1. The document describes various methods for screening nootropic substances, including in vivo, in vitro, and molecular-level experiments. 2. Some key in vivo methods discussed are passive avoidance testing in rats/mice, active avoidance conditioning experiments, and electrophysiological studies like long-term potentiation experiments in hippocampal brain slices. 3. In vitro screening methods outlined involve measuring inhibition of acetylcholinesterase activity from rat brain tissue and butyrylcholinesterase activity from human serum. Molecular-level experiments involve analyzing effects on acetylcholine receptors and neurotransmitter release.

1. SCREENING METHOD OF
NOOTROPICS
NOIDA INSTITUTE OF ENGINEERING AND TECHNOLOGY
(PHARMACY INSTITUTE)
GREATER NOIDA
PRESENTED BY UNDER GUIDENCE
VIKAS MALIK DR. SAUMYA DAS
M.PHARMA.(PHARMACOLOGY) ASSOCIATE PROFESSOR
PTSM-1 NIET,GR. NOIDA

2. CONTENT
 INTRODUCTION 0F MEMORY
 WHAT IS MEMORY ENHANCER/NOOTROPICS
 CLASSIFICATION OF NOOTROPICS
 IN VIVO METHOD
 IN VITRO METHOD
 EXPERIMENT AT MOLECULAR LEVEL

3. MEMORY
•It is defined as the process by which
information is encoded, stored and retrieved
•“Memory” is a slippery concept because it is
still not clear about complete, consensual notion
about the physical nature of its trace.
• The only sure way to grab at such phenomenon
is by measuring behaviors and their
modifications i.e. quantifying it in an indirect
fashion.
• Such approach is called “phenomenological”,
and opposes itself to the so-called “mechanistic”
vision, that departs from previously existent
knowledge about the intrinsic machinery
operating behind the phenomenon

4. MEMORY ENHANCERS/NOOTROPICS
The substance used to enhance cognitive function.
It can increase memory,motivation,mood or anything related to cognition and thought.
They are referred to as “smart” drugs
HISTORY - The term ‘nootropic’ was coined by DR CORNELEU GUERGEA in 1972. It is a Greek
word with combination of ‘nous’-mind and ‘trepein’- to bend.
In 1964,he was the first person to synthesize the first nootropic named
PIRACETAM.

5. Classification of nootropics
 RACETAM(piracetam,oxiacetam) –piracetam is first nootropic agent discovered
 Choline (Acetylcholine Precursors)-(lecithin)
 Acetyl cholinesterase Inhibitors (Over-The-Counter)
 Huperzine A
 Ampakines(aniracetam)
 Herbal
 Bacopa Monneri
 Vinpocetine
 Gingko biloba
 Rhodiola rosea
 Mucuna pruriens – precursor to dopamine
 Gotu Kola
 Other Nootropics
 Sulbutiamine(vitamin b1 derivative)
 Pyritinol
 Citicholine
 L-theanine
 Picamilon
 Phenylethylamine (PEA)
 noopept(peptide)
 Dihydroergotoxine
 Piribedil

6.  IN VIVO METHODS
 PASSIVE AVOIDANCE
 ACTIVE AVOIDANCE
 DISCRIMINATION LEARNING
 CONDITIONED RESPONSE
 STUDY IN MONKEYS
 ELECTROPHYSIOLOGICAL METHODS
 ANIMAL WITH MEMORY DEFICIT
 IN VITRO METHOD
 In vitro inhibition of acetylcholine-esterase activity in rat striatum
 Inhibition of butyrylcholine-esterase activity in human serum
 Cholinesterase inhibition
 EXPERIMENTS AT MOLECULAR LEVEL
 Molecular forms of acetyl cholinesterase from rat frontal cortex and
striatum
 [3H]Oxotremorine-M binding to muscarinic cholinergic receptors in rat
forebrain
 [3H]-N-Methylscopolamine binding in the presence and absence of
Gpp(NH)p
RELEASE OF [3H]ACH AND OTHER TRANSMITTERS FROM RAT BRAIN SLICES

7. 1.PASSIVE AVOIDANCE(aversive task)
It is usually employed to describe experiments in which animal learns to avoid a noxious event by
suppressing a particular behavior
STEP DOWN-Purpose and rationale- An animal(mouse or rat) spends most of the time close to walls
and in corners. When placed on an elevated platform in the center of rectangular compartment, it
steps down immediately to the floor to explore the enclosure and to approach the wall

8. PROCEDURE-
Mice/rat of either sex used. A rectangular box (50x50cm) 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 3 phases:
Familiarization(animal is placed on platform, released after raising the cylinder and latency is
measured. After 10s of exploration, it is returned to home cage)
Learning(immediately after animal descends from platform an unavoidable foot shock applied-
50hz,1.5ma,1s and animal returned to home cage)
Retention test (24 h after the learning trial the animal is again placed on the platform and the
step-down latency is measured. The test is finished when the animal steps down or remains on
the platform (cut-off time: 60 s).
EVALUATION The time of descent during the learning phase and the time during the retention
test is measured. A prolongation of the step- down latency is defined as learning.

9. 2.- ACTIVE AVOIDANCE
Active avoidance learning is a fundamental behavioral phenomenon . As in other instrumental
conditioning paradigms the animal learns to control the administration of the unconditioned stimulus by
appropriate reactions to the conditioned stimulus preceding the noxious stimulus. The first stage of
avoidance learning is usually escape, whereby a reaction terminates the unconditioned stimulus
RUN AWAY AVOIDENCE
PURPOSE AND RATIONALE A straightforward avoidance situation features a fixed aversive gradient which
can be traversed by the animal. The shock can be avoided when the safe area is reached within the
time allocated

10. PROCEDURE
The same box as used in the step-through model can be used in this experiment.
A loudspeaker mounted above the starbox serves for presenting the acoustic conditioned
stimulus
The animal is allowed to explore the whole apparatus for 5 min. The guillotine door is
then closed and the animal is placed into the light starting area. After 10 s the acoustic
CS is applied and the door is simultaneously opened.
Shock is turned on after 5 s.The training is continued until the animal attains the
criterion of 9 avoidances in 10 consecutive trials.
EVALUATION •The time the animal needs to reach the safe area on is recorded. •Also,
the number of errors committed is recorded

11. 3- Electrophysiological methods
LONG-TERM POTENTIATION IN HIPPOCAMPAL SLICES
PURPOSE AND RATIONALE - This procedure is perhaps the most dramatic example of activity-
dependent synaptic plasticity that has yet been identified in the mammalian brain . A brief tetanus
to any one of a number of monosynaptic excitatory pathways in the hippocampus can enhance the
amplitude of evoked responses in the tetanized pathway for hours or days thereafter. The fact that
it occurs in the hippocampus has done much to stimulate interest in LTP as a synaptic model of
memory
PROCEDURE
•Transverse slices, are cut from the hippocampus of guinea pigs which are incubated for 90–120 min
in the recording chamber to allow equilibration with artificial cerebrospinal fluid.
• They are submerged, placed on a nylon mesh and perfused at a flow rate of 2–2.5 ml/min with
oxygenated cerebrospinal fluid having the following composition (in mM): NaCl 124, KCl 3.3, CaCl2
2.5, KH2PO4 1.25, MgSO4 2, NaHCO3 25,7,glucose 10.
• The electrodes are placed into the stratum pyramidale of CA1 or CA3.
• The signal is amplified and stored on magnetic discs for later analysis.
•After the baseline is recorded for 10–20 min, LTP is induced by repetitive stimulation in CA1 and in
CA3 at the same rate and are recorded 0, 10, 20 and 30 min after repetitive stimulation.
EVALUATION The time course of LTP is registered for CA1 and CA3.The mean percent increase in the
amplitude of the population spike from baseline responses after drug application is compared with
controls.

12. 4- STEP THROUGH
PURPOSE AND RATIONALE •This test uses normal behavior of mice and rats. • These animals avoid bright
light and prefer dim illumination. • When placed into a brightly illuminated space connected to a dark
enclosure, they rapidly enter the dark compartment and remain there.

13. PROCEDURE
• The test apparatus consists of a small chamber connected to a larger dark
chamber via a guillotine door.
•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.
• Immediately after the animal enters the dark compartment, the door is shut
automatically and an unavoidable foot shock is delivered.
EVALUATION
•The time to step-through during the learning phase is measured and the time
during the retention test is measured.
• In this test a prolongation of the step-through latencies is specific to the
experimental situation. An increase of the step-through latency is defined as
learning.

14. 1. In vitro inhibition of acetylcholine-esterase activity in rat striatum
PURPOSE AND RATIONALE The purpose of this assay is to screen drugs for inhibition of
acetylcholine-esterase activity. Inhibitors of this enzyme may be useful for the treatment
of Alzheimer’s disease. Acetyl cholinesterase (Ache) which is sometimes called true or
specific cholinesterase, is found in nerve cells, skeletal muscle etc Its distribution in brain
roughly correlates with cholinergic innervations and sub fractionation shows the highest
level in nerve terminals. Recent studies have suggested that Ache inhibitors may also be
beneficial in the treatment of Alzheimer’s dementia

15. PROCEDURE
•A 2 mM stock solution of the test drug is made up in a suitable solvent and q.s. to volume with 0.5 mM
DTNB (di thio nitro benzoic acid)
•Drugs are serially diluted (1 : 10) such that the final concentration (in cuvette) is 10–4 M and screened
for activity. •If active, IC50(half maximal inhibitory conc) values are determined from the inhibitory
activity of subsequent concentrations
TISSUE PREPARATION •Male Wistar rats are decapitated, brains rapidly removed, corpora striata
dissected free, weighed and homogenized in 19 volumes.
•A 25 μl aliquot of this suspension is added to 1 ml of the vehicle or various concentrations of the test
drug and reincubate for 10 min

16. ASSAY •Enzyme activity is measured with the Beckman DU-50 spectrophotometer.
•This method can be used for IC50 determinations and for measuring kinetic
constants
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 %inhibition=slope control-slope drug x
100 slope control

17. 2- In vitro inhibition of butyrylcholine-esterase activity in
human serum
PURPOSE AND RATIONALE
This assay can be used in conjunction with the acetylcholine-esterase
assay to determine the enzyme selectivity of various cholinesterase
inhibitors. Butyrylcholine-esterase (BChE), which is called
pseudocholinesterase, preferentially hydrolyzes butyrylcholine.
PROCEDURE Enzyme Preparation - A vial of lyophilized human serum is
reconstituted in 3 ml of distilled water.
A 25 ml aliquot of this suspension is added to 1 ml of the vehicle or various
concentrations of test drug and pre-incubated for 10 min at 37 °C.
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
%inhibition=slope control-slope drug x 100 slope control

18. EXPERIMENTS AT MOLECULAR LEVEL
Molecular forms of acetyl cholinesterase from rat frontal cortex and striatum PURPOSE AND RATIONALE
Different molecular forms of acetyl cholinesterase can be isolated from animal tissues.
The number of forms isolated, their relative amounts and molecular characteristics depend on the tissue
source and the conditions used for solubilization of the membrane bound enzyme
[3H]Oxotremorine-M binding to muscarinic cholinergic receptors in rat forebrain
PURPOSE AND RATIONALE The purpose of this assay is to determine the binding affinity of potential
cholinomimetic drugs for muscarinic receptors in brain, using an agonist ligand