SCREENING METHODS

OF ANTI ANXIETY
DRUGS
Dr. Jitendra Agrawal
Second year resident

Introduction
o Anxiety is an emotional state caused by the perception of
real or perceived danger that threatens the security of an
individual.
It is a normal human adaptive response to stressful events.
Physiological anxiety transient in nature
Pathological anxiety needs treatment

Physiological and Pathological

Anxiety
Normal

Pathological

Jitter

Panic attacks

Stage-fright

Obsessions,

Nervousness
Worrying

compulsions
Flashbacks, nightmares
Pathological fear

Pathophysiology of anxiety
o Neurotransmitters like GABA, noradrenaline,
serotonin abnormalities anxiety
o Amygdala, temporal lobe, hippocampus and
hypothalamus - involved in anxeity
o Neurochemical theories :
1. Noradrenaline theory
2. Serotonin theory
3. GABA receptor theory

Noradrenaline theory
o ANS of anxious patients- hypersensitive to stimuli.
o Locus coeruleus activates epinephrine release
o Anxiogenics stimulate locus coeruleus firing
o Anxiolytics- inhibits locus coeruleus firing and
decrease noradrenaline activity.

GABA Receptor Theory

o GABA inhibitory neurotransmitter in brain.
o Has inhibitory and regulatory effects on serotonin,
noradrenaline and dopamine.
o GABAA receptor involved in anxiety; decreases
neuronal excitability
o Patients suffering from anxiety disorders have less
level of GABA in cortex.

Serotonin Theory
Abnormalities in serotonin function i.e., release
and uptake plays role in anxiety.
Greater serotonin activity reduces
norepinephrine activity in locus cerulus.
SSRIs increases serotonin levels post
synaptically blocks symptoms of anxiety.

Classification of anxiolytics

Benzodiazepines
alprazolam, chlordiazepoxide , diazepam
Azapirones
Buspirone, Ispapirone, gepirone
SSRI
Citalopram ,Escitalopram ,Fluoxetine
Beta blockers
Propranalol
Sedative antihistaminic
Hydroxyzine

reening methods o
anxiolytics

ANIMAL MODEL FOR ANXIETY

Must add new insight or new dimension to the
process of human anxiety
Should reproduce behavioural and pathological
features of anxiety
Should allow investigation for neurobiological
mechanism that are not easily amenable to study
in man
Permit reliable evaluation of anxiolytic drugs as
well as anxiogenic drugs

 All animal model of anxiety are common in

Induced fear as an analogy to human
anxiety and to give them a basic construct
validity
The degree to which they can be taken as
model of human anxiety is based upon their
response to BZDs

BEHAVIOURAL MODEL
Exteroceptive stimuli model
Novel environment

Open field test

Elevated plus maze
Staircase exploration
Black and white test box
Mirrored chamber

Conditioned aversive procedures

Conflict Procedures
Geller seifters conflict test
Vogels conflict test

Escape avoidance procedure

Social interaction test

 Interoceptive stimuli model

Electrical stimulation of brain
Pharmacological manipulation (drug discrimination
test)

Caffine induced anxiety

Yohimbine induced anxiety
Flumazenil induced anxiety
Pentylenetetrazole induced anxiety
Amphetamine induced anxiety
Cacaine induced anxiety

Foot shock induced agression

Isolation induced agression

Open field test

This behavioral model is base on the induction of anxiety
state ambulation or freezing by exposing the animal to a
highly novel field environment of a high sound and light
As a result their exploratory behavior is inhibited
Open field apparatus consist of circular arena with high
sound and light sources
Simple sterotypy: when animal move from one segment
to another one ambulation score
Complex sterotypy: when animal stands on its hind
limbs- one rearing score
Urination
defecatioN

Elevated Plus Maze Test

o Most widely used method; male mice used
o Anxiolytics decrease anxiety
o increase open arm exploration time

Elevated plus maze

o 2 open arms and 2 closed
arms of 50 10 40cm
dimensions
o Open roof arrangement
o Two open arms are opposite
to each other.
o Maze elevated at 50cm
height.

 Parameters Measured During Next 5

minutes:
o time spent in the open arms
o entries into the open arms
o time spent in the closed arms
o entries into the closed arms
o total arm entries

 Anxiolytic effect indicated by:

o increase in the proportion of time spent in
open arms
o increase in the proportion of entries into open
arms

Staircase exploration
When introduced into a novel environment, rodents
experience a conflict between anxiety and exploratory
behavior manifested by increased vigilance and
behavioral activity
Staircase climibing reflects exploratory or locomotor
activity
Rearing behaviour is an index of anxiety state
The no. of rearing and steps climbed to be recorded
for period of 5 minutes
Decrease in rearing behaviour and increase in steps
climbed is characterisation of anxiolytic effect

Light dark model

o Animals placed in 2 chambered systems, where
they can freely move between a bright and dark
compartment
o Number of crossings between the light and dark
sites is recorded.

o Apparatus - a dark and a

light chamber divided by a
photocell equipped zone.
o This case rests on an activity
monitor which counts total
locomotor activity.

o No. of crossings through the partition

between the light and dark chambers compared
with total activity counts during the 10 min.
o Loco motor activity also monitored.
o Anxiolytics increase locomotor activity and
no. of crossings.

Mirrored chamber
Novel stimulation evokes both exploration and anxiety
and therefore generates approach avoidance conflict
behaviour
It is hypothesised that distortion of readily traversed
environment by a chamber of mirror might produce
aversion to entry
Mice are exposed to the chamber of mirror
Extended latency to enter the chamber of mirror used as a
parameter for anxiety analogy
Anxiolytics reduce this latency in dose dependent manner.

Geller seifters conflict test

Conflict is produced by availability of
reinforcement with punishment
Experimentally induced conflict by
punishing food rewarded behavior
has been used to differentiate
between various psychoactive drugs
by Geller and Seifter

Geller seifters conflict test

Male albino rats with a body weight of 300400 g
are housed individually.
They are food deprived until the body weight is
gradually reduced by approximately 20% of
original and it is maintained at this level by
restricted food diet.
Conditioning is carried out with a flash of light, a
single lever, a liquid dripper, and a grid-floor
connected to a shocker
The animals are trained to lever press for the milk
reward in two distinct response-reward sections.

Geller seifters conflict test

Anxiety or conflict segment (3 min)
a dipper of milk is delivered in response to
each lever press (continuos reinforcement
schedule =CRF),
accompanied by aversive foot-shock
through the grid floor
Creates a conflict between milk reward and
the a painful foot shock

Geller seifters conflict test

nonconflict segment (15 min)
lever presses produce a drop of milk
only at variable intervals of time
from 60 to 210 s with an average
reward of once per 2min
No shocks are administered

Geller seifters conflict test

Four cycles of 15 min nonshock variable
interval segments followed by a three
minute CRF-conflict period phase
The total number of lever presses during
the conflict periods (CRF) and the nonconflict periods (VI) are counted.
Anxiolytic effect :An increase of lever
presses in the conflict periods

Vogel Lick-conflict (Vogel

Punished Drinking)
Rats are deprived
of water for 48 hrs
Then placed in
chamber with
water source
sprague dawley
rats are used in
this model

o number of accepted punishments (electric

shock) are measured during conflict period

Anxiolytic effect :
o increase in the accepted shocks.

Social interaction test

In unfamiliar and
brightly lit
environment, social
interactions are
suppressed.
Anxiolytics
counteract this
suppression.
Animal used : male
sprague dawley rats.

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Electric stimulation of brain

Electrical stimulation of brain aversive areas, in

particular the midbrain central gray, induces defensive
reaction and/or flight behavior in several species
Used as an animal model of anxiety or of panic attack.
Most studies used intracerebral microinjections of
neurotransmitters, their agonists and antagonists to
elucidate the mechanisms of aversive or antiaversive
effects

Foot shock induced aggression

GABA is involved in control and
agrgressive behaviour of animals.
Benzodiazepines are thought to
produce anxiolytic effects by binding
to a specific high affinity site on
GABA-A receptor
So aggressive and fighting behavior
has been extended for GABAergic
anxiolytic drug screening.

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Isolation induced
aggression
o Male mice
subjected to
isolation
develop
aggressive
behavior
towards other
animals of
same sex.

In vitro methods
GABAA receptor binding
GABAB receptor binding
Benzodiazepine receptor: [3H]-flunitrazepam
binding assay
Serotonin (5-HTIA) receptor: binding of [3H]-8hydroxy-2-(di-n-propylamino)-tetralin ([ 3H]DPAT)
Serotonin (5-HTIB) receptors in brain: binding
of [3H]5-hydroxytryptamine ([3H]5-HT)

conclusion
Anxiety disorder is a psychological disorder and associated
with stress, tension, fear and threat about future.

The pathophysiology of anxiety disorder is not fully

understood and hence improper diagnosis leads to increase

morbidity and mortality rates.

Development of the screening methods resulted in

introduction of many new anxiolytic agents.

In future aspects more reliable and easy models for

screening are to be developed.

References
Hand book of experimental
pharmacology. S.K. kulkarni. 3 rd edition
Drug Discovery an
Evaluation:Pharmacological Assays. H.
Gerhard Vogel. 3rd edition
Shenoy et al. Preclinical evaluation of
anxiolytic agents: an overview. Journal
of Pharmaceutical Research and
Opinion.2011 ;1(2):7-22.