The document discusses various types of anxiety disorders and preclinical models used to study anxiety. It describes definitions of anxiety and several anxiety disorders including generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, panic disorders, and phobias. Several commonly used preclinical anxiety models are explained including elevated plus maze test, open field test, light-dark test, staircase exploration, mirrored chamber test, and social interaction test. Evaluation parameters and procedures for each test are provided.

1. Presented by:
Yasmeen Mir
M. Pharm ist year
(Pharmacology-İII)
Roll no.:18155110001

2. Anxiety Disorders
 Definition : Anxiety is a condition of persistent
and uncontrollable nervousness, stress, and worry,
triggered by anticipation of future events,
memories of past events, or ruminations over day-
to-day events, with disproportionate fears of
catastrophic consequences.
 It is characterized by feelings of apprehension,
insecurity, uncertainty or fear.

3.  Generalized Anxiety Disorder(Excessive, unrealistic
worry that lasts six months or more)
 Obsessive-Compulsive Disorder (Persistent, recurring
thoughts or obsessions that reflect exaggerated anxiety
or fears )
 Post-Traumatic Stress Disorder ( Exposure to a
traumatic event)
Panic Disorders (Severe attacks of panic for no
apparent reason)
 Phobias (Extreme anxiety about being judged by
others, or intense fear reaction to a specific object or
situation such as spiders, dogs, or Acrophobia).
Types of anxiety

4. 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 disorders.
 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 phenotypes.

5. This approach has proven to be valuable for screening novel
anxiolytics and testing the pharmacoselectivity of putative anxiolytics .
Emerging genetic technology is the optogenetics. It will be integral to
future basic anxiety research.
optogenetics 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 conditioning.

6. PRECLINICAL EVALUATION OF
ANXIOLYTICS
In-vitro Methods:
1. GABA receptor binding
2. Benzodiazepine receptor: [3H]-flunitrazepam binding
assay
3. Serotonin (5-HT1A) receptor: binding of [3H]-8-hydroxy-
2-(di-n-propylamino)-tetralin ([3H]-DPAT)
4. Serotonin (5-HT1B) receptors in brain: binding of [3H]5-
hydroxytryptamine ([3H]5-HT)

7. The following parameters are calculated:
• Total binding
• Non-specific binding
•Specific binding= total binding– non-specific
binding.

8.  In Vivo Methods
 Methods based on unconditioned
(spontaneous) response:
 Exploratory activity
 Elevated plus-maze (other
Mazes such as Y, X,T, Radial, water
and Zero Maze)
 Open field/Closed field
 Light-dark model (two
compartment box)
 Staircase exploration
 Hole board test
 Mirror chamber test
 Social behavior
 Social interaction
 Isolation induced aggression
 Anticipatory anxiety in mice
 Predator
 Defense test battery in rats
 Human threat (Primates)
 Odor associated avoidance
behavior
 Others
 Novelty suppressed feeding
 Schedule induced polydipsia in
rats
 Marble burying
 Cork gnawing
 Stress induced hyperthermia

9. • Methods based on conditioned (learned) response:
Punishment models:
• Four plate test
• Punishment induced operant behavior
• Conditioned emotional response
Conflict models:
• Vogel punished drinking
• Geller seifter conflict (marmoset, pigeon conflict)
• Shock probe conflict procedure
Respondent conditioned with aversion stimuli:
• Conditioned suppression
• Potentiated startle response
• Electric brain stimulation
Frustration (non-reward) test:
• Shock probe- burying test

10. In Vivo Methods
Validity
• 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.

11. Elevated Plus Maze Test
For selective identification
of anxiolytics and anxiogenic
drugs
Anxiolytics –decrease
anxiety – increase open arm
exploration time.
Anxiogenics – decrease open
arm exploration time.
Most widely used method;
male mice/rats used.

12. Experimental Design
• Group I : control
• Group II : standard
• Group III : test treated with dose x
• Group IV : test treated with dose 2x ….

13. The rats weighing around 200g - housed in pairs
for 10 days prior to testing; 6animals selected for
each group.
Test drug administered 30min prior to
experimentation by i.p route.
The rat is then placed in the centre of the
maze facing one of the enclosed arms.
Animals were allowed to explore in the maze for 5
min. Observations will done from adjacent room via
remote TV camera.

14. 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

15. Evaluation of results:
o Motor activity and open arm exploratory activity determined.
o Values of treated groups expressed as % of control values.
o Benzodiazepines and valproate – decrease motor activity
increase exploratory time.
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.

16. Open-field 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 behavior
patterns, including:
 The tendency to stay on the
periphery of the field without
entering the centre (called
thigmotaxis and often
interpreted as anxious behavior)
 level of defecation and
urination.

17. Anxiolytic agents should lead to increase in this ratio.
 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 periphery.

18. Light – dark model
• Crawley and Goodwin
(1980).
• Mice and rats tend
to explore novel
environment ,but
retreat from
brightly- lit
open field.

19. 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.
Methodology
• Test apparatus- light and dark chamber divided by photocell equipped zone.
• Polypropylene animal cage 44x21x21 cm, darkened with black spray over on-
third of surface.
• 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 dark

20. Male mice with a weight between 18 and 24 g are used
Test drug administered 30min prior to experimentation by
i.p. route.
The animal is placed in the cage and observed for
10 min.

21. Observation
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.
Evaluation:
o Anxiolytics like diazepam & buspirone increase locomotor
activity and no. Of crossings.
o Non anxiolytics - not effective in this model.

22. Staircase exploration

23. • 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 behavior is an index of anxiety state
• The no. of rearing and steps climbed to be recorded for
period of 5 minutes
• Decrease in rearing behavior and increase in steps climbed
is characterization of anxiolytic effect

24. Male mice (Charles River strain) with a weight
between 18 and 24 g are used
Test drug administered 30min prior to
experimentation by s.c. route.
The animal is placed on the floor of the box with
its back to the staircase.(staircase is composed of
five identical
steps 2.5 cm high, 10 cm wide )

25. Mirrored chamber test
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.

27. Social interaction in rats
• 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.

28. Animals placed in groups of 5 each in a perspex open
topped box
1hr before test,2 rats from separate housing treated
with test compound orally
Placed in box with 60W bulb and behavior observed
for 10minutes
social interactions like sniffing, rearing, crawling over
the partner are observed

29. Procedure:
• 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
floor.
• One hour prior to the test, 2 naïve rats from separate housing cages are treated with
test compound orally
• 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 following partner.

30. • 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
test

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

32. Male Mice kept isolated for 6weeks & aggressive behavior
tested.
Male mice accustomed to live together placed in cage of
isolated mice for 5minutes
Isolated mice attacks intruder aggressiveness observed
Drugs given to isolated mice s.c or orally; aggressive behavior
tested at 60, 120,240 minutes (oral route)
If drug active- decrease in aggressiveness Attenuation of
fighting reaction

33. Human threat (primates):
Suarez and Gallup (1982) first demonstrated human
being as a predator. It can be studied in various ways of
avoidance of experimenter. One method involves an
experimenter sitting in a chair in the center of a floor
containing chicks. The proximity of the chicks to the human
is then determined usually by numbering imaginary zones
around the occupied chair. The chicks are then given an
‘avoidance score’ of 1-5 (either in ascending or descending
proximity to the experimenter) based on a total of their
positions over a certain time frame. Another method is
called the ‘box plus experimenter’ method. This test uses
the same premise as the above method; however, during
the box plus experimenter test, the human is seated behind
a wire mesh wall at the end of an arena. The chick is
scored on its approach or avoidance to the experimenter
behaviors. Again, higher avoidance suggests higher fear
levels.

34. Odour associated avoidance behaviour:
The predatory odour avoidance model relies upon
the apparently innate fear that rodents have for the odor
of their natural predators, such as cats and foxes. Rats
tend to avoid such odors and engage in a variety of
defensive behaviours in their presence. Novel methodology for testing is given by
Dielenberg and McGregor (1999). Testing chambers comprised of a rectangular arena
with perspex walls (60 cm x 26 cm x 36 cm) and a metal grid
floor which was raised 2 cm above a tray containing wood shavings. At one end of the
chamber was a small wooden box (21 cm x 24 cm x 22 cm) termed the ‘hide box’. On
the front wall of the hide box was a small square hole (6 cm x 6 cm) that allowed rats to
enter the box. On the opposite wall of the apparatus to the hide box was an
alligator clip positioned 4 cm above the metal grid floor. During testing, a piece of cat
collar was attached to the clip as shown in Fig.8. A domestic cat wore this cat
collar for a period of three weeks before the start of the experiment. Photocell
detectors were placed at opposite ends of the chamber. The placement of the
photocells allowed determination of: i) the amount of time the rats
spent in close vicinity to the cat collar (approach time); and ii) the amount of time
spent in the hide box (hide time). During testing, the room in which the chambers
were situated was illuminated by a 40 W red-light suspended
1.5 m above the apparatus.

36. Vogel punished drinking
 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 diazepam .
 • 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 tongue.
 Rats are deprived of water for 48 hrs
 • Then placed in chamber with watersource
 • sprague dawley rats are used in this model

37. Electric circuit is connected between
drinking tube and floor of cage.
i.p injection of drugs are given; 30min later
rats placed in cage and allowed to drink
water and shock given after 20 licks
For 3minutes next shocks are given for
every 12th lick
No. of shocks delivered in 3min noted for
each animal, no. of shocks received after
treatment noted
a water bottle with metal drinking tube is
fitted to the animal housing

38. o number of accepted punishments (electric
shock) are measured during conflict period
Anxiolytic effect :
o increase in the accepted shocks.
Apparatus
Clear Plexiglas box (38 x 38 cm) has a steel
grid floor.
A water bottle with metal drinking tube at 3
cm above the
grid.
A circuit is connected b/w the drinking tube
and steel
floor, so that the circuit completes if animal
licks the tube.

39. Geller seifter conflict (marmoset,
pigeon conflict)
 .
•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
• Male albino rats with a body weight of 300–400
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.

40. • 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
“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
• 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
non-conflict periods (VI) are counted.
• Anxiolytic effect :An increase of lever presses
in the conflict periods

41. Electric brain stimulation
 • 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

42. 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.

43. The fighting behaviour consists of vocalization, leaping,
running, rearing and facing each other with some attempt to
attack by hitting, biting or boxing
Two mice are placed in a box with a grid floor consisting of
steel rods
A constant current of 0.6 or 0.8mA is supplied to the grid floor
is delivered for 5 s followed by 5 s. intermission for 3 min.
The total number of fights are recorded for each pair during
the 3-min period.

44. Pathological anxiety
(Neurochemically - induced anxiety)
mCPP - [ 1-(3-chlorphenyl) piperazine] a 5HT 1c
agonist.
o mCPP induces hypophagia and
hypo-locomotion , inhibits social interaction,
diminishes exploratory activity in light-dark
box test, hyper therimia etc,.
o Antagonism of these symptoms is used for
screening of anxiolytic drugs.

46. Anticipatory anxiety in mice
 Anticipatory anxiety in mice
 • When group housed mice are removed one by one
 from their former cage, the last mice always have
 a higher body temperature than the 1st one.
 • The anticipatory increase in temperature was
 prevented by prior administration of diazepam &
 buspirone

47. Test drug/solvent administered p.o to all the 18 animals.
Record the basal temp by removing 1st 3 animals/group
after 30 min
Difference in the mean value of last three
animals and basal
value is calculated
Vehicle treated group 1- 1.3˚C

48. Male Sprague
Dawley rats (200-
250g) are housed in
groups of 6;
exposed to 12 hour
light/dark cycle
with free access to
food and water.

49. mCPP Induced Anxiety
-Locomotion Study
• 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.

50. Parameters measured :
o time spent in both sides (horizontal,
vertical activity)
o frequency of motion
o number of transition
Anxiolytic effect :
o increase in parameters measured in
the light/dark box or in number of
transitions if test is active.

51. A method is described for reproducible measurement of ultrasonic
vocalization induced by tail-holding stress in rat pups. The anxiolytic
benzodiazepines, chlordiazepoxide, diazepam, and CL 218872, reduced
the ultrasounds at doses inducing little CNS depressant activity. Gross
behavioral disruption such as sedation (muscimol, prazosin, and
chlorpromazine), tremors (yohimbine), myoclonus (MK212), and
immobility (morphine) resulted in reduction of ultrasounds. Non-
behaviourally active doses of these compound or any doses tested of
mephenesin, amphetamine, amitriptyline, haloperidol, and naloxone did
not affect the ultrasounds. Metergoline inhibited ultrasounds at doses
producing little change in overt behavior. This method is proposed as a
convenient model of anxiety which may also be influenced by central 5-
hydroxytryptamine transmission.