Ruchi Yadav from the Department of Pharmacology presented on screening for anti-Alzheimer activity. There are two types of models: 1) models testing learning and memory using avoidance tests, discrimination learning, and conditioned responses; and 2) transgenic mouse models exhibiting tau pathology or amyloid-beta toxicity. Common learning and memory tests include step-down avoidance, step-through avoidance, and water maze tasks. Transgenic mouse models express mutant human tau or amyloid precursor protein genes linked to Alzheimer's disease.

1. Screening of
AntiAlzheimer activity
Presented By Ruchi Yadav
Department: Pharmacology

2. ALZHEIMER'S DISEASE
 A degenerative brain disease of unknown cause that is the most common form
of dementia, that usually starts in late middle age or in old age, that results in
progressive memory loss, impaired thinking, disorientation, and changes in
personality and mood, and that is marked histologically by the degeneration of
brain neurons especially in the cerebral cortex and by the presence
of neurofibrillary tangles and plaques containing beta-amyloid
 Behavioural problems, such as mood swings and agitation, may also be a part of
the progression of Alzheimer's disease.

3. We have two types of models for screening of anti-
Alzheimer activity
1 ) Models to test learning and memory.
2) Transgenic mouse models of dementia.

4. 1) MODELS TO TEST LEARNING AND MEMORY:
 As there is no definite model for AD, we can use models based on learning
and memory. Learning and acquisition of memory are complex behavioral
phenomenon. Memory deficiency due to aging is difficult to design in
animal models.
 The most commonly used in vivo screening methods for drugs affecting
learning and memory can be considered under following:
I) Inhibitory avoidance methods.
II) Active avoidance test.
III) Discrimination learning.
IV) Conditioned response.

5. 2) TRANSGENIC MOUSE MODELS OF DEMENTIA
I) Tau models.
II) Aβ-tau axis
III) Secretase models
IV) APOE models
V) Axonal transport models

6. I) INHIBITORY AVOIDANCE METHODS
 In these test animal fails to imitate activities and learned habits.
Here animal learn to avoid an unwanted events by suppressing a
particular behavior .
A)Step down test: In open field, rodents prefer to be closed to wall or
in corners. If placed on an elevated platform, they come down to
floor and reach wall or corner. In this both mice and Rat either sex
can be used. Room should be free of noise.
 A rectangular box 50x50x50 with grid floor and movable platform
(7.5x7.5) in the centre of box is used. Floor is connected to shock
device.
 The animal is given a foot shock of 50Hz, 1.5mA, 1s as it descends
from platform.
 Latency to descend is measured. Prolongation of step-down
learning is defined as latency.

7. B) Step through test in rodents:
Rodents prefer to be in dark. If placed in a brightly illuminated area
they rapidly go to dark area. The apparatus has a light chamber
connected to a large dark chamber through a small door. A bulb
(7W/12V) is used in light chamber.
 As the animal enters the dark chamber from light chamber the small
door is closed and in dark chamber animal receives a unavoidable
foot shock of 1mA; 1s to mouse and 1.5mA;2s to rat. An increase in
step through latency is defined as learning.

8. C) Uphill avoidance test in rats: On a slanted surface,
rodents move toward the top known as negative geotaxis. Rats and
mice placed on a tilted platform facing down hill, they turn around
and move up the inclined.
 The apparatus is 50x50cm box with 35cm high opaque plastic walls.
The floor consists of 10mm diameter stainless steel grid bars placed
13mm apart. A shock of 2mA is given to rats when it turns 180° and
I step of climbing. Prolongation of latencies by test drug indicates
positive effect on learning.

9. E) Ibotenic acid-induced impairment of memory:
 Bilateral injection of ibotenic acid into the basal forebrain of rats
produce lesions which leads to memory impairment.
 Water maze, Y-maze, habituation tasks and inhibitory avoidance
tasks with a light and dark compartment apparatus and inhibition of
decrease of cholinesterase activity in the cortex are used to screen
anti Alzheimer drug.

10. II) ACTIVE AVOIDANCE TEST
 To avoid an noxious stimulus, animal may escape to terminate it.
A) Runaway avoidance in rats and mice:
 Animal is placed in a box which is uniformly illuminated and has one small
door. A loud speaker is mounted 50 cm above the start box, and provides
acoustic conditioning stimulus (80db,2000Hz tone.
 5min animal is allowed to explore the whole apparatus. The door is then closed,
after 10s the acoustic condition stimulus is applied and door is opened.
 After 5 min the shock of 1mA;1s for mouse and 1.5mA;2s for rat is
administered after 5s.
 The time required for reaching safe are is measured to assess the efficacy of
drug.

11. B) Shuttle box avoidance test (two way shuttle box):
 Apparatus is rectangular box 50x15cm with 40cm high metal walls and a
grid floor. It is divided into two 25x25 compartments by a wall and a small
door.
 Each compartment is illuminated by 20W bulb. The animal is allowed to
explore the box for 5 mins. Then the door is closed.
 20 s later light is switched on in compartment in which animal is present, the
door is opened, atone(60db) is produced and 5s later a shock is applied in
the light chamber and continued till animal goes to dark chamber.
 The time required by the animal to reach each safe compartment is
recorded.

12. C) Jumping avoidance in one way shuttle box:
 Apparatus is a rectangular box of 40x25 and a grid floor and one goal
area with narrow walls.
 Animal is allowed to explore goal area for 5 min. After that goal is
blocked for 2s an acoustic conditional stimulus (100Hz;85db) is
applied then after 5 min shock of imA;50Hz;0.5s)to animal.
 Animal jumps on the platform.
 Retention is tested on the second day until the animal reaches the
criterion.

13. III) DISCRIMINATION LEARNING
A) Spatial habitual learning:
 Spatial habitual learning means a decrease in reactivity to anovel environment
after repeated exposure to that now familiar environment.
Apparatus: Rectangular chamber of 60x60x40cm for rats and 26x26x40cm for mice made
of painted wood or gray PVC.
 A 25W green or red light electric bulb is placed directly above the maze to achive an
illuminated density of 0.3lx at the centre.
 Rat is placed in the centre or in corner for 5-10 min session and record the following:
i) rearings or vertical activity: the no. of times an animal stood on its hind legs with foreleg in
air or against the wall,
ii) duration of single rearing,
iii ) locomotion

14. B) Spatial learning test in the radial arm maze:
 In radial arm maze the study of spatial reference and working memory process
in rats can be done.
Apparatus:
 It is a wooden elevated 8arm radial maze with arms extending from a central
platform having a diameter of 20cm.
 Each arm is 56cm long and 5cm wide with height of 2cm. Food pellets which
serve as reward at the end of the arm.
 Animal are trained daily to collect food pellets.
 The session terminates after 8 choices and the rat has to obtain the maximum
no. of rewards with least no of errors.

15. C) Spatial learning test in water maze:
 Rats learn to swim in water tank to find to escape platform hidden
under the water.
 A circular water tank filled to a depth of 20cm with water at 25°C.
 Tank is divided into 4 equal quadrants and a small platform at 19cm
height is located in one quadrant.
 Rat has to find the platform to escape.
 Well trained rats take less than 10s.

16. D) Olfactory learning in rats:
 Animal is deprived of water for 48h.
 Apparatus is box 30x30x55 with a photo sensitive cell on the top of
water spout.
 Rats are trained to approach the water spout and to break the light
beam. Responses to the positive order are awarded.
 Session terminates when rat makes 90% correct choices.

17. IV) CONDITIONED RESPONSES
Conditioned nictitating membrane responses in rabbit:
A small loop of surgical nylon is sutured in to the right nictitating membrane. After
one day the rabbit is placed in a restrainer.
Rabbit is filled with a head mount that helps to record the nictitating membrane
response by physical coupling with a length of thread to nylon loop in the
nictitating membrane.

18. 2. TRANSGENIC MOUSE MODELS OF
DEMENTIA
I) Tau models:
 Pre-tangled formation and hyper phosphorylation of tau was
observed.
 Suppression of P301L tau expression in rTg4510 tau transgenic
mice, which normally express the mutant protein at a high level,
reverses behavioral impairments in these mice although NFT
formation continues.
 This suggests that soluble tau rather than NFT is neurotoxic. Both
oligodendrocytes and astrocytes contain filamentous tau inclusions
in patient with FTD.
 Neuronal loss is lower in the B301L tau models than in P301S tau
mouse, consistent with the early onset.

19. Reference
 Animal Models of Alzheimer Disease Frank M. LaFerla and Kim N.
Green Institute for Memory Impairments and Neurological
Disorders, Department of Neurobiology and Behavior, University of
California, Irvine, Irvine, California 92697-4545.
 Drug Discovery and EvaluationPharmacological Assays Co-Editors:
Wolfgang H.Vogel Bernward A. Schölkens Jürgen Sandow Günter
Müller Wolfgang F. Vogel Second Edition.