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SEMINAR ON SCREENING SEMINAR ON SCREENING OF NOOTROPICS OF NOOTROPICS

(COGNITIVE ENHANCERS)(COGNITIVE ENHANCERS)

Presented by:Presented by: Souvik duttaSouvik dutta

11stst M.pharm M.pharm Dept. of PharmacologyDept. of Pharmacology

gautham college gautham college of pharmacyof pharmacy

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CONTENTSCONTENTS1. Introduction to nootropics1. Introduction to nootropics2. Definition of nootropics2. Definition of nootropics3. Definition of Memory3. Definition of Memory4. Parts of brain and its physiology4. Parts of brain and its physiology5. Constitution of Psyche or mind5. Constitution of Psyche or mind6. Indications of nootropics6. Indications of nootropics7. Classification of nootropic agents7. Classification of nootropic agents8. Common mechanism of action8. Common mechanism of action9. Pharmacological actions of the prototype9. Pharmacological actions of the prototype10.Screening models 10.Screening models a. invitro modelsa. invitro models b. invivo modelsb. invivo models

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INTRODUCTION TO NOOTROPICSINTRODUCTION TO NOOTROPICS

The word Nootropics was coined in 1964 The word Nootropics was coined in 1964 by by Dr.Corneliu GiurgeaDr.Corneliu Giurgea..

Its derived from greek wordsIts derived from greek words NOOS-mind tropein-bend/turn.NOOS-mind tropein-bend/turn.

They are referred to as “smart drugs”, They are referred to as “smart drugs”, ”memory enhancers”, ”cognitive enhancers”, ”memory enhancers”, ”cognitive enhancers”, “smart nutrients”.“smart nutrients”.

In the scientific literature they are termed as In the scientific literature they are termed as nootropics. nootropics.

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DEFINITION OF:DEFINITION OF:

a) a) MemoryMemory: it’s a unconscious faculty in which mental : it’s a unconscious faculty in which mental impressions are retained and reproduced in mind. impressions are retained and reproduced in mind. Memory consists of 4 process:Memory consists of 4 process:

(a) learning (b) retention (C) recall (d) recognition (a) learning (b) retention (C) recall (d) recognition memory process depends on objects and events. memory process depends on objects and events.

b) b) DementiaDementia: acquired global impairment of cognitive : acquired global impairment of cognitive functions in absence of clouding of consciousness or functions in absence of clouding of consciousness or motor involvement. Memory, capacity to solve problems motor involvement. Memory, capacity to solve problems of day to day living, social skills, control of emotions are of day to day living, social skills, control of emotions are affectedaffected

c) c) Alzheimer’s diseaseAlzheimer’s disease: A progressive neurodegenerative : A progressive neurodegenerative disorder which affects older individuals. Atropy of cortical disorder which affects older individuals. Atropy of cortical and subcortical area is associated with deposition of beta- and subcortical area is associated with deposition of beta- amyloid protein in form of plaques, and formation of amyloid protein in form of plaques, and formation of neurofibrillary tangles. There is marked cholinergic neurofibrillary tangles. There is marked cholinergic deficiency in brain. deficiency in brain.

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DEFINITION OF NOOTROPICSDEFINITION OF NOOTROPICS

These are the drugs that are used to These are the drugs that are used to improve human cognitive abilities.improve human cognitive abilities.

Intellect , memory and personality of a Intellect , memory and personality of a human are called cognitive functionshuman are called cognitive functions

Nootropic substances include drugs, Nootropic substances include drugs, nutrients and herb with cognitive nutrients and herb with cognitive enhancing effects. enhancing effects.

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Typically nootropics work byTypically nootropics work by Altering the availability of brain’s Altering the availability of brain’s

supply of neurochemicals ie, supply of neurochemicals ie, neurotransmitters, enzymes and neurotransmitters, enzymes and harmones.harmones.

By improving brain’s oxygen supplyBy improving brain’s oxygen supply

By stimulating nerve growthBy stimulating nerve growth

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ANATOMYANATOMY The main parts of brain are: The main parts of brain are:

a) cerebrum b) cerebellum a) cerebrum b) cerebellum c) brain stem .c) brain stem .

Cerebrum is the largest part of brain Cerebrum is the largest part of brain composed of two similar sized cerebral composed of two similar sized cerebral hemispheres. hemispheres.

Each hemisphere is divided into 4 lobes: Each hemisphere is divided into 4 lobes: occipital, parietal, temporal and frontal.occipital, parietal, temporal and frontal.

The cerebrum (association area) is the The cerebrum (association area) is the area responsible for learning, memory and area responsible for learning, memory and identification activities.identification activities.

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CONSTITUTION OF CONSTITUTION OF PSYCHE OR MINDPSYCHE OR MIND

They carry out three functions:They carry out three functions: CognitionCognition : the reception of : the reception of

environmental stimuli.environmental stimuli. AffectAffect : analysing : analysing the information the information

received and formation of reaction received and formation of reaction patterns.patterns.

ConationConation : the actual behaviourial : the actual behaviourial response.response.

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INDICATIONS OF NOOTROPICSINDICATIONS OF NOOTROPICS

Senile dementia of alzheimer type(DAT) Senile dementia of alzheimer type(DAT) and multi infarct dementia (MID) and multi infarct dementia (MID)

Mental retardation in children, learning Mental retardation in children, learning defects, attention deficit disorder.defects, attention deficit disorder.

Common symptoms of elderly ;dizziness Common symptoms of elderly ;dizziness and memory disturbance.and memory disturbance.

Transient ischaemic attack, cerebro Transient ischaemic attack, cerebro vascular accidents like stroke.vascular accidents like stroke.

Sequalae of head injury, ECT, brain Sequalae of head injury, ECT, brain surgery.surgery.

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CLASSIFICATION OF NOOTROPICSCLASSIFICATION OF NOOTROPICS CHOLINERGIC ACTIVATORCHOLINERGIC ACTIVATOR - acetyl –L-carnitine ( ALCAR)- acetyl –L-carnitine ( ALCAR) - piracetam- piracetam - centrophexine- centrophexine

SERATONERGICSSERATONERGICS - theanine- theanine - typtophan- typtophan

DOPAMINERGICSDOPAMINERGICS - L- dopa- L- dopa - phenylalanine. - phenylalanine.

ALZHEIMERS DISEASEALZHEIMERS DISEASE - tacrine- tacrine - donepezil- donepezil - rivastigmine- rivastigmine - galantamine- galantamine

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IMPROVED OXYGEN SUPPLY & BRAIN ENERGYIMPROVED OXYGEN SUPPLY & BRAIN ENERGY

- lipoic acid- lipoic acid

- pyritinol- pyritinol

MEMORY ENHANCERS MEMORY ENHANCERS

- brahmi- brahmi

- vasopressin- vasopressin

MENTAL CONCENTRATION & STAMINAMENTAL CONCENTRATION & STAMINA

- caffine- caffine

- adrafinil- adrafinil

NERVE GROWTH STIMULANT & BRAIN CELL PROTECTIONNERVE GROWTH STIMULANT & BRAIN CELL PROTECTION

- brahmi- brahmi

- ginko biloba.- ginko biloba.

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PIRACETAM (cholinergic activator)PIRACETAM (cholinergic activator) This is a GABA derivative that selectively This is a GABA derivative that selectively

improves the efficiency of higher telencephalic improves the efficiency of higher telencephalic activity by:activity by:

a) enhancement of learning and memorya) enhancement of learning and memory b) facilitation of inter hemisphere information b) facilitation of inter hemisphere information

transfer transfer c) increase tonic cortical control on sub cortical c) increase tonic cortical control on sub cortical

areasareas piracetam has been claimed to improve ATP/ADP piracetam has been claimed to improve ATP/ADP

ratio in telencephalon, stimulate synaptic ratio in telencephalon, stimulate synaptic transmission and to have an anti thrombotic transmission and to have an anti thrombotic effect.effect.

Side effects are gastric discomfort, excitement, Side effects are gastric discomfort, excitement, insomia, dizzinessinsomia, dizziness

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DONEPEZIL ( AD)DONEPEZIL ( AD) This cerebro selective and reversible anti-This cerebro selective and reversible anti-

AChE cognitive as well as non cognitive AChE cognitive as well as non cognitive (activities of daily living ) scores in AD, (activities of daily living ) scores in AD, elevation of Ach level in cortex, especially elevation of Ach level in cortex, especially in neurons tat project from basal fore brain in neurons tat project from basal fore brain to cerebral cortex and hippocampusto cerebral cortex and hippocampus

Its likely to have the greatest impact in Its likely to have the greatest impact in AD among cholinergic agents AD among cholinergic agents

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PPYRITINOLYRITINOL

It’s a vaso active cerebral protective ie, a It’s a vaso active cerebral protective ie, a drug supposed to counteract the cognitive drug supposed to counteract the cognitive and functional defects induced by cerebro and functional defects induced by cerebro vascular insufficiencyvascular insufficiency

Its claimed to activate cerebral Its claimed to activate cerebral metabolism by selectively increasing metabolism by selectively increasing glucose transport across BBB, enhance glucose transport across BBB, enhance cerebral cholinergic transmission and cerebral cholinergic transmission and improve regional blood flow in ischaemic improve regional blood flow in ischaemic brain area brain area

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MECHANISM OF ACTIONMECHANISM OF ACTION

Increasing global/ regional blood flowIncreasing global/ regional blood flow Direct support of neuronal metabolismDirect support of neuronal metabolism Enhancement of neurotransmission.Enhancement of neurotransmission. Improvement of descrete cerebral Improvement of descrete cerebral

function.function. Improve oxygen supply and brain energyImprove oxygen supply and brain energy Nerve growth stimulation and brain cell Nerve growth stimulation and brain cell

protection. protection.

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SCREENING MODELSSCREENING MODELS

INVITRO METHODS:INVITRO METHODS:

a) inhibition of acetyl cholinesterase a) inhibition of acetyl cholinesterase activity in rat striatum.activity in rat striatum.

b) inhibition of butyryl cholinesterase b) inhibition of butyryl cholinesterase activity in human serum.activity in human serum.

c) release of ach and other c) release of ach and other transmitters from rat brain slices. transmitters from rat brain slices.

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IN VIVO METHODSIN VIVO METHODS

1.1. Passive AvoidancePassive Avoidance

2.2. Active AvoidanceActive Avoidance

3.3. Discriminational learningDiscriminational learning

4.4. Conditioned responsesConditioned responses

5.5. Studies in aged monkeysStudies in aged monkeys

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IN- VITRO METHODSIN- VITRO METHODS1.Inhibition of acetyl cholinesterase activity 1.Inhibition of acetyl cholinesterase activity

in rat striatum in rat striatum Purpose and rationalePurpose and rationale

effects of various cholinesterase inhibitors on the effects of various cholinesterase inhibitors on the two major molecular form of acetylcholinesterase two major molecular form of acetylcholinesterase isolated from rat striatum and cerebral cortex. isolated from rat striatum and cerebral cortex.

PROCEDURE The procedure is divided into three main parts: I. Preparation and isolation of molecular forms of AcheII. Assays for the marker enzymes, III. Enzyme inhibition studies.

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ASSAYASSAYBlankBlank: 0.8 ml PO4 buffer/DTNB: 0.8 ml PO4 buffer/DTNB

0.8 ml buffer/Substrate0.8 ml buffer/Substrate

ControlControl: 0.8 ml PO4 buffer/DTNB/Enzyme: 0.8 ml PO4 buffer/DTNB/Enzyme 0.8 ml PO4 buffer/Substrate0.8 ml PO4 buffer/Substrate

DrugDrug: : 0.8 ml PO4 buffer/DTNB/Drug/Enzyme0.8 ml PO4 buffer/DTNB/Drug/Enzyme0.8 ml PO4 buffer/Substrate0.8 ml PO4 buffer/Substrate

EVALUATION

For IC50 determinations: Substrate concentration is 10 mM diluted 1 : 2 in an assay yielding a final concentration of 5 mM. DTNB concentration is 0.5 mM yielding 0.25 mM final concentration 100

% Inhibition = Slope control − Slope drug /Slope Control × 100

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2.In vitro inhibition of butyrylcholine-esterase 2.In vitro inhibition of butyrylcholine-esterase activity in human serumactivity in human serum PURPOSE AND RATIONALE PURPOSE AND RATIONALE

This assay can be used in conjunction with theThis assay can be used in conjunction with the acetylcholine-esterase assay to determine the acetylcholine-esterase assay to determine the enzyme selectivity of various cholinesterase enzyme selectivity of various cholinesterase inhibitors .Butyrylcholine-esterase (BChE), which is inhibitors .Butyrylcholine-esterase (BChE), which is sometimes called pseudocholinesterase, sometimes called pseudocholinesterase, preferentially hydrolyzes butyrylcholine. Thispreferentially hydrolyzes butyrylcholine. This enzyme is found in the highest amounts in serum,enzyme is found in the highest amounts in serum, but its physiological role is not known but its physiological role is not known Ethopropazine and tetra-isopropyl Ethopropazine and tetra-isopropyl pyrophosphoramide are selective inhibitors of pyrophosphoramide are selective inhibitors of butyrylcholinesterase. butyrylcholinesterase.

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Assay Assay

Enzyme activity is measured with spectrophotometer.Enzyme activity is measured with spectrophotometer.Blank: 0.8 ml PO4 buffer/DTNB Blank: 0.8 ml PO4 buffer/DTNB 0.8 ml buffer/Substrate 0.8 ml buffer/Substrate

Control: 0.8 ml PO4 buffer/DTNB/Enzyme Control: 0.8 ml PO4 buffer/DTNB/Enzyme 0.8 ml PO4 buffer/Substrate 0.8 ml PO4 buffer/Substrate

Drug: 0.8 ml PO4 buffer/DTNB/Drug/Enzyme Drug: 0.8 ml PO4 buffer/DTNB/Drug/Enzyme 0.8 ml PO4 buffer/Substrate 0.8 ml PO4 buffer/Substrate

EVALUATION

For IC50 determinations: Substrate concentration is 10 mM diluted 1 : 2 in assay yielding final concentration of 5 mM. DTNB concentration is 0.5 mM yielding 0.25 mM final concentration

% Inhibition =slope control - slope drug/

control slope ×100

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PASSIVE AVOIDANCEPASSIVE AVOIDANCE

1.1. Step downStep down

2.2. Step throughStep through

3.3. Scopolamine induced amnesia in Scopolamine induced amnesia in ratsrats

4. Up hill avoidance4. Up hill avoidance

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I.PASSIVE AVOIDANCEI.PASSIVE AVOIDANCE

1.Scopolamine-induced amnesia in mice1.Scopolamine-induced amnesia in micePURPOSE AND RATIONALEPURPOSE AND RATIONALE

The administration of the antimuscarinic agent scopolamine The administration of the antimuscarinic agent scopolamine

to young human volunteers produces transient memory to young human volunteers produces transient memory deficits .Analogously, scopolamine has been shown to deficits .Analogously, scopolamine has been shown to impair memory retention when given to mice shortly before impair memory retention when given to mice shortly before training in a dark avoidance task .training in a dark avoidance task .

PROCEDURE The scopolamine test is performed in groups of 10 male mice weighing 26–32 g in a one-trial. Five min after i.p. administration of 3 mg/kg scopolamine hydrobromide, each mouse is individually placed in the bright part of a two-chambered apparatus for training. After a brief orientation period, the mouse enters the second, darker chamber. Once inside the second chamber, the door is closed which prevents the mouse from escaping, and a 1 mA, 1-s foot shock is applied through the grid floor. The mouse is then returned to the home cage. Twenty-four hours later, testing is performed by placing

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animal again in the bright chamber. The latency in animal again in the bright chamber. The latency in entering the second darker chamber within a 5 min test entering the second darker chamber within a 5 min test session is measured electronically. Whereas untreated session is measured electronically. Whereas untreated control animals enter the darker chamber in the second control animals enter the darker chamber in the second trial with a latency of about 250 s, treatment with trial with a latency of about 250 s, treatment with scopolamine reduces the latency to 50 s. The test compounds scopolamine reduces the latency to 50 s. The test compounds are administered 90 min before training. A prolonged are administered 90 min before training. A prolonged latency indicates that the animal remembers latency indicates that the animal remembers that it has been punished and, therefore, does avoid that it has been punished and, therefore, does avoid the darker chamber. the darker chamber.

EVALUATION EVALUATION Using various doses latencies after treatment with test Using various doses latencies after treatment with test compounds are expressed as percentage of latencies compounds are expressed as percentage of latencies in mice treated with scopolamine only. in mice treated with scopolamine only.

MODIFICATIONS OF THE METHOD

Amnesia can also be induced by pretreatment with Benzodiazepines.

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2.Up-hill avoidance2.Up-hill avoidancePURPOSE AND RATIONALE PURPOSE AND RATIONALE

Many animal species exhibit a negative geotaxis, i.e. the Many animal species exhibit a negative geotaxis, i.e. the

tendency to orient and move towards the top when placed on tendency to orient and move towards the top when placed on

a slanted surface. When placed on a tilted platform with a slanted surface. When placed on a tilted platform with

head facing down-hill, rats and mice invariably turn around head facing down-hill, rats and mice invariably turn around

and move rapidly up the incline and move rapidly up the incline PROCEDURE Rats of both sex were used and maintained under standard conditions. The experimental apparatus is a 50 × 50 cm box with 35 cm high opaque plastic walls. The box can be inclined at different angles. The floor consists of 10 mm diameter stainless steel grid bars placed 13 mm apart. To deliver the tail-shock, a tail-electrode is constructed, consisting of a wire clip connected to a constant current shock source. The animal is first fitted with the tail-electrode and then placed onto the grid with its nose facing down. During baseline-trials the animal’s latency to make a 180° turn and

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the first climbing response is measured. Thereafter the first climbing response is measured. Thereafter

the animal is returned to its home cage. During the the animal is returned to its home cage. During the

experimental trials the latencies are measured and additionally experimental trials the latencies are measured and additionally a tail-shock (1.5 or 2 mA) was administered contingent on the a tail-shock (1.5 or 2 mA) was administered contingent on the first climbing response after the 180° turn. Immediately after first climbing response after the 180° turn. Immediately after the shock the animal is placed in its home cage. Retest is the shock the animal is placed in its home cage. Retest is performed 24 h later. performed 24 h later.

EVALUATION

The latencies are measured.

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3.Step-down3.Step-downPURPOSE AND RATIONALE PURPOSE AND RATIONALE

An animal (mouse or rat) in an open field spends most An animal (mouse or rat) in an open field spends most

of the time close to the walls and in the corners. When of the time close to the walls and in the corners. When

placed on an elevated platform in the center of a rectangular placed on an elevated platform in the center of a rectangular

compartment, it steps down almost immediately compartment, it steps down almost immediately

to the floor to explore the enclosure and to approach to the floor to explore the enclosure and to approach

the wall. the wall.

PROCEDURE Mice or rats of either sex are used. A rectangular box (50 × 50 cm) with electrifiable grid floor and 35 cm fits over the block. The grid floor is connected to a shock device which delivers scrambled foot shocks. A typical paradigm consists of three phases: (1.) Familiarization: The animal is placed on the platform, released after raising the cylinder, and the latency to descend is measured. After 10 s of exploration, it is returned to the home cage.

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(2.) Learning: (2.) Learning: Immediately after the animal has descended from the Immediately after the animal has descended from the platform an unavoidable footshock is applied (Foot-platform an unavoidable footshock is applied (Foot-shock: 50 Hz; 1.5 mA; 1 s) and the animal is returned shock: 50 Hz; 1.5 mA; 1 s) and the animal is returned to the home cage, to the home cage, (3.) Retention Test: (3.) Retention Test: 24 h after the learning trial the animal is again placed on the24 h after the learning trial the animal is again placed on the platform and the step-down latency is measured. The test isplatform and the step-down latency is measured. The test is finished when the animal steps down or remains on the finished when the animal steps down or remains on the platform (cut-off time: 60 s). platform (cut-off time: 60 s).

EVALUATION EVALUATION

The time of descent during the learning phase and the The time of descent during the learning phase and the time during the retention test is measured. A prolongation time during the retention test is measured. A prolongation of the step-down latency is defined as learning. of the step-down latency is defined as learning.

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4.Step-through4.Step-through

PURPOSE AND RATIONALEPURPOSE AND RATIONALE This test uses normal behavior of mice and rats. These This test uses normal behavior of mice and rats. These

animals avoid bright light and prefer dim illumination. animals avoid bright light and prefer dim illumination.

When placed into a brightly illuminated space connected When placed into a brightly illuminated space connected

to a dark enclosure, they rapidly enter the dark to a dark enclosure, they rapidly enter the dark

compartment and remain there. compartment and remain there.

PROCEDURE Mice and rats of either sex are used. The test apparatus consists of a small chamber connected to a larger dark chamber via a guillotine door. The small chamber is illuminated with a 7 W/12 V bulb. The test animals are given an acquisition trial followed by a retention trial 24 h later. In the acquisition trial the animal is placed in the illuminated compartment at a maximal distance from the guillotine door, and the latency to enter the dark compartment is measured.

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Animals that do not step through the door within a cut-off Animals that do not step through the door within a cut-off time: 90 s (mice) or 180 s (rats) are not used. Immediately time: 90 s (mice) or 180 s (rats) are not used. Immediately after the animal enters the dark compartment, the door is after the animal enters the dark compartment, the door is shut automatically and an unavoidable footshock (Footshock: shut automatically and an unavoidable footshock (Footshock: 1 mA; 1 s – mice; 1.5 mA; 2 s – rat) is delivered. The animal 1 mA; 1 s – mice; 1.5 mA; 2 s – rat) is delivered. The animal is then quickly removed (within 10 s) from the apparatus and is then quickly removed (within 10 s) from the apparatus and put back into its home cage. The test procedure is repeated put back into its home cage. The test procedure is repeated with or without drug. The cut-off time on day 2 is 300 s with or without drug. The cut-off time on day 2 is 300 s (mice) or 600 s (rats), respectively. (mice) or 600 s (rats), respectively.

EVALUATION EVALUATION

The time to step-through during the learning phase is The time to step-through during the learning phase is measured and the time during the retention test is measured. measured and the time during the retention test is measured. In this test a prolongation of the step-through latencies In this test a prolongation of the step-through latencies is specific to the experimental situation. An increase is specific to the experimental situation. An increase of the step-through latency is defined as learning. of the step-through latency is defined as learning.

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II.ACTIVE AVOIDANCEII.ACTIVE AVOIDANCE1.RUN AWAY AVOIDANCE1.RUN AWAY AVOIDANCE

PURPOSE AND RATIONALE PURPOSE AND RATIONALE

A straightforward avoidance situation features a fixed aversive A straightforward avoidance situation features a fixed aversive gradient which can be traversed by the animal. The shock gradient which can be traversed by the animal. The shock can be avoided when the safe area is reached within the can be avoided when the safe area is reached within the time allocatedtime allocated

PROCEDURE Mice or rats of either sex are used and maintained under standard conditions and handled for several days before the experiment. The same box as used in the step-through model can be used. The apparatus is uniformly illuminated by an overhead light source. A loudspeaker, mounted 50 cm above the start-box, serves for presenting the acoustic conditioned stimulus (audiogenerator). The footshock is employed .The animal is allowed to explore the whole apparatus for 5 min.

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The guillotine door is then closed and the animal The guillotine door is then closed and the animal is placed into the light starting area. After 10 s the is placed into the light starting area. After 10 s the acoustic CS is applied and the door is simultaneously acoustic CS is applied and the door is simultaneously opened. Shock is turned on after 5 s. The CS continuous opened. Shock is turned on after 5 s. The CS continuous until the animal reaches the safe area. It is left there for 50 until the animal reaches the safe area. It is left there for 50 70 s (inter trial interval, ITI) before returned to the same 70 s (inter trial interval, ITI) before returned to the same area again. The procedure starts again. The training is area again. The procedure starts again. The training is continued until the animal attains the criterion of 9 avoidancescontinued until the animal attains the criterion of 9 avoidances in 10 consecutive trials. On the next day the procedure is in 10 consecutive trials. On the next day the procedure is repeated until the same learning criterion is reached. The timerepeated until the same learning criterion is reached. The time needed to reach the safe area is measured. needed to reach the safe area is measured.

EVALUATION The time the animal needs to reach the safe area on both days is measured. In addition, the number of errors (not reaching the safe area) is recorded.

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2.Shuttle box avoidance2.Shuttle box avoidance (two-way(two-way shuttle box)shuttle box)

PURPOSE AND RATIONALEPURPOSE AND RATIONALE Compared to runway avoidance, shuttle box avoidance Compared to runway avoidance, shuttle box avoidance

(two-way-shuttle-box) is a more difficult task. Since (two-way-shuttle-box) is a more difficult task. Since

the animal is not handled between trials, the shuttle-the animal is not handled between trials, the shuttle-

box can be easily automatedbox can be easily automated

PROCEDURE Rats of both sex are used and maintained under standard conditions. The apparatus used consists of a rectangular box 50 × 15 cm with 40 cm high metal walls, and an electrifiable grid floor. The box is divided by a wall with a manually or solenoid-operated guillotine door (10 × 10 cm). into two 25 × 15 cm compartments. Each compartment can be illuminated by a 20 W bulb mounted in the hinged Plexiglas lids.

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A fixed resistance shock source with an automatic switch (0.5A fixed resistance shock source with an automatic switch (0.5 s on 1.5 s off) is used. Simple programming equipment s on 1.5 s off) is used. Simple programming equipment provides for automatic delivery of the conditioned stimulusprovides for automatic delivery of the conditioned stimulus (CS) and the unconditioned stimulus (US). The apparatus is (CS) and the unconditioned stimulus (US). The apparatus is placed in a dimly lit room with a masking noise background placed in a dimly lit room with a masking noise background (white noise) of 60 dB. The animal is allowed to explore the(white noise) of 60 dB. The animal is allowed to explore the apparatus for 5 min with the connecting door open and the apparatus for 5 min with the connecting door open and the compartment lights switched off. The guillotine door is then compartment lights switched off. The guillotine door is then closed. After 20 s the light is switched on in the compartmentclosed. After 20 s the light is switched on in the compartment containing the animal, and the door containing the animal, and the door is opened. A tone (CS) is presented and 5 s later the is opened. A tone (CS) is presented and 5 s later the floor shock is applied in the illuminated compartment floor shock is applied in the illuminated compartment and continued until the animal escapes to the dark side and continued until the animal escapes to the dark side of the compartment, the connecting door is closed and of the compartment, the connecting door is closed and the shock discontinued. After a variable intertrial interval the shock discontinued. After a variable intertrial interval (ITI; 30–90 s) the light is switched on in the previous (ITI; 30–90 s) the light is switched on in the previous dark compartment, the door is opened and the dark compartment, the door is opened and the animal is required to cross to the other side. animal is required to cross to the other side.

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EVALUATION EVALUATION

The time the animal needs to reach the safe area on The time the animal needs to reach the safe area on

both days is measured. both days is measured.

The training is continued until the animal reaches the The training is continued until the animal reaches the criterion of 9 avoidances in 10 consecutive trials. criterion of 9 avoidances in 10 consecutive trials. Retention is tested at different intervals after the Retention is tested at different intervals after the original training by retraining the animal to the same original training by retraining the animal to the same criterion again.criterion again.

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Active avoidance – Shuttle box avoidanceActive avoidance – Shuttle box avoidance

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3.Jumping avoidance3.Jumping avoidance (one-way shuttle (one-way shuttle box)box)

PURPOSE AND RATIONALE PURPOSE AND RATIONALE Since a high degree of automation and minimum handling Since a high degree of automation and minimum handling are additional requirements for this model, the are additional requirements for this model, the obvious solution is a simplified one-way avoidance, obvious solution is a simplified one-way avoidance, allowing for the spontaneous or forced return of the allowing for the spontaneous or forced return of the animal to the start. In order to enhance the start-goal animal to the start. In order to enhance the start-goal distinction a vertical gradient is introduced which requires distinction a vertical gradient is introduced which requires the animal to perform a discrete response of an the animal to perform a discrete response of an all-or-none character, such as the jump, which clearly all-or-none character, such as the jump, which clearly differs from the more continuous translational movements differs from the more continuous translational movements required in the usual avoidance tasksrequired in the usual avoidance tasks

PROCEDURE Rats of both sex are used and maintained under standard conditions. The apparatus used consists of a rectangular box 40 × 25 cm with 40 cm high metal walls, an electrifiable grid floor and a Plexiglas ceiling. A 12 × 12 × 25 cm opaque plastic pedestrial, mounted onto one of the narrow walls of the box provides the isolated goal area

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Flush with the horizontal surface of the pedestal moves a Flush with the horizontal surface of the pedestal moves a vertical barrier, which can either be retracted to the rear wallvertical barrier, which can either be retracted to the rear wall of the apparatus to expose the goal area or pushed forward of the apparatus to expose the goal area or pushed forward to block access to the goal completely. The animal is placed to block access to the goal completely. The animal is placed into the apparatus for 5 min with the goal area exposedinto the apparatus for 5 min with the goal area exposed (barrier retracted). The barrier is then moved forwards and (barrier retracted). The barrier is then moved forwards and the goal is blocked for 2 s. The first trial starts by exposing the goal is blocked for 2 s. The first trial starts by exposing the goal area and applying an acoustic CS (1 000 Hz, the goal area and applying an acoustic CS (1 000 Hz, 85 dB). Electric shocks – US (1.0 mA; 50 Hz; 0.5 s) – 85 dB). Electric shocks – US (1.0 mA; 50 Hz; 0.5 s) – are applied 5 s later (once per 2 s), and continued together are applied 5 s later (once per 2 s), and continued together with the CS until the animal jumps onto the platform. with the CS until the animal jumps onto the platform. After 30 s the barrier pushes the animal off the After 30 s the barrier pushes the animal off the platform onto the grid floor. The sequence is repeated platform onto the grid floor. The sequence is repeated until the criterion of 10 consecutive avoidances is until the criterion of 10 consecutive avoidances is reached. Retention is tested on the next day until the reached. Retention is tested on the next day until the animal reaches criterion. animal reaches criterion.

EVALUATION The time the animal needs to reach the safe area on both days is measured. In addition, the number of errors (not reaching the safe area) is recorded.

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III.DISRIMINATIONAL LEARNINGIII.DISRIMINATIONAL LEARNING

1. 1. SPATIAL HABITUAL LEARNINGSPATIAL HABITUAL LEARNINGPURPOSE AND RATIONALE

The open-field test utilizes the natural tendency of rodents to explore novel environments in order to open up new nutrition, reproduction and lodging resources The rate of exploratory behaviors exhibited in an unfamiliar environment is limited through the inherent necessity to avoid potential dangers. The observed behavior therefore is always a compromise between these conflicting interests and is regulated in part by the momentary physiological needs.Spatial habituation learning is defined as a decrement in reactivity to a novel environment after repeated exposure to that now familiar environment. This reduction in exploratory behaviors during re-exposures is interpreted in terms of remembering or recognition of the specific physical characteristics of the environment. The test can be used to examine short-term spatial memory and/or long-term spatial memory

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PROCEDURE PROCEDURE

The open-field apparatus is a rectangular chamber (rats: The open-field apparatus is a rectangular chamber (rats: 60 × 60 × 40 cm, mice: 26 × 26 × 40) made of painted 60 × 60 × 40 cm, mice: 26 × 26 × 40) made of painted wood or grey PVC. A 25 W red or green light bulb is wood or grey PVC. A 25 W red or green light bulb is placed either directly above or beneath the maze to placed either directly above or beneath the maze to achieve an illumination density at the centre of approximately achieve an illumination density at the centre of approximately 0.3 lx. Masking noise is provided by a broad 0.3 lx. Masking noise is provided by a broad spectrum noise generator (60 dB). Prior to each trial, spectrum noise generator (60 dB). Prior to each trial, the apparatus is swept out with water containing 0.1% the apparatus is swept out with water containing 0.1% acetic acid. Housing room and the testing location are acetic acid. Housing room and the testing location are separated and animals are transported to the testing separated and animals are transported to the testing room 30 min before testing. The digitized image of room 30 min before testing. The digitized image of the path taken by each animal is stored and analyzed the path taken by each animal is stored and analyzed post hoc with a semi-automated analysis system.post hoc with a semi-automated analysis system. The rodent is placed on the center or in a corner of the open-The rodent is placed on the center or in a corner of the open-field for 5–10 minute sessions (mice: up to 20 min, because offield for 5–10 minute sessions (mice: up to 20 min, because of the high basal activity level). The animals are re-exposed to the high basal activity level). The animals are re-exposed to the open-field 24 and 96 h after the initial trialthe open-field 24 and 96 h after the initial trial

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EVALUATION EVALUATION

The exploratory behaviours registered are: The exploratory behaviours registered are:

(1) Rearings or vertical activity: the number of (1) Rearings or vertical activity: the number of times an animal was standing on its hind legs times an animal was standing on its hind legs with forelegs in the air or against the wall.with forelegs in the air or against the wall.

(2) The duration of single rearings as (2) The duration of single rearings as

a measure of non-selective attentiona measure of non-selective attention

(3) Locomotion or horizontal activity: the distance in (3) Locomotion or horizontal activity: the distance in centimeters an animal moved. centimeters an animal moved.

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3.Visual discrimination3.Visual discrimination PURPOSE AND RATIONALEPURPOSE AND RATIONALE

Vision is better than any other sensory system for the Vision is better than any other sensory system for the analysis of spatial relationships in the environment of analysis of spatial relationships in the environment of the animal. From the retina to the cerebral cortex, the the animal. From the retina to the cerebral cortex, the organization of the visual system ensures processing organization of the visual system ensures processing of visual information according to simple principles, of visual information according to simple principles, i.e. by fitting the distribution of light over the receptive i.e. by fitting the distribution of light over the receptive surface to elementary geometrical concepts and by comparing surface to elementary geometrical concepts and by comparing these patterns with images stored in the memory. these patterns with images stored in the memory. Experimental studies of pattern discrimination Experimental studies of pattern discrimination must take into account the visual capability of the given must take into account the visual capability of the given species and present the discriminanda under conditions species and present the discriminanda under conditions compatible with light sensitivity and acuity of the eye. compatible with light sensitivity and acuity of the eye. The constructions of the apparatus should ensure that The constructions of the apparatus should ensure that the discriminanda are viewed from one optimum distance the discriminanda are viewed from one optimum distance and for a sufficient period of time. and for a sufficient period of time.

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PROCEDURE PROCEDURE Rats and mice of both sexes are used and maintained under Rats and mice of both sexes are used and maintained under standard conditions. The apparatus consists of a square 10 × standard conditions. The apparatus consists of a square 10 × 10 cm start area separated by a Plexiglas sliding door from 10 cm start area separated by a Plexiglas sliding door from the choice area, which is connected by swing doors to the goalthe choice area, which is connected by swing doors to the goal compartment. The grid floor in the starting and the choice compartment. The grid floor in the starting and the choice areas is electrifiable. The stimulus cards can be attached toareas is electrifiable. The stimulus cards can be attached to the swing doors. The patterns are black on a white the swing doors. The patterns are black on a white background and have different forms. The apparatus is background and have different forms. The apparatus is illuminated by a dim light. The animal is placed into the illuminated by a dim light. The animal is placed into the apparatus with all doors open and allowed to explore it. Then apparatus with all doors open and allowed to explore it. Then it is placed in the start and after 5 s released by it is placed in the start and after 5 s released by raising the Plexiglas door. After another 5 s, electric shocks raising the Plexiglas door. After another 5 s, electric shocks are applied until the animal escapes through either of the are applied until the animal escapes through either of the open doors to the safe goal compartment where it is left for open doors to the safe goal compartment where it is left for some seconds. As soon as this preliminary step is mastered, some seconds. As soon as this preliminary step is mastered, the stimulus cards are inserted, the negative door is locked the stimulus cards are inserted, the negative door is locked and the grid section in front of this door is electrified. The and the grid section in front of this door is electrified. The animal is trained to a criterion. animal is trained to a criterion.

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EVALUATION EVALUATION

The number of correct answers as well as the The number of correct answers as well as the number of trials until the criterion is reached are number of trials until the criterion is reached are counted. counted.

On the next day the animal is retrained to the same criterion and retention is expressed in savings. Another parameter which can be used to evaluate the savings is the cumulative number of errors until the criterion is reached .

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3.Spatial learning in the water maze3.Spatial learning in the water maze PURPOSE AND RATIONALEPURPOSE AND RATIONALE

A task was developed where rats learn to swim in a A task was developed where rats learn to swim in a water tank to find an escape platform hidden under water tank to find an escape platform hidden under the water .As there are no proximal cues the water .As there are no proximal cues to mark the position of the platform, the ability to locate to mark the position of the platform, the ability to locate it efficiently will depend on the use of a configuration it efficiently will depend on the use of a configuration of the cues outside the tank. Learning is reflected of the cues outside the tank. Learning is reflected on the shorter latencies to escape and the decrease on on the shorter latencies to escape and the decrease on the length of the path to find the platform. Although the length of the path to find the platform. Although rodents can find the platform by using non-spatial strategies, rodents can find the platform by using non-spatial strategies, the use of a spatial strategy is the most efficient the use of a spatial strategy is the most efficient way to escape and young animals develop the spatial way to escape and young animals develop the spatial strategy after a small number of trials. strategy after a small number of trials.

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PROCEDURE PROCEDURE Different strains of rats are generally used. The Different strains of rats are generally used. The

apparatus is a circular water tank filled to a depth apparatus is a circular water tank filled to a depth of 20 cm with 25 °C water . Four points equally of 20 cm with 25 °C water . Four points equally distributed along the perimeter of the tank serve distributed along the perimeter of the tank serve as starting locations. The tank is divided in four as starting locations. The tank is divided in four equal quadrants and a small platform (19 cm equal quadrants and a small platform (19 cm height) is located in the centre of one of the height) is located in the centre of one of the quadrants. The platform remains in the same quadrants. The platform remains in the same position during the training days. The rat is position during the training days. The rat is released into the water and allowed 60–90 s. to released into the water and allowed 60–90 s. to find the platform. Animals usually receive 2–4 find the platform. Animals usually receive 2–4 trials per day for 4–5 days until they escape onto trials per day for 4–5 days until they escape onto the platform. Well-trained rats escape in less than the platform. Well-trained rats escape in less than 10 s. 10 s.

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EVALUATION EVALUATION

The latency to reach the escape platform is measured The latency to reach the escape platform is measured during the training days. A free-swim trial is generally during the training days. A free-swim trial is generally performed after the training days where the escape platform performed after the training days where the escape platform is removed and the animal is allowed to swim for is removed and the animal is allowed to swim for 30 s. With the help of a video system, the latency to 30 s. With the help of a video system, the latency to reach the previous position of the platform, the number reach the previous position of the platform, the number of annulus crossings as well as the time the rat spent in of annulus crossings as well as the time the rat spent in the training quadrant are measured. Well-trained rats the training quadrant are measured. Well-trained rats show short latencies, a large number of annulus crossings show short latencies, a large number of annulus crossings and bias to the quadrant where the escape platform and bias to the quadrant where the escape platform was located during the training sessions. was located during the training sessions.

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IV.STUDIES IN AGED MONKEYSIV.STUDIES IN AGED MONKEYS

1.Long-term potentiation in hippocampal 1.Long-term potentiation in hippocampal slicesslices

PURPOSE AND RATIONALEPURPOSE AND RATIONALE

Long-term potentiation (LTP) in the hippocampus is Long-term potentiation (LTP) in the hippocampus is the most dramatic example of activity-dependent the most dramatic example of activity-dependent synaptic plasticity that has yet been identified in synaptic plasticity that has yet been identified in the mammalian brain.. The fact that it occurs in the the mammalian brain.. The fact that it occurs in the hippocampus has done much to stimulate interest in LTP as ahippocampus has done much to stimulate interest in LTP as a synaptic model of memory, since the importance of the synaptic model of memory, since the importance of the hippocampus for memory processing has been evident ever hippocampus for memory processing has been evident ever since the discovery that its bilateral removal in man causes since the discovery that its bilateral removal in man causes a profound impairment in the ability to lay down newa profound impairment in the ability to lay down new memories.memories.The particular popularity of the slice preparation prepared from the rodent hippocampus rests on its lamellar and laminar organization.

5050

PROCEDURE PROCEDURE

Transverse slices, 400 mm thick, are cut from the Transverse slices, 400 mm thick, are cut from the hippocampus of male albino guinea pigs weighing hippocampus of male albino guinea pigs weighing 250–300 g and prepared for electrophysiological recordings. 250–300 g and prepared for electrophysiological recordings. slices are incubated for 90–120 min in the recording chamber slices are incubated for 90–120 min in the recording chamber to allow equilibration with artificial cerebrospinal to allow equilibration with artificial cerebrospinal fluid. They are submerged, placed on a nylon mesh fluid. They are submerged, placed on a nylon mesh and perfused at a flow rate of 2–2.5 ml/min with oxygenated and perfused at a flow rate of 2–2.5 ml/min with oxygenated (95% O2/5% CO2) cerebrospinal fluid having the following (95% O2/5% CO2) cerebrospinal fluid having the following composition (in mM): NaCl 124, KCl 3.3, CaCl2 2.5, KH2PO4 composition (in mM): NaCl 124, KCl 3.3, CaCl2 2.5, KH2PO4 1.25, MgSO4 2, NaHCO3 25,7, glucose 10. The recording 1.25, MgSO4 2, NaHCO3 25,7, glucose 10. The recording chamber is maintained at 33 ±2 °C. The extra cellular chamber is maintained at 33 ±2 °C. The extra cellular population spike is obtained population spike is obtained using glass microelectrodes filled with 2 M using glass microelectrodes filled with 2 M NaCl, The electrodes are placed into the stratum pyramidalNaCl, The electrodes are placed into the stratum pyramidal of CA1 or CA3. The signal is amplified and stored on of CA1 or CA3. The signal is amplified and stored on magnetic discs for later analysis. The evoked responses are magnetic discs for later analysis. The evoked responses are averaged and analyzed off-line using a personal computer. averaged and analyzed off-line using a personal computer.

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The magnitude of the population spike is evaluated The magnitude of the population spike is evaluated by taking the voltage difference between the negative by taking the voltage difference between the negative peak and the following positive peak. Either mossy peak and the following positive peak. Either mossy fibers in the hilus fasciae or commissural/associational fibers in the hilus fasciae or commissural/associational fibers in the stratum radiatum are activated via bipolar, fibers in the stratum radiatum are activated via bipolar, sharpened silver wire electrodes insulated except sharpened silver wire electrodes insulated except for the tips. Constant current pulses (100 ms) are delivered for the tips. Constant current pulses (100 ms) are delivered

with a frequency of 0.2 Hz, only during the test intervals. The stimulation intensity is adjusted to elicit the population spike of about 40% and 80% of its maximal amplitude in CA1 and CA3, respectively. After the baseline is recorded for 10–20 min, LTP is induced by repetitive stimulation of 100 pulses at 20 Hz for 5 s in CA1 and at 50 Hz for 2 s in CA3 at the same strength as for the test pulses. Responses by test pulses are recorded 0, 10, 20 and 30 min after repetitive stimulation. The test drugs are dissolved in the artificial cerebrospinal fluid and applied extracellularly at various concentrations by switching perfusion reservoirs.

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EVALUATION EVALUATION

The time course of LTP is registered for CA1 and CA3. The time course of LTP is registered for CA1 and CA3.

The mean percent increase in the amplitude of the The mean percent increase in the amplitude of the

population spike from baseline responses after drug population spike from baseline responses after drug

application is compared with controls. application is compared with controls.

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REFERENCEREFERENCE Tripathi .K.D.; CNS stimulants and cognition Tripathi .K.D.; CNS stimulants and cognition

enhancers. In: Essentials of medical enhancers. In: Essentials of medical pharmacology ;Edn no 5, Publisher: Jaypee pharmacology ;Edn no 5, Publisher: Jaypee brothers EMCA house , New Delhi.brothers EMCA house , New Delhi.

pp: 435 – 442.pp: 435 – 442.

H.Gerhard Vogel and Wolfgang.H.Vogel.Psychotropic and neurotropic activity,In; Drug Discovery and Evaluation, 5th edition,Springer-Verlag Berlein Heidelberg,Germany, pp-496-548.

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