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Presentation on experimental evaluation of memory enhancers

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basic classification,physiology and anatomy,experimental evaluation of animals(in vitro & in vivo) and humans,latest trends and applications

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Presentation on experimental evaluation of memory enhancers

  1. 1. DR AKHIL AGRAWAL DEPT OF PHARMACOLOGY SKNMC PUNE
  2. 2. It is divided into 4 parts: •INTRODUCTION oMemory(basic physiology and anatomy) oMemory enhancers(basics) •CLASSIFICATION •EXPERIMENTAL EVALUATION oIn vitro (Animals) oIn vivo oIn humans •APPLICATION IN MODERN ERA
  3. 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. 4. TYPES OF MEMORY Based on physiological points: Explicit(recognition) o Concerned with facts o Episodic(events) o Semantic(words,rules,language) o Area of imp-hippocampus Implicit (reflexive) oIncludes skill, habits and conditioned reflexes o Priming(facilitate recognition) oNon-associative learning(single stimulus) oAssociative learning(relation of stimulus) oArea of imp-thalamus
  5. 5. TYPES BASED ON DURATION  IT IS DIVIDED INTO THREE TYPES:  Short(seconds-minutes) o Mechanism-(circuit of reverberating neurons)  Intermediate(days-weeks) o Mechanism-(chemical changes in presynaptic and postsynaptic membrane)  Long(years-lifetime) o Mechanism-(structural changes in synapse)
  6. 6. MEMORY PHASES •The Memory Consolidation Theory, proposed by Müller and Pilzecker is a fundamental paradigm in the psychobiology and neuropharmacology of memory • One consequence of the Consolidation Paradigm is that we can plan our experimental intervention to take place in two or three different moments around memory i.e Formation and Recall. •Since the formation is not an instantaneous process, we may distinguish two formation phases, Acquisition, better known as learning, and Consolidation, the labile phase during which the memory trace will be physically stored • Recall, also known as memory retrieval, takes place during the reexposition to the learning context, with or without previously delivered stimuli Acquisition Consolidation Recall (1 or N-trials) 0 min hours / days
  7. 7. 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.
  8. 8. COGNITIVE ENHANCERS V/S NOOTROPICS COGNTIVE ENHANCERS NOOTROPICS  TEMPORARY  TOLERANCE PRESENT  GENERALISED STIMULANT EFFECT  CAN CAUSE COGNITIVE DAMAGE  MOSTLY NATURAL  EG-CAFFEINE  PERMANENT  NO TOLERANCE  ABSENT  NO  MOSTLY ARTIFICIAL  EG-PIRACETAM
  9. 9. CRITERIA FOR A NOOTROPIC TO FULFILL IN ORDER TO QUALIFY FOR CLASSIFICATION(DR GIURGEA’S CRITERIA)  They should boost memory and learning ability.  They should also strengthen the ability of learned forms of behavior as well as memories to withstand forces such as hypoxiation and electroshock treatment.  They should afford protection to the brain from a variety of injuries, either chemical or physical (for example, due to the ingestion of scopolamine or barbiturates).  They should make the controlling mechanisms of the cortex more effective.  Unlike other kinds of psychotropic drugs, they should have no sedative or stimulant effects, as well as possessing negligible side effects.  They should also have virtually no detectable toxicity.
  10. 10. SKONDIA’S CRITERIA  Dr Skondia was a second researcher to attempt to classify nootropics. His criteria was based on a nootropics metabolic approach more than Dr Giurega’s. Specifically 1. The substance possesses no direct vasoactivity (vasodilatation or vasoconstriction). 2. The substance shouldn’t change basic EEG rhythm. 3. The substance must cross the blood brain barrier. 4. The substance must possess metabolic activity in the human brain. 5. The substance must have little/no side effects. 6. The substance must undergo clinical trials which reveal metabolic cerebral improvement.
  11. 11. HOW IT WORKS  NEUROPLASTICITY  This term describes neural tissue that can easily grow new structures like neurons and synapses. Over time, the ability of our brains to remain flexible and adaptable is key to retaining memory function and staving off the degenerative disorders associated with aging.  The brain needs a steady supply of acetylcholine to remain in a state of high neuroplasticity. This key neurotransmitter is related to all of our major cognitive functions, including mental focus and memory formation  MENTAL ENERGY  It is due to the action of catecholamine hormones dopamine, epinephrine, and nor epinephrine. The body naturally produces these hormones to create mental energy in times of “fight or flight”  They cause vasodilatation indirectly i.e. increase in blood supply to brain.  They also show signs of neuro preservation and neuro protection.  They produce their therapeutic effects by augmenting excitatory synaptic transmission in cortical circuits, primarily through positive modulation of α-amino- 3-hydroxy-5-methyl-4-isoxazole-propionate receptors (AMPARs).
  12. 12. Trends in use of memory enhancers •Nature, an online website ran its own informal survey. 1,400 people from 60 countries responded to the online poll. •Usage of 2 drugs- methylphenidate,modafinil were considered for the poll •Among the users, students formed the major chunk followed by office goers.
  13. 13. Classification of nootropics  RACETAM(piracetam,oxiacetam) –piracetam is first nootropic agent discovered  Choline (Acetylcholine Precursors)-(lecithin)  Acetyl cholinesterase Inhibitors (Over-The-Counter)  a) 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
  14. 14. CLASSFICATION OF MEMORY ENHANCERS 1) DRUGS WITH SOME CLINICAL BENEFIT a)Drugs increasing central cholinergic activity(for treatment of AD)- (tacrine,galantamine and donezepil) b) Nootropics c)NMDA receptor(glutamate receptor) antagonist-(memantine) 2) Clinical benefit is uncertain- a)cerebral vasodilators(pyritinol) b)dopamine agonist(piribedil) c)others(ginkgo biloba) DRAWBACKS OF AVAILABLE DRUGS- • Mechanism of action of these drugs are not clear • Therapeutic benefit is also limited • Possible cerebral steal phenomenon • Prolonged use may cause serious side-effects • Diversity in different classes and so poorly classified
  15. 15. Safety of nootropics Common Side Effects Include:  Headache  Jitteriness  Insomnia  Gastrointestinal Problems  There are many other side-effects depending upon individual drug.  Roughly half of users reported unpleasant side effects, and some discontinued use because of them.  As the frequency drug decreases, the frequency of side-effects decrease.
  16. 16. Limitations •Observing an effect in one behavioral task of a specific drug applied into certain brain structure does not warrant that other behavioral tasks would be equally affected •A more diffused, wide-embracing systemic treatment drug is harder to interpret. Since this treatment may simultaneously target very different CNS Structures. •All scientific conclusions obtained from experimental animals cannot be extrapolated to human clinical cases.
  17. 17. IN VIVO METHODS  PASSIVE AVOIDANCE  ACTIVE AVOIDANCE  DISCRIMINATION LEARNING  CONDITIONED RESPONSE  STUDY IN MONKEYS  ELECTROPHYSIOLOGICAL METHODS  ANIMAL WITH MEMORY DEFICIT
  18. 18. 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 o 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
  19. 19. oPROCEDURE-  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 10 s 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).
  20. 20. 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. MODIFICATION •Most common modification is to induce amnesia in animals. •There are different methods to do so including (i) electroconvulsive shock, (ii) scopolamine, (iii) alcohol, (iv) CO2 • Ricceri studied the effect of nerve growth factor on passive avoidance learning and retention
  21. 21. CRITICAL ASSESSMENT •There are some critical parts in the experimental procedure: (i) Placing the animal on the platform, since the tendency of the animal to escape the human hand may shorten the step-down latencies. (ii) Another important point is the timing of the electric shock. It must not be applied at the first contact of the animal with the floor, since the light touch with the forelimbs does not cause the required shock intensity. (iii) It is also necessary to keep the room sound-proof, and this can be done by using a white noise generator (60–70 dB). LIMITATION The variability of this method is relative high, therefore, it is necessary to test large groups of animals (minimum 10 animals per group).
  22. 22. 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. 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.
  23. 23. 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. MODIFICATIONS OF THE TEST •This test procedure is employed in different modifications.To test drugs usually several test-groups can be tested. • (i) CXC-group: vehicle – without amnesia – vehicle; (ii) DXD-group: drug – without amnesia – drug; (iii) CAC-group: vehicle – amnesia – vehicle; (iv) DADgroup: drug – amnesia – drug; (v) CAD-group: vehicle – amnesia – drug; (vi) DAC-group: drug – amnesia – vehicle. •Itokazu showed that l-dopa caused memory deficit in mice by this method and hence recommended as a model in human dementia •LIMITATION AND CRITICAL ASSESMENT Same as previous experiment
  24. 24. TWO COMPARTMENT TEST PURPOSE AND RATIONALE •A rodent in an open field tends to enter any recesses in the walls and to hide there. • When placed into a large box, connected through a narrow opening with a small dark compartment, the animal rapidly finds the entrance into the small chamber, enters it and spends most of its time there. The times spent in the large and small compartments are measured. •The latency of the first entrance into the dark chamber and the number of crossings from one compartment into the other can be used as auxiliary criteria. PROCEDURE •Same as in step down experiment CRITICAL ASSESSMENT AND LIMITATION Same as in step down experiment
  25. 25. UPHILL AVOIDANCE PURPOSE AND RATIONALE  Many animal species exhibit a negative geotaxis, i.e. the tendency to orient and move towards the top when placed on a slanted surface. When placed on a tilted platform with head facing down-hill, rats and mice invariably turn around and move rapidly up the incline PROCEDURE  The experimental apparatus is a 50 × 50 cm box with 35 cm high opaque plastic walls.  The animal is first fitted with the tail-electrode to give shock and then placed onto the grid with its nose facing down. During baseline trials the animal’s latency to make a 180° turn and initiate the first climbing response is measured.  During the experimental trials the latencies are measured and additionally a tail-shock (1.5 or 2 mA) was administered contingent on the first climbing response after the 180° turn.
  26. 26. EVALUATION The latencies are measured CRITICAL ASSESSMENT OF THE METHOD Its most obvious advantage is that it can be administered to animals debilitated in sensory-motor coordination by pharmacological or surgical treatments that would preclude use of other inhibitory (passive) avoidance tasks. LIMITATION Though it is a useful to the existing arsenal of inhibitory avoidance methods, it is still highly variable and hence large number of animals are required
  27. 27. Trial-to-criteria inhibitory avoidance  PURPOSE AND RATIONALE  As animals experience different sensitivity to the foot shock punishment applied in the dark area, immediately after the first trial the animal is returned to the lighted area to evaluate if the task has been acquired.  A criteria is established to determine the learning of the test, usually requiring the animal to remain in the lighted area for a period of 30–60 s.  PROCEDURE  The animals are trained in the same way as in the step-through version. They are placed in the lighted compartment and after they entered with the four paws into the dark area, the door is closed and a mild foot shock is delivered.  The number of trials to attain criteria are counted as an indication of the speed of acquisition.
  28. 28. EVALUATION •The animals are placed in the lighted area, the door opened and the latency to step with the four paws into the dark area is recorded. • A cut-off latency of 180 or 300 s is usually imposed. CRITICAL ASSESSMENT OF THE TEST •This modification of the classical inhibitory avoidance procedure is useful to determine the effect of drugs on acquisition as amnesic drugs significantly increase the number of trials to reach the criterion.(eg-diazepam,scopolmine) •Once the animals have been trained to a predetermined criteria any effect of the drug on the retention trial can be associated to drug effects on consolidation rather on the acquisition process (any nootropic-piracetam) MODIFICATIONS OF THE TEST This test can also be performed as a continuous trial to criterion. In this procedure the door is not closed after the animal enters the dark area and upon delivery of the shock the animal can escape to the lighted area. LIMITATION Same as in uphill avoidance method
  29. 29. Scopolamine-induced amnesia in mice PURPOSE AND RATIONALE  The administration of the antimuscarinic agent scopolamine to young human volunteers produces transient memory deficits  Analogously, scopolamine has been shown to impair memory retention when given to mice shortly before training in a dark avoidance task PROCEDURE  Five min after i.p. administration of 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 foot shock is applied through the grid floor.  The latency in entering the second darker chamber in untreated control animals is 250s, whereas treatment with scopolamine reduces latency to 50 s.  A prolonged latency indicates that the animal remembers that it has been punished and, therefore, does avoid the darker chamber.
  30. 30. CRITICAL ASSESSMENT OF THE METHOD •In spite of the fact that the pathogenesis of primary degenerative dementia (Alzheimer’s disease) in man has been only partially elucidated, the scopolamine amnesia test is widely used as primary screening test for so-called anti-Alzheimer drugs.(eg-Rivastigmine) MODIFICATIONS OF THE METHOD •Lenègre investigated the effects of piracetam on amnesias induced by scopolamine, diazepam and electroconvulsive shock. •Sparks and schreurs reported that trace amounts of copper in water induced B-amyloid plaques and learning deficit in rabbit model for Alzheimer's disease LIMITATION The neuropathology of Alzheimer's disease is not confined to only cholinergic system
  31. 31. Cognitive deficits on chronic low dose MPTP-treated monkeys PURPOSE AND RATIONALE  Röltgen and Schneider described the effect of chronic dose of (MPTP) exposure on cognitive functions in monkeys.  These animals develop cognitive deficits and difficulties in performing previously learned tasks, such as delayed response, delayed alternation and object retrieval, as well as dopamine depletions in several brain areas without severe Parkinsonian signs as found after high MPTP doses (methyl phenyl tetrahydropyridine)
  32. 32. PROCEDURE •Adult monkeys are trained to perform a delay response task while seated in a restraining chair placed inside a sound attenuating modified Wisconsin General Test apparatus. • The monkey sits behind an opaque screen that when raised allows access to a sliding tray that contains recessed food wells. • Monkeys are trained to retrieve a raisin from one of the food wells after observing the experimenter baits the well. •Animals are trained until performance with a 5s delay is 90% correct or better for at least 5 consecutive days •Once the animals are performing at criterion level,MPTP is administered intravenously in doses ranging from 0.05 mg/kg at the start of the study to 0.20 mg/kg. •Animals receive cumulative doses up to 60 mg over periods up to one year. Pharmacological data are obtained after animals consistently show at least a 15% performance deficit on delayed response
  33. 33. EVALUATION •Delayed response performance after drug administration is compared with matched control performance obtained on the same day prior to drug administration The total number of correct responses as well as the number of mistakes and ‘no response’ errors are tabulated for each session. •Date are then expressed as mean (± standard deviation) performance. All animals serve as their own controls. Statistical analysis consists of analysis of variance, repeated measures design, with post hoc comparisons (Bonferroni t- test).
  34. 34. 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
  35. 35. Run away aviodance 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 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.
  36. 36. EVALUATION •The time the animal needs to reach the safe area on is recorded. •Also, the number of errors committed is recorded CRITICAL ASSESSMENT AND LIMITATION Same as in step down method
  37. 37. Shuttle box avoidance (two-way shuttle box) PURPOSE AND RATIONALE  The animal learns that a random stimulus (a tone, the CS) is a reliable predictor for a coming aversive experience , and can prompt an evasive action in order to avoid it, i.e., it moves to the other side of the shuttle box (the CR) when the stimuli predict aversive events.  Since there is the possibility to learn how to escape, this task may be classified as an operant conditioning, i.e., the animal must learn the relation between CS and US in order to anticipate US with a CR (escape) and avoid it.
  38. 38. PROCEDURE •The apparatus used consists of a rectangular box with high metal walls, and an electrifiable grid floor. •Simple programming equipment provides for automatic delivery of the conditioned stimulus (CS) and the unconditioned stimulus (US). The animal is allowed to explore the apparatus for 5 min with the connecting door open and the compartment lights switched off. •The guillotine door is then closed. After 20 s the light is switched on in the compartment containing the animal, and the door is opened. • A tone (CS) is presented and 5 s later the floor shock is applied in the illuminated compartment and continued until the animal escapes to the dark side of the compartment 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) are recorded
  39. 39. CRITICAL ASSESSMENT OF THE METHOD •The task is rather difficult due to the lack of a permanent safe area •Lack of a simple instrumental response •Presence of a variable aversive gradient • Increased weight of emotional factor. MODIFICATIONS OF THE METHOD Salmi described a computer-assisted two way avoidance conditioning equipment for rats. LIMITATION •Compared to runway avoidance, shuttle box avoidance is a more difficult task. • Since the animal is not handled between trials, the shuttle box can be easily automated
  40. 40. Jumping avoidance (one-way shuttle box) PURPOSE AND RATIONALE  It is a simplified one-way avoidance, allowing for the spontaneous or forced return of the animal to the start.  In order to enhance the start-goal distinction a vertical gradient is introduced which requires the animal to perform a discrete response of an all-or-none character, such as the jump, which clearly differs from the more continuous translational movements required in the usual avoidance tasks
  41. 41. PROCEDURE •The apparatus used consists of a rectangular box with high metal walls, electrifiable grid floor and a Plexiglas ceiling. • Flush with the horizontal surface of the pedestal moves a vertical barrier, which can either be retracted to the rear wall of the apparatus to expose the goal area or pushed forward to block access to the goal completely. •The animal is placed into the apparatus for 5 min with the goal area exposed.The barrier is then moved forwards and the goal is blocked for 2 s. • The first trial starts by exposing the goal area and applying an acoustic . Electric shocks – US are applied 5 s later , and continued together with the CS until the animal jumps onto the platform . •After 30 s the barrier pushes the animal off the platform onto the grid floor.
  42. 42. EVALUATION The time the animal needs to reach the safe area on both days is measured. In addition, the number of error (not reaching the safe area) is recorded. CRITICAL ASSESSMENT OF THE METHOD In automated procedures extinction is more rapid, especially when short inter test intervals are used LIMITATION A high degree of automation and minimum handling is required
  43. 43. DISCRIMINATION LEARNING  In the experiments described above the animals have no choice between the conditioned stimuli. They have only one conditioned stimulus.  The experiments can be classified either as simultaneous or successive discrimination paradigms.
  44. 44. Spatial habituation learning PURPOSE AND RATIONALE(classical non-aversive and non-associative task.)  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.  Spatial habituation learning is defined as a decrement in reactivity to a novel environment after repeated exposure to that now familiar environment.  The test can be used to examine short-term spatial memory and/or long-term spatial memory
  45. 45. PROCEDURE •The open-field apparatus is a rectangular chamber made of painted wood or grey PVC. •The rodent is placed on the center or in a corner of the open-field for 5–10 minute sessions EVALUATION The exploratory behaviors registered are: (1) Rearing or vertical activity: the number of times an animal was standing on its hind legs with forelegs in the air o against the wall. (2) The duration of single rearing a a measure of non-selective attention (3) Locomotion or horizontal activity: the distance in centimeters an animal moved.
  46. 46. MODIFICATIONS OF THE TEST •In order to assess emotionality parameters the aversivity of the open-field can be varied by increasing the size and/or the illumination density of the apparatus . • The emotional behaviors registered are: (1) Corner time: the time spent in the 4 corner squares (2) Wall time: the time an animal spent close to the wall as a measure for thigmotaxis (scanning the walls of the apparatus with the vibrissae). (3) Center time: the time spent in the center of the open field . (4) Defecation: number of boli deposited. (5) Freezing: the time the animal stays completely immobile except for movements associated with respiratory activity . CRITICAL ASSESSMENT OF THE TEST The open-field paradigm is a well validated, simple and time economical test, which has been widely used to examine the neurobiological foundations sub serving spatial memory, general activity and emotionality in rodents with different approaches including: lesions, drugs, electrophysiology, neuroanatomy, and neurogenetics LIMITATION The training session is a quick procedure, this task is not simple to be automated
  47. 47. Spatial discrimination PURPOSE AND RATIONALE  In the simplest case of discrimination learning the animal distinguishes between two symmetric stimulus response sets, the equal probability of which has been changes by differential reinforcement events. Position of the cues with respect to the animal’s body defines the CS+ and CS–.  Usually right-left discrimination is employed. PROCEDURE  The apparatus used is usually a simple T- or Y-maze, with an electrifiable grid floor. The last 10 cm of each arm are separated from the rest of the apparatus by a swing-door which prevent the animal from seeing the food cup or the plastic sheet covering the grid in the goal area.  In an aversively motivated spatial discrimination learning the animal is trained to escape and/or to avoid foot shocks by always going to the right.  Then the animal is placed on the start and after 5 s electric shocks (0.5 s, 50 Hz, 1.0 mA) are applied at 3 s intervals.  After a 60 min interval the safe goal area is shifted to the other arm of the maze and the discrimination is reversed
  48. 48. EVALUATION •Errors are scored. An error means that the animal enters the wrong arm with all four legs. • During retention the number of trials until the animal makes correct choices are counted. CRITICAL ASSESSMENT OF THE METHOD •The ecology of rodents makes these animals specially proficient in spatial discrimination learning, which is usually mastered in a few trials. MODIFICATIONS OF THE TEST •Barnes and others introduced the radial maze as a modification of spatial discrimination. •This method is now well established and widely used to test for any new cognitive enhancer. LIMITATION There are many initial errors due to inability of the animal to remember the correct solution but rather to the tendency of the animal to explore alternative pathway
  49. 49. Spatial learning in the radial arm maze PURPOSE AND RATIONALE  Olton and co-workers have developed a spatial discrimination task for rodents that has been extensively used in learning and memory studies, and that has served as the basic task for one of the most important theories on the role of the hippocampus .  The rat uses spatial information provided by the distal cues in the room to efficiently locate the baited arms. The radial arm-maze allows the study of spatial reference and working memory processes in the rat.  In reference memory procedures, information is useful for many sessions/days and may usually be needed during the entire experiment.  On the contrary, working memory procedures have a major temporal component as the information presented in the maze is useful for one session but not for subsequent ones; the rat has to remember the information during a delay interval . Correct choices in the radial arm- maze are rewarded by food.
  50. 50. PROCEDURE •The apparatus is a wooden elevated eight-arm radial maze with the arms extending from a central platform 26 cm in diameter. • Food pellets (reward) are placed at the end of the arms. During the test, rats are fed once a day and their body weights maintained at 85% of their free feeding weight to motivate the rat to run the maze. •The session is terminated after 8 choices and the rat has to obtain the maximum number of rewards with a minimum number of errors. EVALUATION The number of errors (entries to non-baited arms) are counted during the session
  51. 51. MODIFICATIONS OF THE TEST Depending on the hypothesis being tested in the radial arm-maze (1) animals can be trained extensively and then receive specific brain lesions (hippocampus) after recovery from the surgery the rats are re-trained to determine the cognitive ability or the speed of recovery as these lesions severely disrupt processing of spatial information (2) In working memory studies animals are forced to obtain reward in specific arms , and after a time delay they have to either return to the same arms or to avoid these arms and obtain the food in the rest of the arms . (3) Animals are trained to find food in only one arm, and after a delay they are required to return to the same arm.
  52. 52. CRITICAL ASSESSMENT OF THE TEST •The radial arm-maze has been widely used to determine the neurobiological mechanisms underlying spatial learning in rodents and to evaluate the effect of drugs. •The deleterious effects of scopolamine, benzodiazepines, haloperidol, ketamine, PCP, as well as the facilitator effects of nicotine, pramiracetam, picrotoxin, naloxone have been reported in the literature LIMITATION • One of the disadvantages of the test is that hypothalamic lesions or the anorectic effect of certain drugs (amphetamine) affect the appetitive nature of the maze and animals do not master the maze for this reason.
  53. 53. Visual discrimination PURPOSE AND RATIONALE  Vision is better than any other sensory system for the analysis of spatial relationships in the environment of the animal. The organization of the visual system ensures processing of visual information according to simple principles, i.e. by fitting the distribution of light over the receptive surface to elementary geometrical concepts and by comparing these patterns with images stored in the memory.  Visual pattern recognition is one of the most challenging problems of contemporary neurophysiology and experimental psychology, with significant implications for mathematical and technical modeling of perceptual phenomena
  54. 54. PROCEDURE • The apparatus consists of a square separated by a Plexiglas sliding door from the choice area, which is connected by swing doors to the goal compartment. •The animal is placed into the apparatus with all doors open and allowed to explore it. Then it is placed in the start and after 5 s release by raising the Plexiglas door. • After another 5 s, electric shocks are applied until the animal escapes through either of the open doors to the safe goal compartment where it is left for some seconds. CRITICAL ASSESSMENT AND LIMITATION OF THE METHOD The method is time consuming and only useful to address a specific hypothesis
  55. 55. Spatial learning in the water maze PURPOSE AND RATIONALE(explicit associative memory)  A task was developed where rats learn to swim in a water tank to find an escape platform hidden under the water.  As there are no proximal cues to mark the position of the platform, the ability to locate it efficiently will depend on the use of a configuration of the cues outside the tank.  Learning is reflected on the shorter latencies to escape and the decrease on the length of the path to find the platform PROCEDURE  The apparatus is a circular water tank filled to a depth of 20 cm with 25 °C water. The tank is divided in four equal quadrants and a small platform is located in the centre of one of the quadrants.  The rat is released into the water and allowed 60–90 s to find the platform.
  56. 56. EVALUATION The latency to reach the escape platform is measured during the training days. MODIFICATIONS OF THE TEST •In the “cue” version of the water maze, the platform is clearly visible over the water and allows the evaluation of the motor and motivational aspects of the rat under study, as the animals should easily find the visible platform in 10–15 s. • To evaluate working memory processes in the water maze, the rat can be trained to find a new platform position and later its performance is tested after a short delay
  57. 57. MODIFICATIONS OF THE METHOD •Nitta found an impairment of the performance of the water maze task in rats treated by infusion into the cerebral ventricle for 14 days with β-amyloid protein which consisted of senile plaques of Alzheimer’s disease. •It is recommended as a model for alzeimer’s disease CRITICAL ASSESSMENT OF THE TEST •This test allows the researcher to study working and reference memory processes, and to dissociate the memory deficits induced by brain lesions or drug injections from the motor, motivational or sensory deficits. •This test has been used to study the effect of drugs on memory , the participation of the hippocampus, amygdala and catecholamine depletions, as well as recovery of function . LIMITATION It has comparitvely less specificity as was once believed
  58. 58. Water maze 8 arm radial maze Advantages  No motivational problems – problem cases are easily tested.  Olfactory cues are not present.  Animals learn readily and quickly. Disadvantages  Differences in platform finding latency are the product of learning and motor performance; it is not possible to subtract one from another  Working memory cannot be tested independently.  A videotracking system is essential for evaluating the swim tracks of animals.  A fairly large pool has to be installed in a fairly dedicated room Advantages  Distinguishes between motor impairment and spatial learning  Different memory types can be tested: working memory, long-term (reference) memory, motor (egocentric) memory.  A tracking program is not essential.  Cheap, simple set-up, which can be dismantled and easily stored after use  Animals must be well-handled and motivated to perform the tasks Disadvantages  Appetitive task: the animals must be mildly food-deprived to be motivated to look for baited arms  Training the animals usually takes much more time than water maze
  59. 59. Olfactory learning PURPOSE AND RATIONALE  Odors provide rodents with important information on the environment and the learning of successive olfactory discrimination problems in rats is closely related to the acquisition rules of higher primates.  Odor-reward associations are learned in few trials as odors exert more discriminative control over other sensory modalities like tones or lights .  Animals have to learn to discriminate an arbitrary designated positive odor (i.e., banana) from a negative one (i.e., orange) to receive a reward. Procedure  The olfactory apparatus is a rectangular box with a photosensitive cell mounted on top of the water spout/odor outlet.  Rats are trained to approach the water spout and to brake the light beam. Responses to the positive odor are rewarded with water while responses to the negative odor results in the presentation of a light flash.  Sessions are terminated when the rat makes 90% correct choices or after 400 trials.
  60. 60. EVALUATION •The animal is rewarded with 0.05 ml of water when it brakes the beam to the positive odor or when it does not respond to the negative odor. •Results are reported as % correct responses or as a logit transformation of the %correct/incorrect response ratio CRITICAL ASSESSMENT OF THE TEST •The anatomical connections from the olfactory bulbs to cortical as well as subcortical areas are fairly well known, and brain lesions that impair olfactory discrimination learning could be used as models of amnesia. •Systemic injections of scopolamine disrupts the performance of rats during long delays with no effect on immediate recall, data that are consistent with the effect of scopolamine in humans •The test can also be used to evaluate the cognitive effects of drugs such as ACTH analogs that facilitate the storage of olfactory information
  61. 61. MODIFICATIONS OF THE TEST •A T-maze with controlled access to the reward compartment was developed , that allows the test to evaluate working memory processes after time delays •Willer examined the effects of a competitive NMDA agonist on the ability of rats to acquire potentiated aversions to the odor element of a taste-odor compound LIMITATION It requires lot of training for animals
  62. 62. Aversive discrimination in chickens PURPOSE AND RATIONALE  One to 2-days old chicks have been extensively used to study learning and memory. The chicks are trained to discriminate between an aversive and a non-aversive stimulus in a single 10–15 s learning trial.  Following this learning trial, retention is monitored over a comprehensive range of learning retention intervals to chart the course and to differentiate possible stages of memory formation.
  63. 63. PROCEDURE • In a single passive avoidance learning trial, chicks are pre-trained to peck at a 4 mm chromed bead, dipped in water and presented for 10 s. •In a discrimination paradigm, chicks are trained to avoid a red bead and are tested on red and blue beads successively and discrimination ratios registered. •Twenty different chicks are used for each data point. Retention in both paradigms is indexed by the proportion of chicks avoiding the aversive bead, with the provision that in the discrimination paradigm is indicated by the avoidance of the red bead and pecking on the blue bead.
  64. 64. EVALUATION Discrimination ratios of treated animals after various time intervals following administration of drugs are statistically compared with saline treated controls. CRITICAL ASSESSMENT OF THE METHOD The test has been used with slight modifications successfully by many authors in several research groups MODIFICATIONS OF THE TEST •Bourne compared the taste and odor avers an methyl anthranilate with the odorless quinine as averants. •Clements and Bourne and Stamatakis injected drugs, e.g., GABA agonists or α2-noradrenergic agonists and antagonists, directly into the intermediate median hyperstriatum ventral of day-old chicks, prior to training on a chrome bead dipped in either methyl anthranilate or quinine.
  65. 65. Conditioned response Conditioned nictitating membrane response in rabbits PURPOSE AND RATIONALE  The rabbit’s classically conditioned eye blink response has become a widely used model system for studying associative learning in mammals and to find drugs potentially useful in the treatment of age-related memory disorders (alzeimer’s disease) Apparatus and general procedure  A small loop of surgical nylon is sutured into the right nictitating membrane, and the surrounding hair is removed. One day later, the rabbit is placed in a Plexiglas restrainer, and two stainless-steel wound clips are applied to the skin over the parietal region.  The transducer assembly converts nictitating membrane movements into electrical signals that are subjected to an analog to digital conversion using a 5-ms sampling rate and a resolution of 0.06 mm actual membrane extension.  During the course of the experiment, two stimuli are employed as conditioned stimulus: a) a 1000-ms, 1-kHz, 84-dB tone ; b) a 1000-ms, intermittently presented light produced by interruption of the house lights at 10 Hz to yield a change in illumination, measured at the eye level of the rabbit from 32.11 to 8.01.  The unconditioned stimulus is a 100ms,3mA,60Hz shock delivered to wound clips by constant shock generator
  66. 66. DRUGS Drug solutions or saline are injected subcutaneously into the cervical area of the rabbit via an infusion pump at a rate of 3 ml/min, 30 min before behavioral testing PROCEDURE •Experimentally naive rabbits are randomly assigned in equal numbers to each of the treatments (n = 10 per treatment). • The experiment consists of two phases: •Phase 1 is an adaptation day followed by 9 days of acquisition training. •Each acquisition session consists of 30 tone-shock and 30 light-shock trials presented in a randomized sequence within 10 trial blocks, with the restriction that no more than three consecutive tone or light trials can occur. • On each conditioned stimulus –unconditioned stimulus trial, the offset of the 1 000-ms tone or light conditioned stimulus occurs simultaneously with the onset of the 100-ms unconditioned stimulus. • A response is defined as at least a 0.5-mm extension of the nictitating membrane.
  67. 67. EVALUATION The data are analyzed by repeated measures analyze of variance and Tukey tests. CRITICAL ASSESSMENT OF THE METHOD The rabbit nictitating membrane response has been used widely to study the effects of drugs on learning MODIFICATIONS OF THE TEST •Several authors used a corneal air puff as unconditioned stimulus •Scavio studied post-training effects of several drugs, such as amphetamine, chlorpromazine, ketamine, and scopolamine, on the acquisition and extinction of the rabbit’s conditioned membrane response. •Solomon described behavioral paradigm for delay, trace, and long-delay conditioning. Various drugs influence the nictitating membrane response in rabbits, such as cocaine , sodium pentobarbital , haloperidol , MDA (3,4-methylenedioyamphetamine)
  68. 68. Automated learning and memory model in mice PURPOSE AND RATIONALE  Vanover and Barrett developed and validated pharmacologically an automated, relatively rapid, and reproducible behavioral model of learning and memory using an autoshaped procedure in mice. PROCEDURE  For experimental sessions, mice are placed in enclosures containing specially designed operant chambers equipped with a recess for dipper accessibility on one wall of the chamber and two additional smaller holes on either side of dipper well.  The dipper can be raised into the dipper well for a sucrose solution reinforcer presentation. Each recess has a photocell monitoring nose-poke responses.  Five to 9 mice per group are tested in a 2-day procedure designed to measure acquisition and retention of nose-poke response under an autoshaping schedule of reinforcement.
  69. 69. • During the autoshaping procedure which is used to measure acquisition and retention of the response reinforcement for nose-poking a tone is added. This tone sounds on a variable-interval schedule of presentation and stays for either 6 s or until a nose-poke in the dipper well is made before the end of the 6 s period, at which time the tone is turned off and a dipper with sucrose solution is presented •A dipper well nose-poke response made during the presence of the tone is counted as reinforced response. • Each sessions lasts for 2 h or until 20 reinforcers have been earned, whichever comes first. •Drugs in various doses or vehicle are administered i.p. immediately before the session. •The latency of the response to the 10th reinforcer is considered as a measure of acquisition and retention, because all mice have been exposed to all possible intervals(in random order) by the 10th tone.
  70. 70. EVALUATION •The mean and standard error of the mean are calculated for every group. Two-tailed Student t-tests are used to compare two independent groups on any one dependent variable. •One way analyses of variance (ANOVA) are conducted across groups for effects of dose on acquisition (day 1 performance). One way analyses of covariance (ANCOVA) are conducted across groups for effects of dose for retention (day 2 of performance) with the performance on day 1 as the covariate. • When an ANOVA or ANCOVA shows statistical significance, post-hoc Duncan’s multiple range tests are conducted with every group compared with vehicle. LIMITATION If a mouse fails to give a nose poke response,still it is given dipper at the termination of tone.it may lead to false positive results
  71. 71. Studies in aged monkeys PURPOSE AND RATIONALE  Nonhuman primates have the closest taxonomic relationship to humans, sharing many morphologic and physiologic similarities in the central nervous system.  They offer additional advantages for neurobehavioral animal models of aging in that many of the behavioral processes thought to be affected by aging (e.g. reaction time, learning and memory) can be studied easily PROCEDURE  The apparatus developed specifically for the series of studies used to develop the primate model was the Automated General Experimental Device (AGED),  The AGED is a totally automated, computer-controlled testing system, whose prominent feature consists of a 3 × 3 matrix of stimulus response (SR) panels. Each SR panel is hinge mounted directly in front of the reinforcement well so that when a panel is pushed, a red switch is magnetically activated and a reinforcement well is exposed.  Both colored and patterned stimuli can be projected onto the SR panels. A plastic partition with a stimulus window and armholes separates the monkey from the SR matrix.
  72. 72. EVALUATION The monkey must remember the stimulus location to get a reinforcement. Number of correct answers will be counted as well as the time until the monkey answer correctly. CRITICAL ASSESSMENT OF THE METHOD •This system provides an accurate and objective mean of collecting data under a number of behavioral paradigms. •Further, this system provides experimental control over a number of variables that might confound behavioral measures MODIFICATIONS OF THE METHOD Cai and Arnstein described dose-dependent effects of Dopamine D1 receptor agonists on spatial working memory in aged monkeys. LIMITATION •Ethical issues •Catching procedures are risky •Mandatory presence of technician is time consuming and costly
  73. 73. 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
  74. 74. 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.
  75. 75. MODIFICATIONS OF THE METHOD •Fujii studied the effects of an adenosine a1 receptor antagonist, 8 cyclopentyltheophylline, on the reduction of LTP in ca1 neurons of hippocampal slices. •Behnisch and reymann employed slices of hippocampal area ca1 in the rat to test the hypothesis that the activation of metabotropic glutamate receptors during tetanization is necessary for the maintenance of long-term potentiation. LIMITATION •They lack normal synaptic connections with other neurons, may be functioning under conditions greatly different than normally seen in the living organism • It is not certain that the "extracellular" and "intracellular" media used do not constitute a totally artificial environment that would not be relevant to a living neuron in vivo. • The lack of normally circulating agents such as steroids, hormones, plasma proteins, and other colloidal substances could lead to drastic changes in the function of the molecules and channels under study
  76. 76. ANIMALS WITH MEMORY DEFICIT MEMORY DEFICIT AFTER CEREBRAL LESIONS PURPOSE AND RATIONALE  Cerebral lesions have been used as a method of determining the involvement of particular brain area in performing a particular function  By training an animal for a certain task, then lesioning a specific area(inj of ibotenic acid in basal forebrain),one can determine whether that area of brain is necessary to perform that function  Mostly neurotoxins are tested esp excitatory amino acid neurotransmitters such as glutamate.
  77. 77. OTHER FORMS OF THE SAME TEST- •Cognitive deficit after cerebral ishemia •Strains with hereditary memory deficit •Genetically modified animals LIMITATION •It requires a lot of skill and expertise. •Takes a lot of time •Specificity of lesion and co relating with effects is difficult
  78. 78. In vitro methods 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
  79. 79. 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
  80. 80. 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 LIMITATION •Good lab set up is required which is costly •It requires lot of skill and precision
  81. 81. 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. It is found in the highest amounts in serum, but its physiological role is not known 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 AND LIMITATION  Same as previous experiment
  82. 82. Ex vivo cholinesterase inhibition PURPOSE AND RATIONALE  This assay is used to determine the dose-response relationship and duration of action of cholinesterase inhibitors in vivo. Cholinesterase inhibitors, including physostigmine and tacrine have been shown to improve cognitive functions in Alzheimer’s disease.  Physostigmine is a potent, but nonselective inhibitor of cholinesterase and has a short duration of action. Tacrine also inhibits both acetylcholine- esterase (true) and butyrylcholine-esterase, but is more potent as an inhibitor of the pseudo-enzyme .  Ideally, a dose-response for cholinesterase inhibition is determined first.  Then a dose which gives a reasonable effect (>50% inhibition if possible) is chosen to do the time-course experiment.  The effects on brain acetyl cholinesterase activity are examined in striatal tissue, using 5 mM acetylthiocholine as a substrate
  83. 83. PROCEDURE Substrate in buffer a) 198 mg acetylthiocholine chloride (10 mM) b) q.s. to 100 ml with 0.05 M phosphate buffer, pH 7.2 (reagent 1) DRUG TREATMENT •Groups of four male Wistar rats are dosed i.p. with vehicle or the test drug. •For the initial dose response study, the rats are given varying doses of drug based on toxicity reported in primary overt effects testing and sacrificed at 1 h after dosing. •The animals are observed and the occurrence of cholinergic signs is noted ( salivation, diarrhea and chromodacryorrhea). •For the time-course study, a dose of the test drug is given which gave significant inhibition of cholinesterase activity TISSUE PREPARATION Same as previous experiment
  84. 84. ASSAY •Enzyme activity is measured with the Beckman DU-50 spectrophotometer. •Substrate concentration is 10 mM diluted 1 : 2 in the assay yielding final concentration of 5 mM. EVALUATION The percent inhibition at each dose or time is calculated by comparison with the enzyme activity of the vehicle control group. %inhibition=slope control-slope drug x 100 slope control MODIFICATIONS OF THE METHOD •Antagonism of physostigmine-induced lethality in mice has been used by Gouret as a general indicator of central or peripheral anticholinergic activity. •A low dose of physostigmine can be used for detecting procholinergic activity
  85. 85. 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
  86. 86. [3H]-N-Methylscopolamine binding in the presence and absence of Gpp(NH)p PURPOSE AND RATIONALE The assay differentiates the interaction of muscarinic agonists and muscarinic antagonists with 3H-N-methylscopolamine (3H-NMS)-labeled receptors in cerebellar tissue based on the selective effect of guanine nucleotides on the affinity of muscarinic agonists for the receptor STIMULATION OF PHOSPHATIDYLINOSITOL TURNOVER IN RAT BRAIN SLICES PURPOSE AND RATIONALE •The purpose of this assay is to determine the ability of test compounds to stimulate the turnover of phosphatidylinositol (PI) in brain tissue. • This assay can be used to determine agonist activity at a number of CNS receptors known to be linked to the PI response. A major interest is the evaluation of muscarinic cholinergic receptors
  87. 87. RELEASE OF [3H]ACH AND OTHER TRANSMITTERS FROM RAT BRAIN SLICES PURPOSE AND RATIONALE •Electrically stimulated release of [3H]ACh is used as a biochemical screen for agents which may possibly enhance or inhibit release of [3H]ACh through a direct muscarinic interaction or other indirect interactions. •Muscarinic autoreceptors have been shown to have a role in the regulation of ACh release in several areas of the CNS • Direct stimulation of muscarinic receptors with muscarinic agonists or indirect stimulation with acetyl cholinesterase inhibitors decreases ACh release evoked by either increased potassium concentration or electrical stimulation.
  88. 88. Experiments on nootropics on humans  The use of a scopolamine model to study the potential nootropic effects of aniracetam and piracetam in healthy volunteers ABSTRACT-  In this study 26 healthy volunteers received scopolamine 0.7 mg subcutaneously on seven occasions at least a week apart.  Following this, over the seven occasions, a range of oral and intravenous dose regimens were administered including aniracetam 2 mg intravenously, 100 mg intravenously, 200 mg intravenously, 1500 mg per os and piracetam 2400 mg per os  At 60 min, scopolamine produced marked and significant decrements in all of the measures of memory and information processing.  Aniracetam 1500 mg was able to significantly antagonize decrements on both memory and information processing tasks.  Overall, the findings demonstrate that aniracetam 1500 mg can antagonize cognitive decrements produced by cholinergic blockade in healthy volunteers, and suggest that the drug possesses nootropic properties.
  89. 89. Giaguinto and colleagues gave 12 healthy humans 5mg Valium orally at 10PM the night before their experiment. • The next morning they were given either I.V. Oxiracetam or saline in a double blind crossover experiment. •Oxiracetam strongly decreased the excessive delta activity while simultaneously strongly increasing alpha activity, and also induced a modest increase in beta activity.  Itil and co-workers (1986) treated four groups of 15 patients suffering mild to moderate dementia with either Oxiracetam or placebo for three months. •The double blind study used Oxiracetam in doses of 800, 1600 and 2400mg daily. Quantitative EEG data indicated that in patients with dementia, Oxiracetam had a mode of action similar to other vigilance enhancing compounds. The majority of patients who had abnormal slow EEG patterns before treatment showed a "normalization" of their brain waves- i.e. a decrease in slow (delta and theta) and an increase in alpha waves.
  90. 90. Mindus and colleagues reported the results of a double blind crossover trial with 18 healthy middle aged people based on medical records and administration of several intelligence tests •Most of the subjects were in intellectually demanding jobs, but had reported a slight reduction for some years in their capacity to retain or recall information. •After four weeks of 4.8 grams per day Piracetam, subjects were switched to placebo for four weeks, while the original placebo group then received Piracetam for four weeks. •Results of a series of paper and pencil tests, as well as computerized tests to measure perceptual motor reactions, showed a clear benefit of Piracetam over placebo. LIMITATION •A major complication that is raised in memory research is its fallible nature in humans. • Our ability to store and process what is going on around us relies on memory being a constructive process. • The technologies explained above may show areas of activation associated with certain behaviors, but without any idea of lesion location, it is difficult to pinpoint exactly what part of the brain relates to which behavioral deficits observed
  91. 91. Indications of cognitive enhancers  Degenerative changes with ageing  For treating diseases-alzheimer disease,huntington disease,adhd,parkinson disorder  Memory loss due to brain injury  As a study aid  For work and business  For social acuity  For health  Eugeroics (armodafinil)– wakefulness promoting agents they are clinically prescribed for narcolepsy,shift work disorder  It can even reduce the risk of stroke and repair brain damage from physical or chemical trauma like alcohol consumption and concussion  Myoclonus-piracetam is approved in UK
  92. 92. BIBLIOGRAPHY  HANS GERHARD VOGEL 3RD EDITION;DRUG DISCOVERY AND EVALUATION:DRUG EFFECTS ON PHARMACOLOGICAL ASSAYS; LEARNING AND MEMORY ;PAGE 878-942  SK GUPTA 1ST EDITION;DRUG SCREENING METHODS;DRUGS FOR LEARNING AND MEMORY;PAGE 117-130  KD TRIPATHI 7TH EDITION;ESSENTIALS OF MEDICAL PHARMACOLOGY;CNS STIMULANTS AND COGNITION ENHANCERS;PAGE488-491  HL SHARMA 2ND EDITION;PRINCIPLES OF PHARMACOLOGY;DRUG THERAPY FOR NEURODEGENERATIVE DISEASES;PAGE 532-543  SK SRIVASTAVA 1ST EDITION VOL1;MEDICAL PHARMACOLOGY;CNS STIMULANTS,ANALEPTICS AND COGNITIVE ENHANCERS;PAGE 763-767  BRANDEN MAHER ET AL;POLL RESULTS –LOOK WHO’S DOPING;NATURE INTERNATIONAL JOURNAL OF SCIENCE

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