LECTURE 1 INTRODUCTION INTO RESEARCH SKILLS IN BEHAVIORAL NEUROSCIENCE (REMCO
MOLENHUIS)
Outline – Research in Behavioral Neuroscience
● Historical introduction
○ Example: studies into Autism Spectrum Disorders (ASD)
○ Correlation and causation
○ Observational versus experimental research
● The use of model systems
○ Experimental manipulation
○ Readout (e.g. molecular, cellular, behavioral)
After this lecture you should be able to:
- note the difference between correlation and causation
- give examples of common observational and experimental approaches used in biomedical research - describe the differences
between observational and experimental studies
- explain some of the advantages of using animal models in genetic research
Autism Spectrum Disorder (ASD) difficult to study
- Defined based on behavioral symptoms, broad clinical definition
- Lack of known causal factors
- Poor access to brain tissue
Understanding biological mechanisms as a first step towards more effective intervention strategies
Some advantages of using animal (= in vivo) models ?
1. Causality can be tested by experimental design
2. Control over environmental and genetic background
3. Relevant tissue (e.g. brain) can be obtained
4. Developmental aspects can be studied (life-span)
Storks deliver babies?!
Or the bigger the population, the more likely to spot storks?
Short story is that the bigger the population, the more eyeballs available to spot storks...
Correlation / causation?
Observation studies
● Case-control studies
● Genetic association studies (e.g. GWAS)
● Cohort studies (e.g. LifeLines)
Experiments
● Experimental manipulations:
○ Cellular models: “in vitro”
○ Animal models: “in vivo”
● Clinical trials (randomized)
Correlation ≄ causation
Know these ↓↓
RESEARCH SKILLS IN LIFE SCIENCES B&N TRACK 1
, LECTURE 2 RESEARCH SKILLS: GENES AND BEHAVIOR (REMCO MOLENHUIS)
Genetic association studies (‘’forward genetics’’)
Behavioral trait → which genotypes?
Gene manipulation experiments (‘’reverse genetics’’)
Effect on behavioral traits? ← genotype
Outline - genes and behavior
● Genetic association studies
○ Family studies (e.g., Mendelian disorders)
○ Population genetics (e.g., case-control, behavioral differences)
● Genetic manipulation experiments
○ Genetic manipulation (e.g., gene knock-out, CRISPR/Cas9)
○ Behavioral readouts
○ Conditional gene manipulation (e.g., spatio-temporal control)
you should be able to:
- describe methods that are commonly used in behavioral genetic association studies, in families (rare mutations) and populations (common genetic
variation)
- describe strategies for manipulating genes in experimental model systems, incl. for conditional expression
- explain some of the advantages behavioral genetic research in animal models
Family is not heritability
Heritable due to genes?
Familial due to environment?
Twin studies can be used dissect genetic and environmental influences
Monozygotic twins - share environment and genes
Dizygotic - share environment
- Monozygous twins (MZ) share ~100% of their genes, while dizygotic twins (DZ)
share only ~50%.
- Twin designs allow to assesses the genetic and environmental influence on a trait
using twin pairs.
Twin studies indicate genetic contribution to behavioral disorders
- Estimate heritability by assessing whether MZ twins are more similar (“concordant”)
than DZ twins.
- Heritability = 2 x (rMZ – rDZ)
- Autism: > 80%
- Reading disability: 40%
You do not need to know formula, just understand
Note: Heritability always depends on the environmental context in which it was
measured (e.g. see Wikipedia on Flynn-effect) !!
3 200 000 000 nucleotides of DNA (human genome)
Linkage study (1990s): searching genetic locus for aggressive behavior in family
aggressive/violent behaviour in a Dutch family was linked to defect in MAOA gene:
1. Only present in males suggests X-chromosome.
2. Investigations of genetic markers and ‘haplotypes’ unique for affected family members.
3. Mutation likely inherited via combination of markers and pointing at marker for MAOA gene
4. Disturbed monoamine metabolism observed in urine analysis.
Using “Sanger sequencing” technique (1977) to read the full implicated DNA sequence
‘’in each of five affected males, a point mutation was identified in the eighth exon of the MAOA structural gene, which changes a glutamine to a
termination codon.”
RESEARCH SKILLS IN LIFE SCIENCES B&N TRACK 2
, Family studies 1993 - now
Family studies using Whole Exome Sequencing (2010-now)
Using “next-generation sequencing” to read all exomes at once:
- 180,000 exons
- 30 million base pairs (1% of genome)
Used to identify mutations in families:
- rare inherited mutations
- “de novo” mutations (only found in affected child)
Also used in clinical practice!
Mendelian disorders vs. common genetic traits
- Mendelian disorders in families are due to mutation with large effect (i.e., rare gene
mutations)
- Behavior in populations is influenced by many variants with small effects (i.e., common
genetic variation)
Genome-wide association study (GWAS) to identify common genetic variation (e.g., schizophrenia)
1. Comparing Schizophrenia patients with healthy controls for ~600.000 genetic makers across the entire genome.
2. GWAS requires extremely large sample sizes as the effect-sizes for these markers (i.e. SNPs) are often small.
3. Significant markers (i.e. SNPs) are correlated with the actual causal variant, but not necessarily causal!
Genetic association studies can also be used across species
Measuring behavior in peromyscus mice
Burrowing behaviors in peromyscus are heritable
genetic associations for burrowing behaviors
- Multiple loci explaining difference in tunnel length – 3 cm each
- Locus explaining probability to make escape tunnel
Mice with human-like mutation in monoamine oxidase (MAO-A) display enhanced aggression
From genetic disorders to animal models
- Animal models focus on genes disrupted by rare mutations
- Study in fixed genetic and environmental background
- No differences in general brain morphology in Fmr1 KO mice
- increased testicular weight and abnormal exploratory behaviors in Fmr1 KO mice
- Impaired Morris Water Maze Performance in Fmr KO mice ->
- Lack of preference for a conspecific mouse in Fmr1 KO mice
RESEARCH SKILLS IN LIFE SCIENCES B&N TRACK 3
,Faster and more precise generation of genetically modified animals with Crispr/Cas9 (since 2013)
1. Cocktail with CRISPR/Cas9 can be injected directly into zygotes: very fast!!
2. CRISPR RNA with variable targeting sequence (crRNA) allows binding to target DNA
3. Single-stranded donor oligonucleotides (ssODN) contains alternative sequence
This technique allows to:
● Create efficient gene knockin or knockout
● Create point mutations
● Modify sequence segments
Crispr/Cas9 has also enabled genetic manipulation of ‘exotic’ species, such as monogamous prairie voles
When prairie voles choose a mate, there’s no turning back—the “love chemical” oxytocin increases in their brains and they
devote themselves to only each other.
Oxytocin receptor knockout causes reduced preference for social novelty in male prairie voles
Genes are often expressed in multiple tissues and brain regions: how to deal with this?
Flex switches enable endless possibilities to switch genes on/off at the desired timepoint (=temporal) or tissue (=spatial)
Conditional gene KO mouse models can be achieved using the Cre-
loxP system
A conditional KO mouse is an animal in which the gene of interest is:
- Inactivated only in specific cell types in a certain tissue; other
cell types and tissues exhibit an unmodified, functional gene
expression (= Tissue-specific KO model)
- Temporally inactivated at a given time-point in embryonic,
post-natal or adult animals following injection with Tamoxifen
(= Temporal KO model)
RESEARCH SKILLS IN LIFE SCIENCES B&N TRACK 4
, LECTURE 3 STRESS RESEARCH IN BEHAVIORAL NEUROSCIENCE (BAUKE BUWALDA)
Stress: body response
Stress response is adaptive, makes it possible to deal with the situation
GODFATHERS OF STRESS RESEARCH
- Hans Selye -> general adaptation syndrome (whatever you confront an animal with, they always respond with a similar
physiological response to that stimulus)
Introduced stress in field of physiology
- Walter Bradford Cannon -> homeostasis (active regulation)
Sympatho-adrenergic system (-> immediate fight-flight response)
Medulla:
catecholamines
Synthesis of catecholamine hormones
Stress: adrenaline
↓
Release energy (glucose and free fatty acids)
↓
‘’Fight or flight’’
HPA-axis
Acute stress is highly adaptive
HPA-axis and SAM activation prepare and support behavioral response
Stress can sometimes have maladaptive consequences ...
If stressor is uncontrollable and unpredictable, it is a dangerous stress
RESEARCH SKILLS IN LIFE SCIENCES B&N TRACK 5
MOLENHUIS)
Outline – Research in Behavioral Neuroscience
● Historical introduction
○ Example: studies into Autism Spectrum Disorders (ASD)
○ Correlation and causation
○ Observational versus experimental research
● The use of model systems
○ Experimental manipulation
○ Readout (e.g. molecular, cellular, behavioral)
After this lecture you should be able to:
- note the difference between correlation and causation
- give examples of common observational and experimental approaches used in biomedical research - describe the differences
between observational and experimental studies
- explain some of the advantages of using animal models in genetic research
Autism Spectrum Disorder (ASD) difficult to study
- Defined based on behavioral symptoms, broad clinical definition
- Lack of known causal factors
- Poor access to brain tissue
Understanding biological mechanisms as a first step towards more effective intervention strategies
Some advantages of using animal (= in vivo) models ?
1. Causality can be tested by experimental design
2. Control over environmental and genetic background
3. Relevant tissue (e.g. brain) can be obtained
4. Developmental aspects can be studied (life-span)
Storks deliver babies?!
Or the bigger the population, the more likely to spot storks?
Short story is that the bigger the population, the more eyeballs available to spot storks...
Correlation / causation?
Observation studies
● Case-control studies
● Genetic association studies (e.g. GWAS)
● Cohort studies (e.g. LifeLines)
Experiments
● Experimental manipulations:
○ Cellular models: “in vitro”
○ Animal models: “in vivo”
● Clinical trials (randomized)
Correlation ≄ causation
Know these ↓↓
RESEARCH SKILLS IN LIFE SCIENCES B&N TRACK 1
, LECTURE 2 RESEARCH SKILLS: GENES AND BEHAVIOR (REMCO MOLENHUIS)
Genetic association studies (‘’forward genetics’’)
Behavioral trait → which genotypes?
Gene manipulation experiments (‘’reverse genetics’’)
Effect on behavioral traits? ← genotype
Outline - genes and behavior
● Genetic association studies
○ Family studies (e.g., Mendelian disorders)
○ Population genetics (e.g., case-control, behavioral differences)
● Genetic manipulation experiments
○ Genetic manipulation (e.g., gene knock-out, CRISPR/Cas9)
○ Behavioral readouts
○ Conditional gene manipulation (e.g., spatio-temporal control)
you should be able to:
- describe methods that are commonly used in behavioral genetic association studies, in families (rare mutations) and populations (common genetic
variation)
- describe strategies for manipulating genes in experimental model systems, incl. for conditional expression
- explain some of the advantages behavioral genetic research in animal models
Family is not heritability
Heritable due to genes?
Familial due to environment?
Twin studies can be used dissect genetic and environmental influences
Monozygotic twins - share environment and genes
Dizygotic - share environment
- Monozygous twins (MZ) share ~100% of their genes, while dizygotic twins (DZ)
share only ~50%.
- Twin designs allow to assesses the genetic and environmental influence on a trait
using twin pairs.
Twin studies indicate genetic contribution to behavioral disorders
- Estimate heritability by assessing whether MZ twins are more similar (“concordant”)
than DZ twins.
- Heritability = 2 x (rMZ – rDZ)
- Autism: > 80%
- Reading disability: 40%
You do not need to know formula, just understand
Note: Heritability always depends on the environmental context in which it was
measured (e.g. see Wikipedia on Flynn-effect) !!
3 200 000 000 nucleotides of DNA (human genome)
Linkage study (1990s): searching genetic locus for aggressive behavior in family
aggressive/violent behaviour in a Dutch family was linked to defect in MAOA gene:
1. Only present in males suggests X-chromosome.
2. Investigations of genetic markers and ‘haplotypes’ unique for affected family members.
3. Mutation likely inherited via combination of markers and pointing at marker for MAOA gene
4. Disturbed monoamine metabolism observed in urine analysis.
Using “Sanger sequencing” technique (1977) to read the full implicated DNA sequence
‘’in each of five affected males, a point mutation was identified in the eighth exon of the MAOA structural gene, which changes a glutamine to a
termination codon.”
RESEARCH SKILLS IN LIFE SCIENCES B&N TRACK 2
, Family studies 1993 - now
Family studies using Whole Exome Sequencing (2010-now)
Using “next-generation sequencing” to read all exomes at once:
- 180,000 exons
- 30 million base pairs (1% of genome)
Used to identify mutations in families:
- rare inherited mutations
- “de novo” mutations (only found in affected child)
Also used in clinical practice!
Mendelian disorders vs. common genetic traits
- Mendelian disorders in families are due to mutation with large effect (i.e., rare gene
mutations)
- Behavior in populations is influenced by many variants with small effects (i.e., common
genetic variation)
Genome-wide association study (GWAS) to identify common genetic variation (e.g., schizophrenia)
1. Comparing Schizophrenia patients with healthy controls for ~600.000 genetic makers across the entire genome.
2. GWAS requires extremely large sample sizes as the effect-sizes for these markers (i.e. SNPs) are often small.
3. Significant markers (i.e. SNPs) are correlated with the actual causal variant, but not necessarily causal!
Genetic association studies can also be used across species
Measuring behavior in peromyscus mice
Burrowing behaviors in peromyscus are heritable
genetic associations for burrowing behaviors
- Multiple loci explaining difference in tunnel length – 3 cm each
- Locus explaining probability to make escape tunnel
Mice with human-like mutation in monoamine oxidase (MAO-A) display enhanced aggression
From genetic disorders to animal models
- Animal models focus on genes disrupted by rare mutations
- Study in fixed genetic and environmental background
- No differences in general brain morphology in Fmr1 KO mice
- increased testicular weight and abnormal exploratory behaviors in Fmr1 KO mice
- Impaired Morris Water Maze Performance in Fmr KO mice ->
- Lack of preference for a conspecific mouse in Fmr1 KO mice
RESEARCH SKILLS IN LIFE SCIENCES B&N TRACK 3
,Faster and more precise generation of genetically modified animals with Crispr/Cas9 (since 2013)
1. Cocktail with CRISPR/Cas9 can be injected directly into zygotes: very fast!!
2. CRISPR RNA with variable targeting sequence (crRNA) allows binding to target DNA
3. Single-stranded donor oligonucleotides (ssODN) contains alternative sequence
This technique allows to:
● Create efficient gene knockin or knockout
● Create point mutations
● Modify sequence segments
Crispr/Cas9 has also enabled genetic manipulation of ‘exotic’ species, such as monogamous prairie voles
When prairie voles choose a mate, there’s no turning back—the “love chemical” oxytocin increases in their brains and they
devote themselves to only each other.
Oxytocin receptor knockout causes reduced preference for social novelty in male prairie voles
Genes are often expressed in multiple tissues and brain regions: how to deal with this?
Flex switches enable endless possibilities to switch genes on/off at the desired timepoint (=temporal) or tissue (=spatial)
Conditional gene KO mouse models can be achieved using the Cre-
loxP system
A conditional KO mouse is an animal in which the gene of interest is:
- Inactivated only in specific cell types in a certain tissue; other
cell types and tissues exhibit an unmodified, functional gene
expression (= Tissue-specific KO model)
- Temporally inactivated at a given time-point in embryonic,
post-natal or adult animals following injection with Tamoxifen
(= Temporal KO model)
RESEARCH SKILLS IN LIFE SCIENCES B&N TRACK 4
, LECTURE 3 STRESS RESEARCH IN BEHAVIORAL NEUROSCIENCE (BAUKE BUWALDA)
Stress: body response
Stress response is adaptive, makes it possible to deal with the situation
GODFATHERS OF STRESS RESEARCH
- Hans Selye -> general adaptation syndrome (whatever you confront an animal with, they always respond with a similar
physiological response to that stimulus)
Introduced stress in field of physiology
- Walter Bradford Cannon -> homeostasis (active regulation)
Sympatho-adrenergic system (-> immediate fight-flight response)
Medulla:
catecholamines
Synthesis of catecholamine hormones
Stress: adrenaline
↓
Release energy (glucose and free fatty acids)
↓
‘’Fight or flight’’
HPA-axis
Acute stress is highly adaptive
HPA-axis and SAM activation prepare and support behavioral response
Stress can sometimes have maladaptive consequences ...
If stressor is uncontrollable and unpredictable, it is a dangerous stress
RESEARCH SKILLS IN LIFE SCIENCES B&N TRACK 5
