Screening of Anxiolytic Agents in
For MD Pharmacology
Dr. Advaitha M V
• Definition : Anxiety is defined as a subjective
sense of unease, dread, or foreboding.
• It is the most prevalent psychiatric illnesses in
the general community, are present in 15–20%
of medical clinic patients.
Preclinical anxiety models
• The goal of anxiety models is to produce a
form of abnormally elevated anxiety.
• It should more closely resemble, by
definition, the pathological nature of human
• Anxiety models are Models that generate
lasting or permanently heightened anxiety.
This can be achieved by acutely or chronically
subjecting animals to stressors before testing.
OR identifying genetic populations (inbred
and selectively bred strains) or engineering
mutant mice with innate anxiety-like
• This approach has proven to be valuable for
screening novel anxiolytics and testing the
pharmaco selectivity of putative anxiolytics .
• Emerging genetic technology is the
optogenetics. It will be integral to future basic
• It is the combination of genetic and optical methods
to control specific events in targeted cells of living
tissue in the millisecond timescale.
• Optogenetic approaches have been used to map
neural circuits in the amygdala that contribute to fear
• However , in general , it is only simple tests or
assays which transiently evoke an anxiety-like
behaviour in rhodents is commonly employed.
The five main anxiety disorders with preclinical
tests which resembles human anxiety
• Generalized anxiety disorder (GAD)----MOST
OF THE TESTS
• Post-traumatic stress disorder (PTSD)…….
FEAR CONDITIONED TESTS
• Panic disorder………… ELEVATED T MAZE
MODEL and MOUSE DEFENSE TEST BATTERY
• Obsessive compulsive disorder (OCD)……
• Social anxiety disorder (SAD)…… SOCIAL
PRECLINICAL EVALUATION OF
• In-vitro Methods
• In-vivo Methods
• Our main focus here would be In-vivo.
The following parameters are calculated:
• Total binding
• Non-specific binding
• Specific binding= total binding– non-specific
In Vivo Methods
• Face validity : A feature that is assessed (for a
test or model of anxiety) by determining how
closely the model or test resembles human
anxiety symptoms .
• Predictive validity : whether the model or
test reliably responds to clinically efficacious
anti- anxiety medications .
• Construct validity : whether the degree to
which the model or test recruits the same
underlying neurobiology as implicated in
human anxiety .
Elevated plus –maze (EPM) test
This has widely validated to measure anxiety to
• The test is based on the natural aversion of
mice for open and elevated areas, as well as
• natural aversion on their natural spontaneous
exploratory behavior in novel environments.
• The apparatus consists of open arms and
closed arms, crossed in the middle
perpendicularly to each other, and a center
• Mice are given access to all of the arms and
are allowed to move freely between them.
• The number of entries into the open arms.
(Entry an arm is defined as animal placing all
four paws onto the arm. All tests are taped by
using a video camera)
• The time spent in the open arms are used as
indices of open space-induced anxiety.
Anxiolytic drugs specifically increase the
number of entries into the open arms and the
time spent there
Anxiogenic drugs specifically decrease the
• The total entries score and total distance are
Useful index of general activity.
The percentages of entries and time spent in
each arm constitute the index of primary
• The rats (200–250 g body weight) are housed
in pairs for 10 days prior to testing in the
• During this time the rats are handled by the
investigator on alternate days to reduce stress.
• 6 rats are taken for each group.
• Thirty min after i.p. administration of the test
drug or the standard, the rat is placed in the
center of the maze facing one of the enclosed
• Duration of test is 5 min where all data is
• Test should be conducted in sound proof
• After each test, the maze is carefully cleaned
up with wet tissue paper (10% ethanol
• Anxiolytic treatments do not by
themselves increase exploration
in the tests but they decrease the
stress-induced inhibition of
• Crawley and goodwin (1980)
• Mice and rats tend to explore novel
environment ,but retreat from brightly lit
• In a two chambered system, where animal can
freely move between brightly lit open field
and dark corner, they show more crossings
between two chambers and more locomotor
activity after treatment with anxiolytics.
• Test apparatus- light and dark chamber
divided by photocell equipped zone.
• Polypropylene animal cage 44x21x21 cm,
darkened with black spray over on-third of
• Partition containing 13cm long x 5cm high
opening separates the dark one third from
bright two thirds of the cage
• Cage rests on an animex activity monitor,
which counts the locomotor activity.
• Four sets of photocells across the partition,
automatically counts movement through the
partition and clocks time spent in light and
• Naïve male mice or rats are placed in cage
• Animals are treated 30 min before the
experiment with test or vehicle
intraperitoneally and are observed for 10 min.
• Groups of 6-8 animals are used for each dose.
• Evaluation: number of crossings through the
partition between the light and dark chamber
are compared with total activity counts during
• Critical assessment of the method: anxiolytics
like diazepam, pentobarbital, meprobamate,
produce dose dependent increase in crossings
• Test is relatively simple with no painful stimuli
• Purpose and rationale : In an unfamiliar and
brightly lit environment, the normal social
interaction of rats ( sniffing, nipping,
grooming) is suppressed.
• Anxiolytics counteract this suppression.
• Male sprague-dawley rats (225-275g) are
housed in groups of 5 animals
• Apparatus: perspex open-topped box (51x51x
20cm) with 17x17cm marked areas on the
• One hour prior to the test, 2 naïve rats from
separate housing cages are treated with test
• They are placed into the box (with 60W bright
illumination 17cm above) and their behaviour
is observed over a 10 min period.
• 2 types of behaviour can be noted:
• Social interaction between the animals is
determined by timing the sniffing of partner,
crawling under or climbing over partner and
• Exploratory motion is measured as the
number of crossings of the lines marked on
the floor of the test box.
• Six pairs are used for each dose
• Evaluation: 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
• This test, originally designed by Hall on rats.
• It consists of placing an animal in an unknown
environment with surrounding walls, so as to
observe a number of behaviour patterns,
The tendency to stay on the periphery of the
field without entering the centre (called
thigmotaxis and often interpreted as anxious
level of defecation and urination.
• The open field floor is divided into squares
• Animals are tested individually, always being
placed in the same position.
• Duration of test is usually 5 min.
• Higher levels of anxiety should mainly lead to
decreases in the ratio ‘number of squares
visited in centre/number of squares visited on
• Anxiolytic agents should lead to increase in
• This test is a well-known method used in rats,
designed by Vogel et al.
• Recently, this test has been reported to
successfully detect anxiolytic-like action of
• In this test, thirsty animals gain water reward
through a water spout, but at the expense of
receiving a mild electric shock delivered to the
• Licking in untreated/controls is suppressed
due to the conflict.
• Anxiolytics release this suppressed behaviour
and decrease the conflict.
• Diazepam and pentobarbital produces a
significant anti-conflict effect, which means
These drugs increases the number of electric
shocks the mice received during the test
• But drugs like baclofen, buspirone,
chlorpromazine and also haloperidol did not
produce anti-conflict effects.
Resident-intruder aggression test
Purpose and rational: To study the effects of
drugs in a test for offensive aggression, the
isolation-induced resident intruder aggression
model in the rat.
• Procedure: Sprague-Dawley rats weighing 250
to 450 g are housed in a light-dark (12L:12D)-,
temperature (ca. 22°C)- and humidity (ca.
Resident male rats (about 450 g) are tested in
their home cages for aggression against a
smaller (250 g) male intruder
• They are treated by intraperitoneal injection
of test drug or saline 15 min before the test.
• The resident female is removed from the cage
30 min prior to the start of the test period.
• After placing the intruder rat in the territorial
cage, the behavior of the resident male is
• The time until the first attack (in seconds),
number of attacks, and duration of each
attack (in seconds) are recorded for the next
15 min by a blind observer.
• Different behavioral elements are scored and
grouped into 7 behavioral categories:
offensive, exploration, social interest,
inactivity, avoidance, body care, defense.
• Evaluation: Paired and unpaired t-tests are
used for comparisons of means of absolute
Other Anti- Aggressive tests
• FOOT- SHOCK INDUCED AGGRESSION IN MICE
• ISOLATION INDUCED AGGRESSION
• MATERNAL AGGRESSION IN RATS
Anticipatory anxiety in mice
• Purpose and rationale: when group-housed
mice are removed one by one from their
home cage, the last mice removed have
always higher rectal temperatures than those
• This phenomenon is caused by anticipatory
fear for an aversive event .
• Increase in temperature prevented by prior
treatment with diazepam and buspirone
• Procedure: groups of 18 male albino swiss
mice ( 25-30g) are housed in constant room
temperature and relative humidity, for atleast
• Test drugs or standard (diazepam) are
administered orally in various doses to groups
of 18 mice prior to the test.
• Thirty min later, first 3 mice are removed from
the cage, rectal temperature recorded by
inserting a silicone lubricated thermistor
probe (2mm diam)
• Average temperature of these 3 mice is taken
as basal value
• Mice number 4 to 15 are simply removed and
again returned to the cage.
• Thereafter body temperature is determined in
the remaining three animals.
• The difference of the mean value of these
mice and the basal values is calculated as
• Vehicle treated test groups display increases
of 1.1 to 1.3 °C.
• The mean increase values of treated groups
are compared by ANOVA statistics with the
mCPP induced anxiety in rats
• Purpose and rational: The metabolite of the
antidepressant drug trazodone is
1- (3-chlorphenyl)piperazine (= mCPP).
• It is 5- HT1C/1B/2C agonist.
• It has been shown to be anxiogenic both in
man and in rats.
• The compound induces hypophagia and
hypolocomotion, inhibits social interaction in
rats, diminishes exploratory activity of rats in
the open field test
• In the light-dark box test induces
• Antagonism against these symptoms has been
proposed as a screening model for anxiolytic
• Purpose and rational: Placing a hungry rat
into an unfamiliar environment with access to
food results in a suppression of feeding
behavior relative to the condition when the
test environment is familiar.
• This effect has been termed hyponeophagia
and occurs because of the novelty of the test
• The avoidance of novel foods is termed food
• Both hyponeophagia and food neophobia
have been assumed to measure emotionality
or anxiety by eliciting a conflict situation
arising from a fear of the novel setting and
foods, and the drive to eat.