4 Methods of Research in

Psychopharmacology
4 Methods of Research in Psychopharmacology

• “Learn not to accept data without a

critical appraisal of the procedures
that were employed to obtain the
results and the reinterpretations these
new data would compel of the data
previously available.”
Neuropsychopharmacology, 9th edition
Methods of Research in Psychopharmacology

“Cocaine leads to tolerance” “Cocaine leads to sensitization”

• Independent variable (IV)

• Dependent variable (DV)
• Operational Definitions of IV and DV
• how the researcher decides to measure the variables
in the study
• Different operational definitions of the IV and DV may
produce profoundly different conclusions
Evaluating Animal Behavior

Behavioral measures (in animals):

• Understand neurochemical basis of
behavior and drug-induced changes in
that behavior
• Develop animal models of psychiatric
disorders
• Screen drugs in preclinical settings
Evaluating Animal Behavior

Advantages of animal studies:

• Rigorous control of environment
• Histories and genetic backgrounds are
well known
• Use of methods that are unethical in
humans
Rodent Behavioral Tests
 Evaluating Animal
Behavior: Operant
Conditioning, Pain,
Learning and Memory
Evaluating Animal Behavior: Operant Conditioning

Operant conditioning
• Animals learn to respond to obtain
rewards or avoid punishment
• Behaviors that are reinforced: likely
to be repeated
• Behaviors that are punished: not
likely to be repeated

Schedule of reinforcement
§ Fixed-ratio (FR): reinforcement
is delivered after a fixed # of
responses Skinner Box
§ Fixed-interval (FI):
reinforcement after a fixed
amount of time
Evaluating Animal Behavior: Measures of Analgesia

Analgesia: reduction of perceived

pain without loss of consciousness

• Tail-flick test
§ measures response of the
animal to heat applied to the tail
• Hot plate test
§ latency to respond to a thermal
stimulus applied to paws>>>
time it takes the mouse to lick
the hind paw, vocalize, or jump
from the hot plate surface

https://www.ottoenvironmental.com/content/images/thumbs/0011530_ugo-basile-hotcold-plate.jpeg
Measuring Pain in Humans

Methods: Eighteen healthy volunteers had two Ouch!

series of rectal balloon distensions
performed on two separate days.
Individualized balloon pressure, corresponding to
pain detection threshold or to the maximum
possible distension (30 psi), was used.
Evaluating Animal Behavior: Learning and Memory
Radial arm maze (RAM)
• Hungry rat allowed to explore maze
• piece of food at the end of each arm
• learn to visit each arm only once on a given day
§ must remember which arms were visited previously
§ External visual cues as guides
§ Sensitive to hippocampal lesions

cue
Evaluating Animal Behavior: Learning and Memory

Morris Water Maze cue

§ circular pool of water
(opaque)
§ External visual
cues as guides
§ Sensitive to cue
hippocampal lesions
Evaluating Animal Behavior: Learning and Memory

Barnes Maze

http://i01.i.aliimg.com/photo/v16/119311479/Barnes_Maze.jpg

• External visual cues as guides

• No swimming involved—an unnatural behavior for rodents
Evaluating Animal Behavior: Learning and Memory

Novel object recognition

• animals are given several minutes to explore two objects

https://knowingneurons.files.wordpress.com/2013/01/novel-object-recog-test1.jpg?w=610
Evaluating Animal Behavior: Learning and Memory

Delayed Response Test

•working memory (sensitive to PFC lesions)
Evaluating Animal Behavior: Learning and Memory

Caveat for all tests of learning and memory:

§ often do not determine whether altered
responses are due to drug-induced changes in:
§ attention or motivation
§ encoding, consolidation or retrieval of the
memory
§ Effects on motor or sensory ability
 Evaluating Animal
Behavior: Anxiety-Like
Behaviors
Evaluating Animal Behavior: Anxiety

Kind of
a big
deal! Nature Reviews Drug Discovery 4, 775-790 (September 2005)
Evaluating Animal Behavior: Anxiety

Open Field Box Elevated Plus Maze

Elevated Zero Maze Light-Dark Box

Nature Reviews Drug Discovery 4, 775-790 (September 2005)

• A natural conflict situation occurs when a rodent is exposed to an unfamiliar

environment
Evaluating Animal Behavior: Fear
Fear Conditioning
• Conditioned emotional
response: presentation of a
signal (a light or tone) is
followed by an unavoidable
electric shock
• Contextual vs Cued
• Contextual: return to same
box 1 day later>>>>leads to
enhanced “freezing”
• Cued: 1 day later, put in
different box and play
signal>>>>leads to
enhanced “freezing”
Evaluating Animal Behavior: OCD-Like Behaviors

Marble-Burying
Angoa-Perez et al., 2012
Evaluating Animal Behavior: Impulsive Aggression

Resident-Intruder Test
Angoa-Perez et al., 2012
 Evaluating Animal
Behavior: Depression-Like
Behaviors
Evaluating Animal Behavior: Depression
Depression

Kind of
a big
deal!
Nature Reviews Drug Discovery 4, 775-790 (September 2005)
Evaluating Animal Behavior: Depression

Forced Swim Test

“Behavioral despair”
Evaluating Animal Behavior: Depression

Tail Suspension Test

Evaluating Animal Behavior: Depression

Sucrose Preference Test: Anhedonia

10%
H2 O
sucrose

http://neurobehaviour.lunenfeld.ca/DEFAULT.ASP?page=Depression
Preclinical Models of Depression

Chronic, mild unpredictable stress

•exposes rodents to a series of physically stressful events for
several weeks
§ Resulting behaviors: cognitive impairments, anxiety-like
behaviors, social withdrawal, and anhedonia
§ Chronic, rather than acute antidepressant treatment required
to reverse the behaviors
§ Higher validity to the human condition

Al-Hasani, et al., 2013

Preclinical Models of Depression

Maternal Separation

•simulates early life stress

•Young animals separated
from their mothers for brief
periods daily during the first
few weeks of life
•Tested as adults
•Produces long-term changes
in CRF function that may
predispose to depression https://pharmns.med.uky.edu/animal-model-early-life-stress-maternal-separation

later in life
 Evaluating Animal
Behavior: Schizophrenia
and ASD-like Behaviors
Preclinical Models of Schizophrenia

Amphetamine-induced
hyperkinesia:
• High doses of Amph produces
symptoms similar to
schizophrenia
§ characteristic stereotyped
sniffing, licking, and gnawing
and increased locomotor
activity
• used for years to screen http://www.psychogenics.com/amphetamineinduced.html

potential antipsychotic drugs

Preclinical Models of ASD

Crawley 3-Chamber Box

 Evaluating Animal
Behavior: Drug Rewards
and Addiction
Evaluating Animal Behavior: Drug Rewards

Drug Reward and

Reinforcement
•FR (fixed ratio) schedules
§ Reinforcement delivered
after a fixed # of
responses

•FI (fixed interval) schedules

§ Reinforcement delivered
after a fixed amount of
time
Evaluating Animal Behavior: Drug Rewards

Self-administration
• Used to study abuse
potential in humans
• Breaking point: point at
which effort required
exceeds reinforcing value

Caine, et al., 1993

Evaluating Animal Behavior: Drug Rewards

Intracranial self-stimulation
(ICSS)
§ animal self-administers
a weak electric current
to brain reward areas
§ Threshold: current
value (defined by
experimenter) at which
animal continually self-
administers
http://www.cerebromente.org.br/n18/history/self-stimulation.jpg
Evaluating Animal Behavior: Drug Rewards

Conditioned Place
Preference (CPP)
•conditioned association
between drug effect and
environment

Swerdlow et al., 1989

Evaluating Animal Behavior: Drug Effects

Drug discrimination testing

•with conditioning, animal learns to discriminate
between drug and saline treatment based on internal
cues produced by the drug
Techniques for Assessing
the CNS
Neurobiological Techniques for Assessing the CNS
Optogenetics
•Insertion of genes into neurons that confer light responsiveness to
control events within those neurons

http://stanford.edu/group/dlab/media/images/cannula-setup.jpg

http://sitn.hms.harvard.edu/flash/2016/optogenetics-can-chronic-pain-be-treated-with-light/
Multiple Neurobiological Techniques for Assessing the CNS

Implanted Microelectrodes
• implanted into a single cell
(intracellular recording)
• or into the extracellular fluid
near a single cell
(extracellular recording)
Multiple Neurobiological Techniques for Assessing the CNS

In vivo microdialysis
§ measure neurotransmitter
released in a specific brain
region while the subject is
actively engaged in
behavior
Neurobiological Techniques for Assessing the CNS
Locating and quantifying NTs and
receptors

§“Soup” method (quantification)

• tissue sample is isolated and
ground (homogenate)
• allows the study of the number of
receptors in a brain region and
affinity for drugs

§“Slice” method (spatial localization)

• uses intact slice of tissue
• visualize distribution of receptors
in the brain
Neurobiological Techniques for Assessing the CNS
Positron emission tomography (PET)
§ maps distribution of an injected radioactively labeled
substance
§ used to determine location of radioactively labeled
drugs
§ Also to locate areas of brain activity when person is
performing cognitive tasks
Neurobiological Techniques for Assessing the CNS

Functional MRI (fMRI)

• Oxygenated hemoglobin
has a different magnetic
resonance signal than
oxygen-depleted
hemoglobin
• Active brain cells need
more O2>>>blood flow
increases http://40.media.tumblr.com/6f71ad724884cda0161a68b0d269bf09/tumblr_mh124iY2lA1rwjinmo1_1280.jpg
Genetic Engineering
Genetic engineering

Genetic engineering
§ DNA of an organism is altered (knockouts or knock-
ins) or a foreign gene is added to the DNA of an
organism (transgenic)
Genetic engineering

Knockout mice
§ lack gene for making a particular
protein
§ Constitutive (germ-line), site-
specific (cre-lox), or inducible (tet
on/off)
Genetic engineering

Knock-in Mice
§ Point mutations
§ Targets a single
nucleotide to produce a
single a.a. substitution
§ May produce a loss or
gain of function
§ Allows study of a
specific mutation
Genetic engineering

Transgenic mice
• Human genes inserted
into mice
• models for
neurodegenerative
diseases (e.g., HD,
ALS, AD)
Genetic engineering

Knockdowns

RNAi mice

• shRNA molecules
• Allows assessment of
lowered levels of gene product
• May be a more valid
representation of the human
condition than a KO
§ Why?
Genetic engineering

(CRISPR/Cas9)
Clustered Regularly Interspaced Short Palindromic Repeats

Single Step genome editing

https://www.jax.org/news-and-insights/jax-blog/2016/february/three-crispr-approaches-for-mouse-genome-editing

• new mutations can be directly generated in a genetic background of choice

• eliminates the need, time, and resources to backcross mutations from one genetic
background to another
• New mutations can be added to existing mouse strains that already carry desired
mutations, reducing the time and costs to generate double and triple mutant mice
Genetic engineering

CRISPR/Cas9: Designer Babies!

Genetic engineering

CRISPR/Cas9: Designer Mutant Babies

• Here we report significant on-target mutagenesis, such as large

deletions and more complex genomic rearrangements at the targeted
sites in mouse embryonic stem cells, mouse hematopoietic progenitors
and a human differentiated cell line
• The observed genomic damage in mitotically active cells caused by
CRISPR– Cas9 editing may have pathogenic consequences
Genetic engineering
Genetic engineering

Potential problems with genetically modified mice:

1. Behaviors are controlled by multiple genes
2. Compensation by other genes
3. Altered gene function occurs in all tissues at all
stages of development
4. Environmental factors affect gene expression:
epigenetics
5. Inbred lines