memory and learning drug screening

1. SCREENING:
Drugs Affecting
Learning & Memory
SHREYA GUPTA

2. INTRODUCTION
 Learning and memory are closely related concepts.
 Learning is the acquisition of skill or knowledge,
 Memory is the expression of what you’ve acquired.
Another difference is the speed with which the two things
happen,
 If one acquires the new skill or knowledge slowly and
laboriously, that’s learning
 If acquisition occurs instantly, that’s making a memory
https://www.apa.org/topics/learning/

3. LEARNING
 Process of acquiring new, or modifying existing
knowledge, behaviours, skills, values, or preferences
 Relatively permanent change in behaviour as a result of
experience
 Types:
1. Classical conditioning
2. Operant conditioning
3. Observational learning

4. TYPES OF LEARNING

5. TYPES OF LEARNING

6. TYPES OF MEMORY

7. TYPES OF MEMORY
LONG TERM MEMORY:
1. Explicit memory/ Declarative memory
 Consciously recalled, deals with facts/events
 Memories can be episodic, relate to experiences or
'episodes' in your life
 Or, they are semantic, relating to facts or general knowledge
 Affected by neurodegenerative diseases such as Alzheimer’s
disease

8. TYPES OF MEMORY
LONG TERM MEMORY:
2. Implicit memory/ Procedural memory-
 Unconscious memory, deals with skills/tasks
 May be procedural, involving learned motor skills—learning
how to ride a bike
 Or, result from priming, which occurs when exposure to one
stimulus influences your brain’s response to another.
 For example, in word-judging tasks, participants identify pairs
of associated words such as bread–butter faster than non-
associated pairs such as BREAD–DOCTOR.

9. TYPES OF MEMORY
SHORT TERM MEMORY:
 Enables the brain to remember small amount of information
for a short period of time
 Shortest type of memory is working memory, which can last
just seconds, used to hold information in brain while we
engage in other cognitive processes.
 A person’s working memory capability is one of the best
predictors of general intelligence, as measured by standard
psychological tests

10. MEMORY PATHWAY

11. AREAS OF BRAIN

12. NEUROTRANSMITTERS
 ACETYLCHOLINE: increases neuronal depolarization in cortex,
increases memory formation
 GLUTAMATE: Enhance signal transduction & Synaptic plasticity
 DOPAMINE: Increase in Limbic region &hippocampus
Increases memory
 GABA: may be involved in long-term object
recognition memory and working memory.
 SEROTONIN: linked to emotional and motivational aspects of
human behaviour

13. LEARNING DISABILITIES
 Dyslexia
 Dysgraphia
 Dyspraxia
 Dysphasia
 Central Auditory Processing Disorder
 Non-Verbal Learning Disorder

14. MEMORY DISORDERS
DEMENTIA:
 Alzheimer's disease
 Dementia with Lewy Bodies (DLB)
 Frontotemporal Dementia (FTD)
 HIV Dementia
 Normal Pressure Hydrocephalus (NPH)
 Vascular Dementia
AMNESIA
 Korsakoff's Syndrome
 Transient Global Amnesia (TGA)

15. DEMENTIA vs AMNESIA
DEMENTIA AMNESIA
Impairment of cognition, causing
functional decline, severe impairment
in memory, judgement, orientation,
cognition
Memory impairment with no other
cognitive impairments
Mostly irreversible May be reversible
Aging is risk factor Aging not risk factor
Slow and gradual
Uncertain of beginning point
Sudden onset of memory loss
Causes: brain trauma
Neurodegeneration
Genetics
Concussion
Trauma
PTSD
Alcoholism

16. DRUGS DEHANCING MEMORY
 Benzodiazepines
 Statins
 Antiseizure drugs
 Opioids
 Nonbenzodiazepine sedative-
hypnotics (Z- drugs)
 Anticholinergics
 Antihistamines (first
generation)
 Antipsychotics
 Alcohol

17. DRUGS ENHANCING MEMORY
 Amphetamines
 Methylphenidate
 Modafinil, armodafinil
 Caffeine, nicotine
 Guanfacine
 Clonidine
 Atomoxetine
 Piracetam
 Rivastigmine
 Tacrine
 Donepezil
 Galantamine
 Memantine
 Pramipexole

18. NEED FOR SCREENING
 Although over 400 trials have been conducted in people
for dementia (Alzheimer’s disease), almost no new drugs
have been brought to the market
 One of major hurdle is the development of appropriate
animal models for dementia
 Successful development of animal models may well
depend upon our ability to accurately reproduce specific
pathophysiological or etiologic factors
https://www.nature.com/articles/d41586-018-05722-9

19. In Vitro Tests
 In Vitro Inhibition of
Acetylcholine Esterase Activity
in Rat Striatum.
 In Vitro Inhibition of
Butyrylcholine-Esterase Activity
in Human Serum
 Uncompetitive NMDA
Receptor Antagonism
 Molecular Forms of
Acetylcholinesterase from Rat
Frontal Cortex and Striatum
 Release of ACh and Other
Transmitters from Rat Brain
Slices
 Oxotremorine Binding to
Muscarinic Cholinergic
Receptors in Rat Forebrain
 Inhibition of Respiratory Burst
in Microglial
Cells/Macrophages
 Stimulation of
Phosphatidylinositol Turnover
in Rat Brain Slices

20. In Vivo Tests
 Passive avoidance
 Active avoidance
 Discrimination testing
 Animals with memory deficits
 Upcoming tests

21. PASSIVE AVOIDANCE
1. Step down method
2. Step through method
3. Scopolamine-Induced Amnesia in Mice

22. IN-VIVO METHODS:
Inhibitory/Passive Avoidance
 Evaluates inhibition to imitate activities or learned habits
 “Passive avoidance” describes experiments in which the
animal learns to avoid a noxious event by suppressing a
particular behavior.

23. STEP-DOWN METHOD
PURPOSE AND RATIONALE
 An animal (mouse or rat) in an open field spends most of the
time close to the walls and in the corners
 When placed on an elevated platform in the center of a
rectangular compartment, it steps down almost immediately to
the floor to explore the enclosure and to approach the wall

24. STEP-DOWN METHOD
PROCEDURE
 Mice or rats of either sex are
used
 A rectangular box (50 x 50 cm)
with electrifiable grid floor
 Grid floor is connected to a shock
device, which delivers scrambled
foot shocks
 Wooden platform (10 × 7 × 1.7 c
m) in the center of the grid floor

25. STEP-DOWN METHOD
Paradigm consists of three phases:
 Familiarization: animal is placed on platform, and latency to
descend is measured. Returned to home cage after 10s of
exploration
 Learning: Immediately after animal has descended from the
platform an unavoidable foot shock is applied (50Hz;1.5mA; 1s)
and animal is returned to the 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.
 Test is finished when the animal steps down or remains on the
platform (cut off time: 60 s).

26. STEP-DOWN METHOD
EVALUATION
 The time of descent during the learning phase and the time
during the retention test is measured
 Prolongation of the step-down latency is defined as
learning.

27. STEP – THROUGH METHOD
PURPOSE AND RATIONALE
 This test uses normal behaviour
of mice and rats.
 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

28. STEP – THROUGH METHOD
PROCEDURE
 Mice and rats of either sex are used
 Apparatus: small illuminated (7W bulb) chamber connected to
a larger dark chamber via a door
 The test animals are given an acquisition trial followed by a
retention trial 24 h later
 Acquisition trial: animal is placed in the illuminated
compartment at a maximal distance from door, latency to enter
the dark compartment is measured
 Animals that do not step through the door within a cut-off
time: 90 s (mice) or 180 s (rats) are not used

29. STEP – THROUGH METHOD
 Immediately after the animal enters the dark compartment,
door is shut automatically and an unavoidable foot shock
(1mA; 1 s – mice; 1.5mA; 2 s – rat) delivered.
 Animal is then quickly removed (within 10 s) from the
apparatus and put back into its home cage.
 Retention trial: Test procedure is repeated with or without
drug, cut-off time on day 2 is 300 s (mice) or 600 s (rats),
respectively

30. STEP – THROUGH METHOD
EVALUATION
 The time to step-through during the learning phase is
measured and the time during the retention test is measured.
 An increase of the step-through latency is defined as learning.

31. STEP – THROUGH METHOD
Modifications of step through
 Unilateral ibotenic acid lesions in the right nucleus
basalis as a rat model of Alzheimer disease
 L-DOPA caused memory deficits in mice, recommended
this as a model for human dementia.

32. CRITICAL ASSESSMENT OF BOTH
METHODS
 High variability, it is necessary to test large groups of animals
(minimum 10 animals per group)
 Tendency of the animal to escape contact with the human
hand as it is placed on platform may shorten the step-down/
step through latencies
 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.
 The duration and intensity of the shock should be constant

33. SCOPOLAMINE-INDUCED
AMNESIA IN MICE
PURPOSE AND RATIONALE
 Scopolamine has been shown to impair memory retention
when given to mice shortly before training in a dark avoidance
task
 Based on ability of cholinergic agonist drugs to reverse the
amnesic effects of scopolamine in animals and humans

34. SCOPOLAMINE-INDUCED
AMNESIA IN MICE
PROCEDURE
 Test is performed in groups of 10 male NMRI mice weighing
26–32 g in a one-trial
 5min after i.p. administration of 3 mg/kg scopolamine
hydrobromide, each mouse is individually placed in the bright
part of 2 chambered apparatus for training.
 After a brief orientation period, mouse enters the second,
darker chamber. Once inside the second chamber, the door is
closed, a 1mA,1-s foot shock is applied through the grid floor,
mouse is then returned to the home cage

35. SCOPOLAMINE-INDUCED
AMNESIA IN MICE
 24 hours later, testing is performed by placing the animal again
in the bright chamber
 Latency in entering second darker chamber within a 5 min test
session is measured
 Untreated control animals enter the darker chamber in the
second trial with a latency of about 250 s, while treatment with
scopolamine reduces the latency to 50s
 Test compounds are administered 90min before training. A
prolonged latency indicates that the animal remembers that it
has been punished and, therefore, does avoid the darker
chamber.

36. SCOPOLAMINE-INDUCED
AMNESIA IN MICE
EVALUATION
 Using various doses, latencies after treatment with test
compounds are expressed as percentage of latencies in mice
treated with scopolamine
 Scopolamine amnesia test is widely used as primary screening
test anti-Alzheimer drugs

37. DRUGS INDUCING AMNESIA
 N-methyl-d-aspartate antagonists phencyclidine, ketamine, and
dizocilpine
 Chlordiazepoxide and dizocilpine combination in mice
 Pre-treatment with benzodiazepines
 Lipopolysaccharide-intoxicated mice as a model for Alzheimer’s
disease.
 Induction of learning and memory impairment in mice by
treatment with 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine
(MPTP).
 Trace amounts of copper in water induce β-amyloid plaques and
learning deficits in a rabbit model of Alzheimer’s disease

38. ACTIVE AVOIDANCE
1. Runaway avoidance
2. Shuttle box avoidance (2-way shuttle box)
3. Jumping avoidance (1- way shuttle box)

39. IN-VIVO METHODS:
Active Avoidance
 Active avoidance learning is a fundamental behavioural
phenomenon
 Learning defined by appropriate reactions to the conditioned
stimulus preceding the noxious stimulus
 It usually allows animal to escape which terminates the
conditioned stimulus

40. 1. RUNAWAY AVOIDANCE
PURPOSE & RATIONALE
 Straightforward avoidance situation features a fixed
aversive gradient(shock) can be traversed by the animal
 The shock can be avoided when the safe area is reached
within the time allocated

41. RUNAWAY AVOIDANCE
PROCEDURE
 Mice or rats of either sex used
 Animal is placed in a box which is uniformly illuminated and has
one small door to the safe area
 A loud speaker is mounted 50 cm above the start box, and
provides acoustic conditioning stimulus (80db,2000Hz tone)
 5min animal is allowed to explore the whole apparatus.
 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.

42. RUNAWAY AVOIDANCE
 CS continues until the animal reaches the safe area. It is left
there for 50–70 s (intertrial interval, ITI) before returned to the
same area again
 Training is continued until the animal attains the criterion of 9
avoidances in 10 consecutive trials

43. 2. SHUTTLE BOX AVOIDANCE
(2-way shuttle box)
PROCEDURE
 Rats of both sex are used
 Apparatus used consists of rectangular box 15 cm x 40 cm high
metal walls and electrifiable grid floor
 It is divided into two 25x25 compartments by a wall and a
small door
 Each compartment can be illuminated by a 20w bulb mounted in
lid
 Fixed resistance shock source with an automatic switch (0.5 s on
1.5 s off) is used for automatic delivery of the conditioned stimulus
(CS) and the unconditioned stimulus (US)

44. SHUTTLE BOX AVOIDANCE
(2-way shuttle box)
 Animal allowed to explore the apparatus for 5 min with the
connecting door open and the compartment lights switched off
 Door is then closed
 After 20 s light is switched on in the compartment containing the
animal, and the door is opened. A tone (CS, 80 dB, 2000Hz) is
presented and 5 s later floor shock(US) is applied in the
illuminated compartment and continued until the animal escapes
to the dark side of the compartment
 The connecting door is closed and the shock discontinued

45. SHUTTLE BOX AVOIDANCE
(2-way shuttle box)
 After a variable intertrial
interval (ITI; 30–90 s) light
is switched on in the
previous dark
compartment,
 The door is opened and
animal is required to cross
to the other side
 The training is continued
until the animal reaches
the criterion of 9
avoidances in 10
consecutive trials.

46. 3. JUMPING AVOIDANCE
(1- way shuttle box)
PROCEDURE
 Apparatus is a rectangular box of 40x25 and a grid floor and one
goal area (platform) with narrow walls
 Animal is allowed to explore goal area for 5 min.
 After that goal is blocked for 2s and acoustic conditional stimulus
(100Hz; 85db) is applied, after 5 min shock (1mA; 50Hz;0.5s)
applied
 Animal jumps on the platform. After 30 s the barrier pushes the
animal off the platform onto the grid floor
 Sequence is repeated until the criterion of 10 consecutive
avoidances is reached. Retention is tested on the second day

47. AVOIDANCE TESTING
EVALUATION
 The time the animal needs to reach the safe area on 2
consecutive days measured
 Number of errors (not reaching the safe area) recorded.
CRITICAL ASSESSMENT OF THE METHOD
 Automated method since no manual handling of animal
between trials
 Early extinction of response may be seen if intertrial intervals
are short

48. DISCRIMINATION
LEARNING
1. Spatial habitual learning
2. Spatial learning in radial arm maze
3. Olfactory learning

49. 1. SPATIAL HABITUAL LEARNING
PURPOSE & RATIONALE
 Uses open-field test to utilize natural tendency of rodents to
explore novel environments in order to open up new nutrition,
reproduction and lodging resources
 Spatial habituation learning: Decrement in reactivity to a novel
environment after repeated exposure to that now familiar
environment
 This reduction in exploratory behaviour during re-exposures is
interpreted in terms of remembering or recognition of the
specific physical characteristics of the environment

50. SPATIAL HABITUAL LEARNING
Test can be used to examine:
 Short-term spatial memory (within-trial reductions in
exploratory activity)
 Long-term spatial memory (between-trial reductions in
exploratory activity after a retention interval of 24, 48 or 96 h
after the initial exposure)

51. SPATIAL HABITUAL LEARNING
PROCEDURE
 Open-field apparatus: rectangular
chamber (rats: 60 x 60x40 cm, mice:
26x26x40 cm) made of painted wood or
grey PVC
 25W green or red light electric bulb is
placed directly above the maze to
achieve an illuminated density of 0.3lx at
the centre
 Rodent is placed on the center or in a
corner of the open-field for 5–10 minute
sessions
 Animals are re-exposed to the open field
24 and 96 h after the initial trial

52. SPATIAL HABITUAL LEARNING
EVALUATION
The exploratory behaviours registered are:
 Rearing or vertical activity: the number of times an animal was
standing on its hind legs with forelegs in the air or against the
wall
 Duration of each rearing
 Locomotion or horizontal activity: the distance in centimetres
an animal moved

53. 2. SPATIAL LEARNING IN THE RADIAL
ARM MAZE
PURPOSE & RATIONALE
 Allows the study of spatial reference and working memory
processes in the rat
 Rat uses spatial information to efficiently locate the baited
arms.

54. SPATIAL LEARNING IN THE RADIAL
ARM MAZE
 Apparatus is wooden elevated
eight-arm radial maze with
arms extending from a central
platform 26 cm in diameter
 Each arm is 56 cm long, 5 cm
wide, 2 cm high rails along
the length of the arm
 Maze is well illuminated
 Food pellets (reward) placed
at the end of the arms

55. SPATIAL LEARNING IN THE RADIAL
ARM MAZE
 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.
 Animals are trained on a daily basis in the maze to collect the
food pellets
 Session is terminated after 8 choices and the rat has to obtain
the maximum number of rewards with a minimum number of
errors
 Number of errors (entries to non-baited arms) are counted
during the session.

56. SPATIAL LEARNING IN THE RADIAL
ARM MAZE
EVALUATION
 Used to determine the neurobiological mechanisms underlying
spatial learning in rodents and to evaluate the effect of drugs
 Deleterious effects: scopolamine, atropine, ethanol,
benzodiazepines, haloperidol, ketamine, PCP
 Facilitatory effects: physostigmine, nicotine, picrotoxin,
naloxone have been tested

57. 3. OLFACTORY LEARNING
PURPOSE AND RATIONALE
 Odours provide rodents with important information on
environment
 Odour reward associations exert more discriminative control over
other sensory modalities like tones or light
 Animals have to learn to discriminate an arbitrary designated
positive odour (i. e., banana) from a negative one (i. e., orange) to
receive a reward

58. OLFACTORY LEARNING
PROCEDURE
 Animal is deprived of water for 48h
 Apparatus is box 30x30x55cm with a photo sensitive cell on the
top of water spout
 Rats are trained to approach the water spout and to break the
light beam
 Responses to the positive order are awarded
 Session terminates when rat makes 90% correct choices.

59. OLFACTORY LEARNING

60. OLFACTORY LEARNING
EVALUATION
 Animal is rewarded with 0.05 ml of water when it breaks the
beam to the positive odor or when it does not respond to the
negative odor
 Incorrect responses (“no go” to the positive odor or “go” to the
negative odor are followed by a flash and a longer intertrial
interval).
 Results are reported as the % correct responses or as a
%correct/incorrect response ratio.

61. OLFACTORY LEARNING
CRITICAL ASSESSMENT OF THE TEST
 Anatomical connections from olfactory bulbs to cortical and
subcortical areas are known, and brain lesions that impair
olfactory discrimination learning could be used as models of
amnesia
 Systemic injections of scopolamine, PCP impair acquisition of
odour discriminations

62. ANIMALS WITH MEMORY
DEFICITS
1. Memory Deficits After Cerebral Lesions
2. Cognitive Deficits After Cerebral Ischemia
3. Naturally occurring models
4. Transgenic models

63. 1. MEMORY DEFICITS AFTER
CEREBRAL LESIONS
 Cerebral lesions used as a method of determining involvement
of a particular brain area in performing a particular function.
 By training an animal to perform a certain task, then lesioning
a specific brain area, one can determine whether that area of
the brain is necessary or sufficient to perform that function

64. MEMORY DEFICITS AFTER
CEREBRAL LESIONS
ASPIRATIVE ELECTRICAL CHEMICAL
• Sucking out the tissue of
interest.
• Lesions not selective with
respect to tissue type.
• Neurons, support cells,
and fibers coursing
through the area are all
eliminated
• Electrical lesions are also
non-selective
• They destroy all tissue types
in the lesion area
• studies in monkeys :
1. model of episodic memory
impairment in with fornix
transection
2. memory functions studied
with lesions of the
hippocampus and adjacent
cortex
• Injecting chemical
agents into specific
brain sites.
• Induce more selective
lesion, typically
destroying cell bodies
and dendrites while
sparing axons

65. MEMORY DEFICITS AFTER
CEREBRAL LESIONS
Chemically induced cerebral lesions:
 Most commonly used neurotoxins in memory research are
glutamate, and glutamate analogs such as ibotenate,
NMDA, kainate and AMPA
 Intracerebroventicular injection of streptozotocin,in a rats,
causes prolonged impairment of brain glucose and energy
metabolism
 Cyclooxygenase-2 inhibitor into the dorsal hippocampus
attenuates memory acquisition in rats
 MPTP-treated rhesus monkeys.

66. 2. COGNITIVE DEFICITS AFTER
CEREBRAL ISCHEMIA
PURPOSE & RATIONALE
 Impairment of cerebral
metabolism induced by
reduced blood supply induces
cognitive deficits
 In the brain of Mongolian
gerbils, complete forebrain
ischemia can be produced by
occluding both common
carotid arteries resulting in
amnesia

67. COGNITIVE DEFICITS AFTER
CEREBRAL ISCHEMIA
PROCEDURE
 Male Mongolian gerbils (50–70 g)used, anesthetized by i.p.
pentobarbital injection.
 Both common carotid arteries are exposed, occluded for 5 or
10min with miniature aneurysm clips
 In sham operated controls, carotids exposed, not occluded
 24 h after occlusion, each animal is placed in the bright part of
a light/dark chambered apparatus for training.
 After a brief orientation period, the gerbil enters the second,
dark chamber.

68. COGNITIVE DEFICITS AFTER
CEREBRAL ISCHEMIA
 Once inside the second chamber, the door is closed, a 100V, 2-
s foot shock is applied through the grid floor.
 The gerbil is then returned to the home cage.
 Testing is repeated 24 h later by placing the animal again in
the bright chamber
 Latency in entering the dark chamber within a 5-min test
session is measured
 Latency compared with sham operated controls is decreased
depending on the duration of ischemia.
 After drug treatment, an increase of latency before entering
the dark compartment indicates good acquisition.

69. 3. NATURALLY OCCURING
MODELS
 Normal and aged rodents: Normal aging
 Senescence-accelerated mouse(SAM):
signs of advanced senescence, such as reduced activity, hair loss,
skin coarseness, peri-ophthalmic lesions, age-associated increase
in hippocampal Aβ and behavioural impairment, and shortened
life span

70. 4. TRANSGENIC MOUSE MODELS
OF DEMENTIA
 Human Tau models
 Tg2576 model of AD
 PS1 FAD models
 APP23 model
 Secretase models
 APOE models
 Axonal transport models

71. UPCOMING MODELS OF
ALZHEIMER'S DISEASE
• Non human- Primate models
• Mini Brains

72. 1. NON-HUMAN PRIMATE
MODELS
 Marmoset model of
Alzheimer’s disease: by using
CRISPR to insert mutations into
the gene PSEN1 in fertilized
eggs, acquire amyloid plaques in
7 years
https://www.nature.com/articles/d41586-018-05722-9
Macaques : injecting them with brain
tissue from humans, which leads the
animals to develop plaques and tau
tangles, as well as cognitive impairment

73. 2. MINI BRAINS
 3D tissue models of the brain
 Small organoids are grown from stem cells over period of a
few weeks,
 Enable large numbers of drugs to be evaluated in a short
amount of time
 HMS in Boston, Massachusetts (2014) grew human neural stem
cells containing APP and PSEN1 mutations in a 3D culture
system supported by a gel matrix

74. MINI BRAINS
• Tissue contained neurons
that deposited amyloid-β
into the gel, and plaques
formed after 5 to 6 weeks.
• A week or two later, the
elusive tau tangles also
appeared
https://www.nature.com/articles/d41586-018-05722-9

75. CONCLUSION

76. THANK
YOU

77. References
 Drug Discovery and Evaluation Pharmacological Assays Co-Editors: Wolfgang
H.Vogel Bernward A. Schölkens Jürgen Sandow Günter Müller Wolfgang F.
Vogel Second Edition.
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10.1002/msj.20159. PMID: 20101721; PMCID: PMC2925685.
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 https://www.nature.com/articles/d41586-018-05722-9
 https://www.hopkinsmedicine.org/neurology_neurosurgery/centers_clinics/me
mory_disorders/conditions/dementia.html
 https://www.sciencedirect.com/topics/medicine-and-dentistry/avoidance-
response