitrazepam binding assay
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PURPOSE AND RATIONALE
 [3H]-flunitrazepam have specific binding sites in CNS membrane preparations
that satisfy the criteria for pharmacological receptors, e.g. saturability,
reversibility, stereo selectivity and significant correlation with in vivo activities
of the antianxiety drugs
PROCEDURE:
Reagents
 [Methyl-3H]-Flunitrazepam.
 Clonazepam HCl
Tissue preparation
 Male Wistar rats are decapitated and the brains rapidly removed.
 The cerebral cortices are removed, weighed and homogenized with a
Potter-Elvejhem homogenizer in 20 volumes of ice-cold 0.32 M sucrose.
 This homogenate is centrifuged at 1000 g for 10 min. The pellet is discarded
and the supernatant is centrifuged at 30 000 g for 20 min.
 The resulting membrane pellet is resuspended in 40 volumes of 0.05 M Tris
buffer.

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Assay
1 ml 0.05 M Tris buffer, pH 6.9
560 l Hμ 2O
70 l 0.5 M Tris buffer, pH 6.9μ
50 l 3H-Flunitrazepam 20 l vehicleμ μ
 0.1 mM Clonazepam
300 l tissue suspensionμ
 The tubes containing [3H] –flunitrazepam , buffer, drugs and water are then
incubated at 0–4 °C in an ice bath
 A 300µl aliquot of the tissue suspension is added to the tubes at 10 sec intervals.
 The tubes are then incubated at 0-4ºC for 20 min and the assay stopped by
vacuum filtration through Whatman filters. This step is performed at 10 sec
intervals.
 Each filter is immediately rinsed with three 5ml washes of ice-cold buffer.
 The filters are counted in 10 ml of liquid scintillation counting cocktail.
EVALUATION
 The percent inhibition at each drug concentration is the mean of triplicate
determinations.
 IC50 calculations are performed using log-probit analyses.

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SCREENING OF ANTISTRESS
ACTIVITY

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STRESS
Stress represents reaction of a body to stimuli that tends to disturb its normal
physiological equilibrium or homeostasis.
Common symptoms of stress include problems with sleep, depression,
anxiety, fatigue, and lethargy.
BEHAVIORAL DEPRESSION
Generalized and chronic stress induces behavioral
depression.
Methods used to assess depressive behavior are:
•SWIM STRESS INDUCED ‘BEHAVIORAL
DESPAIR’
•LEARNED “HELPLESSNESS” TEST

31. SWIM STRESS INDUCED "BEHAVIORAL
DESPAIR"
Purpose and Rationale:
 Rats are forced to swim in a restricted
space from which they cannot escape
induce a characteristic behavior of
immobility
 This behavior is a state of despair which
can be reduced by antistress agents.
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METHOD
Male Sprague- Dawley rats(160-180g)are
individually forced to swim inside a vertical
cylinder
Rats are initially highly active
After 2-3 min activity begins to subside
immobility or floating
After 5-6 min, immobility reaches a plateau

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 After 15 min animals are removed, dried& returned to cages.
 Again after 24 hour the test is repeated and immobility is measured during a 5
minute test .
 Animal is judged to immobile when animal remains floating passively in water
with head in upright position and nose just above surface.
 The test drug is administered 1hr prior to the test
Evaluation:
Duration of immobility is
measured in control and test.
A decrease in immobility
time in comparison to the
control is measured.
Antistress effect: Statistically
significant decrease in immobility.

33. LEARNED “HELPLESSNESS” TEST
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PURPOSE AND RATIONALE
Animal is exposed to inescapable & unavoidable electric shock in one situation later
fail to escape shock in a different situation when escape is possible
METHOD
 Male Sprague –Drawly rats(300g) is used.
 Learned helplessness is produced by exposure to electric shock 0.7mA; 10s
of shock/min repeatedly given for 1 hr.
 A box with grid floor is taken.
At 20cm height above the floor ,a platform can be inserted through one side wall to
allow jump up escape.
The platform is not available during training
 At the beginning of trial, after the appropriate treatment with the drug, the
platform is pushed in the box and a 0.4mA shock initiated.
 If an escape response occur, animal is allowed to be on the platform for 10 sec
and then returned to the grid floor.
 10 trials with an interval for 20 sec are given.
EVALUATION:
A drug is considered to be effective if the animal helplessness is reduced and the
number of failures to escape is decreased.

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