In vivo methods Methods based on unconditioned

(spontaneous) response:o Exploratory activity

Elevated plus-mazeWater maze test Light-dark model (two compartment box)

Staircase explorationo Social behaviour

Social interactionIsolation induced aggressionAnticipatory anxiety in mice

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Methods based on conditioned (learned) response:oConflict models

Vogel punished drinking/ Vogel’s lick conflict model

Chemical induced methodmCPP induced anxiety in rats

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Elevated Plus Maze Test

o Most widely used method; male mice/rats used.

o For selective identification of anxiolytic and anxiogenic drugs

o Anxiolytics –decrease anxiety – increase open arm exploration time

o Anxiogenics – decrease open arm exploration time.

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o 2 open arms and 2 closed arms of 50 x 10 x 40cm dimensions

o Open roof arrangement

o Two open arms are opposite to each other.

o Maze elevated at 50cm height above the ground.

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Experimental Design

• Group I : control• Group II : standard • Group III : test treated with dose

x • Group IV : test treated with dose

2x ….

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Animals were allowed to explore in the maze for 5 min. Observations will done from adjacent room via remote TV camera.

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 entries8

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 and 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

o Circular tank of 100-150 cm diameter with 20-30 cm wall

o A platform of 9-10 cm hidden 2-3 cm below water level

o Add titanium dioxide susp. to make water opaque.

o Temp : 23˚C.

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Anti – anxiety test(light – dark model)

o Rodents – have exploratory activity

o Animals placed in 2 chambered systems, where they can freely move between a brightly –lit open field and a dark corner.

o After treatment with anxiolytic - show more crossings between the two chambers and more locomotor activity.

o Number of crossings between the light and dark sites is recorded. 11

Methodology o Apparatus - a dark and a light chamber divided by a photocell equipped zone.

o Polypropylene animal cage (44 x 21 x 21 cm) is darkened with black spray over 1/3rd of its surface.

o A partition containing 13cm(l) ,5 cm (h) opening is used for separating the dark one-third of the cage.

o This case rests on an activity monitor which counts total locomotor activity. 12

o An electronic system consisting of 4 sets of photocells across the partition.

o It automatically counts movements

through the partition and records the time spent in the light and dark compartments.

o Animals- treated 30 min before the test with drugs or vehicle given i.p. placed in the cage and observed for 10 min.

o Groups of 6-8 animals used for each dose. 13

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. 14

Isolation induced aggression

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

o Compounds tested for their ability to suppress this isolation induced aggression.

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Animal used: Male NMRI strain mice

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Evaluation of results:

o No. of animals with complete suppression of aggressiveness.

o Reaction time noted.

o Graduated scale of inhibition of aggressiveness is established.

o Results of test group animals is compared with the control group results.

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Social Interaction In Rats

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Methodology

Parameters measured : o exploration, sniffing, rearing, social

contacts, sexual behaviour, attack, fighting, biting ,defensive posture, immobility and climbing over the partner.

Evaluation :o Values of treated partners compared

with data from control animals – ANOVA and t - test used.

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o 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.

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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

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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

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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.

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.25

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.

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Vogel Lick-conflict (Vogel Punished Drinking)

Source of anxiety: thirsty, native rats are

administered shocks while licking water.

Animals used: Sprague dawley rats.

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Apparatus

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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.

Methodology

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Parameters measured:o number of accepted punishments

(electric shock)

Anxiolytic effect :o statistically significant increase

in the accepted shocks.

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Staircase apparatus

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