ANIMAL MODELS FOR BRAIN FUNCTION AND
DISORDERS
INTRODUCTION LECTURE – Judith Homberg
Purpose of the course
• Theoretical-practical understanding of conducting animal experiments, from A to Z.
• Complementary to animal welfare course
• Critical if you aim for an internship focusing on animal research and insightful when aiming to
understand papers about animal experiments
• Discussions of pro’s and con’s of animal experiments and designs (the ideal experiment is not
existing, choices have to be made), training of critical thinking about experiment design
• Different animal species for animal research
• Overview of state-of-the-art technologies: neuroimaging, opto- and chemo genetics,
electrophysiology, EEG, tracing and intracerebral pharmacological manipulations, surgery
methods, fluorescent immunohistochemistry, transparent brain, electrophysiology
Learning goals
• Be able to choose the best animal species for the purpose of your experiment
• Be able to decide how to design your experiment and which controls you need
• Insight into practicalities related to animal experiments
• Knowledge of state-of-the-art neurotechnologies
• Overview of methods that can be used for data analysis
• Understanding of neurobiological mechanisms underlying major brain disorders and how
mechanisms have been elucidated
• Insight into the translational value of animal-derived data
Why animal models?
• Law dictating testing efficacy and toxicology of new drugs
• Detailed mechanistic understanding of health and (brain) disorders -> design new treatments
(also for animals self)
• Fundamental understanding of (neuro)biology
• Controlling factors: genetics, environment, short life span
Experimental animals
• Mice & rats, yeast & nematodes, dogs & cats, monkeys, guinea pigs & rabbits, zebrafish & fruit
files
o Mice & rats are not too simple, but not too complicated
• Rat: behavioral pharmacology/toxicology, more expensive, higher cognitive functions, bigger
(more tissue), social (easy to handle), outbred (more variance) -> more into phenotyping
• Mouse: genetic tool, cheaper (less housing space), lower cognitive functions, smaller (less tissue),
less social, inbred (genetically more similar) -> more into pathways
• Biggest difference between rats and humans is that the cortex in human is a lot more complex /
development more
• You have to be aware of that you don’t know 100% how an animal is feeling, when you test a
disorder in animals
,Validatory of animal models
• Face validity: similarity in symptoms
o Evaluation is very subjective
• Predictive validity: similarity in drug effects
o There are substantial individual differences in responsivity to drugs in humans, which are not
taken into account. Furthermore, several drugs are not specific for the treatment of one
disorder.
• Construct validity: similarity in neural substrates
o How do we know whether an animal model has construct validity if mechanisms are not
known in humans?
Reliability
• How robust are your data?
• Reproducibility of data: If effects can be found in different labs it is more likely that the effect has
a biological meaning. If effects can only found under very specific conditions in the lab and it very
dependent on the researcher who conducts the experiment, the biological meaning can be
questioned.
o Replication of data is not popular, requires the use of more animals for the same type of
experiment and is not likely to be funded because not innovative. However, replication is
very important for science Animal Welfare course 2 wk
Article 9
The EU Law on animal experiments
• Rules you have to follow, with 3 main points: R’s Writing DEC application Instantie voor dierwelzijn (IvD)
• Lot of paperwork before you can conduct an animal experiment 6m Dierexperimenten commissie (DEC)
DEC approved Centrale Commissie Dierproeven (CCD)
The Three R’s and Research
•
Writing workprotocol Instantie voor dierwelzijn (IvD)
Replace: avoid using animals whenever possible 2-4 wk
o Alternatives can be human research, cel systems or computer models Workprotocol approved
o There are no alternatives for measuring behaviour and studying
invasive brain mechanisms. Living organisms with a reasonable complex behavioural
repertoire and brain comparable to humans are needed
• Reduce: when it is impossible to avoid-using animals use the least number possible
o Reasonable group sizes, having small groups (even if effects are significant) is not of benefit
for publication
o Researchers also want to use themselves as low as possible number of animals (but
sufficient for scientific quality/publication) because of the work and costs
o G*power calculate group sizes
• Refine: use the least number of animals, while ensuring the greatest respect for the animal
-> Minimize animal suffering, improve animal welfare and enhance quality of the data collected
o Cage enrichment, social housing
o Surgery: anesthesia, pain killers, antibiotics, (sterility, surgery assistant)
o Extensive handing of the animals
o Avoidance of food/water restriction when possible
o Application of stress only when needed, e.g. to measure stress-related behaviour
• A lot of pressure of Dutch government to replace animal models, but sometimes animals are not
replaceable, for example COVID pandemic -> public discussion. But also responses from other
fields, such as neuroscience. → From silence to openness and transparency in the
communication about animal research & researchers from different universities are more united.
• What can we do better? Social dynamics (more females), caping, diet, room, researcher/stuff
,ANINAL MODELS IN TRANSLATIONAL RESEARCH ON STRESS-
RELATED DISORDERS – Benno Roozendaal
Animal models of emotional memory
• PTSD characterized by strong traumatic memories (several other psychiatric disorders also have
strong emotional memories: phobias, drug addiction, OCD, et cetera)
• In an animal model of PTSD you could submit animals to severe stress (trauma) and look at
changes in behavior or in the brain? -> Is that really a good model of PTSD?
• Is PTSD really characterized by strong memory or by an aberrant processing of this information,
leading to unwanted retrieval, generalization and impairment in extinction?
o Only a small percentage of humans that experience severe trauma will develop PTSD, but
they will remember the traumatic event -> most animal models don’t take that to account ->
Most drug treatments based on animal models of anxiety disorders do not work in patients:
mismatch → What is wrong: is the animal model wrong or is it not well translated to human
situation?
• My general approach: study emotional memory in animals, basic mechanisms, effects of drugs on
different aspects of learning and memory and neural mechanisms involved -> based on that you
can make predications of how it works in humans
Emotional arousal induces activation of stress hormone systems
• Stress -> release of stress hormones:
epinephrine and norepinephrine
from adrenal medulla -> accelerates
heartbeat, dilates bronchi, inhibits
peristalsis, etc.; same time activation
of glucocorticoids from adrenal
cortex (cortisol in humans and
corticosterone in rodents)
o They don’t activate in exactly
the same manner, but there is
overlap
• Glucocorticoids can cross the blood-
brain barrier and directly affect the brain, while epinephrine influences the brain indirectly
through vagus nerve activation.
• Glucocorticoids have a negative feedback mechanism -> inhibit own production
Interaction between glucocorticoids and noradrenergic system
• Inhibitory avoidance: normally rat don’t like light and goes to the
dark, but now the get a shock in the dark area -> Next time: how
long takes it before rat gets to the dark?
o Dexamethasone (glucocorticoid): latency is higher, so better
memory of the fear of the dark room is created
o Atenolol, Zinterol, Propranolol (beta-blocker; blocks
norepinephrine receptor) in amygdala at the same time:
block the effect
-> Glucocorticoid need norepinephrine in the amygdala to have
its effect (only effect on emotions, because interaction plays critical role)
• Emotional training induces the release of norepinephrine in the basolateral amygdala
, Glucocorticoid effects on memory consolidation require noradrenergic system
• Do glucocorticoid effects on memory interact
with emotional arousal?
• Object task: Mouse placed into a cage with
two novel objects (not emotional task,
because we want to know if emotional task is
crucial for glucocorticoid effects) ->
corticosteroid -> putting mouse in the case
with old object and new object (not interested
in old object normally)
o Putting mouse in a novel cage is always
stressful and therefore release of
norepinephrine, so 2 groups: habituation
group and no habituation group
• Without habituation, so norepinephrine: corticosteroids enhance discrimination
• With habituation: corticosteroids do not enhance discrimination/memory
• Propranolol (beta-blocker): blocks effect of corticosteroid
• Yohimbine (norepinephrine stimulant): corticosteroid able to enhance memory
-> Corticosteroids needs norepinephrine, so emotional arousal, to have an effect
Stress hormones activate the amygdala
• Cortisol we always have in our blood, very high levels in the morning and later in the day lower
levels. This is important to keep in mind when studying effects of cortisol.
• Cortisol and adrenaline both activate the amygdala directly or indirectly, which facilitating stores
memory in other brain areas. So, if both hormones interact, this makes the memory stronger
Does emotional arousal (and stress hormones) also influence the accuracy or specificity of
memory?
• Inhibitory avoidance discrimination task: training
in one box -> training other box where is foot
shock delivery -> novel box
• Vehicle group: equal latencies between shock and
non-shock group when there is 1 min interval
between shock and placing in novel box
o Interval 2 minutes: difference is measured ->
animals remember in which box they got a shock
• Yohimbine (norepinephrine stimulant) causes that animals know better in which box they got a
shock -> specific memory starker
• Corticosterone causes higher latency for both boxes, so the memory is stronger but not specific
-> Yohimbine and corticosterone have same effect on
strength but not on accuracy of the memory!
• What is the long-term fate of these memories?
• Context with no shock -> context with shock -> NE
infusion into amygdala -> muscimol into
hippocampus -> retention test: context with no
shock or context with shock or novel context
• In control animals the memory of the shock
disappears, but when NE is administered right after
training the memory stays active longer. This effect
stays after 28-days (memory stores in prefrontal
DISORDERS
INTRODUCTION LECTURE – Judith Homberg
Purpose of the course
• Theoretical-practical understanding of conducting animal experiments, from A to Z.
• Complementary to animal welfare course
• Critical if you aim for an internship focusing on animal research and insightful when aiming to
understand papers about animal experiments
• Discussions of pro’s and con’s of animal experiments and designs (the ideal experiment is not
existing, choices have to be made), training of critical thinking about experiment design
• Different animal species for animal research
• Overview of state-of-the-art technologies: neuroimaging, opto- and chemo genetics,
electrophysiology, EEG, tracing and intracerebral pharmacological manipulations, surgery
methods, fluorescent immunohistochemistry, transparent brain, electrophysiology
Learning goals
• Be able to choose the best animal species for the purpose of your experiment
• Be able to decide how to design your experiment and which controls you need
• Insight into practicalities related to animal experiments
• Knowledge of state-of-the-art neurotechnologies
• Overview of methods that can be used for data analysis
• Understanding of neurobiological mechanisms underlying major brain disorders and how
mechanisms have been elucidated
• Insight into the translational value of animal-derived data
Why animal models?
• Law dictating testing efficacy and toxicology of new drugs
• Detailed mechanistic understanding of health and (brain) disorders -> design new treatments
(also for animals self)
• Fundamental understanding of (neuro)biology
• Controlling factors: genetics, environment, short life span
Experimental animals
• Mice & rats, yeast & nematodes, dogs & cats, monkeys, guinea pigs & rabbits, zebrafish & fruit
files
o Mice & rats are not too simple, but not too complicated
• Rat: behavioral pharmacology/toxicology, more expensive, higher cognitive functions, bigger
(more tissue), social (easy to handle), outbred (more variance) -> more into phenotyping
• Mouse: genetic tool, cheaper (less housing space), lower cognitive functions, smaller (less tissue),
less social, inbred (genetically more similar) -> more into pathways
• Biggest difference between rats and humans is that the cortex in human is a lot more complex /
development more
• You have to be aware of that you don’t know 100% how an animal is feeling, when you test a
disorder in animals
,Validatory of animal models
• Face validity: similarity in symptoms
o Evaluation is very subjective
• Predictive validity: similarity in drug effects
o There are substantial individual differences in responsivity to drugs in humans, which are not
taken into account. Furthermore, several drugs are not specific for the treatment of one
disorder.
• Construct validity: similarity in neural substrates
o How do we know whether an animal model has construct validity if mechanisms are not
known in humans?
Reliability
• How robust are your data?
• Reproducibility of data: If effects can be found in different labs it is more likely that the effect has
a biological meaning. If effects can only found under very specific conditions in the lab and it very
dependent on the researcher who conducts the experiment, the biological meaning can be
questioned.
o Replication of data is not popular, requires the use of more animals for the same type of
experiment and is not likely to be funded because not innovative. However, replication is
very important for science Animal Welfare course 2 wk
Article 9
The EU Law on animal experiments
• Rules you have to follow, with 3 main points: R’s Writing DEC application Instantie voor dierwelzijn (IvD)
• Lot of paperwork before you can conduct an animal experiment 6m Dierexperimenten commissie (DEC)
DEC approved Centrale Commissie Dierproeven (CCD)
The Three R’s and Research
•
Writing workprotocol Instantie voor dierwelzijn (IvD)
Replace: avoid using animals whenever possible 2-4 wk
o Alternatives can be human research, cel systems or computer models Workprotocol approved
o There are no alternatives for measuring behaviour and studying
invasive brain mechanisms. Living organisms with a reasonable complex behavioural
repertoire and brain comparable to humans are needed
• Reduce: when it is impossible to avoid-using animals use the least number possible
o Reasonable group sizes, having small groups (even if effects are significant) is not of benefit
for publication
o Researchers also want to use themselves as low as possible number of animals (but
sufficient for scientific quality/publication) because of the work and costs
o G*power calculate group sizes
• Refine: use the least number of animals, while ensuring the greatest respect for the animal
-> Minimize animal suffering, improve animal welfare and enhance quality of the data collected
o Cage enrichment, social housing
o Surgery: anesthesia, pain killers, antibiotics, (sterility, surgery assistant)
o Extensive handing of the animals
o Avoidance of food/water restriction when possible
o Application of stress only when needed, e.g. to measure stress-related behaviour
• A lot of pressure of Dutch government to replace animal models, but sometimes animals are not
replaceable, for example COVID pandemic -> public discussion. But also responses from other
fields, such as neuroscience. → From silence to openness and transparency in the
communication about animal research & researchers from different universities are more united.
• What can we do better? Social dynamics (more females), caping, diet, room, researcher/stuff
,ANINAL MODELS IN TRANSLATIONAL RESEARCH ON STRESS-
RELATED DISORDERS – Benno Roozendaal
Animal models of emotional memory
• PTSD characterized by strong traumatic memories (several other psychiatric disorders also have
strong emotional memories: phobias, drug addiction, OCD, et cetera)
• In an animal model of PTSD you could submit animals to severe stress (trauma) and look at
changes in behavior or in the brain? -> Is that really a good model of PTSD?
• Is PTSD really characterized by strong memory or by an aberrant processing of this information,
leading to unwanted retrieval, generalization and impairment in extinction?
o Only a small percentage of humans that experience severe trauma will develop PTSD, but
they will remember the traumatic event -> most animal models don’t take that to account ->
Most drug treatments based on animal models of anxiety disorders do not work in patients:
mismatch → What is wrong: is the animal model wrong or is it not well translated to human
situation?
• My general approach: study emotional memory in animals, basic mechanisms, effects of drugs on
different aspects of learning and memory and neural mechanisms involved -> based on that you
can make predications of how it works in humans
Emotional arousal induces activation of stress hormone systems
• Stress -> release of stress hormones:
epinephrine and norepinephrine
from adrenal medulla -> accelerates
heartbeat, dilates bronchi, inhibits
peristalsis, etc.; same time activation
of glucocorticoids from adrenal
cortex (cortisol in humans and
corticosterone in rodents)
o They don’t activate in exactly
the same manner, but there is
overlap
• Glucocorticoids can cross the blood-
brain barrier and directly affect the brain, while epinephrine influences the brain indirectly
through vagus nerve activation.
• Glucocorticoids have a negative feedback mechanism -> inhibit own production
Interaction between glucocorticoids and noradrenergic system
• Inhibitory avoidance: normally rat don’t like light and goes to the
dark, but now the get a shock in the dark area -> Next time: how
long takes it before rat gets to the dark?
o Dexamethasone (glucocorticoid): latency is higher, so better
memory of the fear of the dark room is created
o Atenolol, Zinterol, Propranolol (beta-blocker; blocks
norepinephrine receptor) in amygdala at the same time:
block the effect
-> Glucocorticoid need norepinephrine in the amygdala to have
its effect (only effect on emotions, because interaction plays critical role)
• Emotional training induces the release of norepinephrine in the basolateral amygdala
, Glucocorticoid effects on memory consolidation require noradrenergic system
• Do glucocorticoid effects on memory interact
with emotional arousal?
• Object task: Mouse placed into a cage with
two novel objects (not emotional task,
because we want to know if emotional task is
crucial for glucocorticoid effects) ->
corticosteroid -> putting mouse in the case
with old object and new object (not interested
in old object normally)
o Putting mouse in a novel cage is always
stressful and therefore release of
norepinephrine, so 2 groups: habituation
group and no habituation group
• Without habituation, so norepinephrine: corticosteroids enhance discrimination
• With habituation: corticosteroids do not enhance discrimination/memory
• Propranolol (beta-blocker): blocks effect of corticosteroid
• Yohimbine (norepinephrine stimulant): corticosteroid able to enhance memory
-> Corticosteroids needs norepinephrine, so emotional arousal, to have an effect
Stress hormones activate the amygdala
• Cortisol we always have in our blood, very high levels in the morning and later in the day lower
levels. This is important to keep in mind when studying effects of cortisol.
• Cortisol and adrenaline both activate the amygdala directly or indirectly, which facilitating stores
memory in other brain areas. So, if both hormones interact, this makes the memory stronger
Does emotional arousal (and stress hormones) also influence the accuracy or specificity of
memory?
• Inhibitory avoidance discrimination task: training
in one box -> training other box where is foot
shock delivery -> novel box
• Vehicle group: equal latencies between shock and
non-shock group when there is 1 min interval
between shock and placing in novel box
o Interval 2 minutes: difference is measured ->
animals remember in which box they got a shock
• Yohimbine (norepinephrine stimulant) causes that animals know better in which box they got a
shock -> specific memory starker
• Corticosterone causes higher latency for both boxes, so the memory is stronger but not specific
-> Yohimbine and corticosterone have same effect on
strength but not on accuracy of the memory!
• What is the long-term fate of these memories?
• Context with no shock -> context with shock -> NE
infusion into amygdala -> muscimol into
hippocampus -> retention test: context with no
shock or context with shock or novel context
• In control animals the memory of the shock
disappears, but when NE is administered right after
training the memory stays active longer. This effect
stays after 28-days (memory stores in prefrontal
