Research Skills in Life Sciences
Behavior and Neuroscience
Lecture 1 – Molenhuis & Buwalda
Introduction Into BN Research Skills
Learning goals at the end of the course
- Describe research techniques commonly used in neuroscience
- Explain the possibilities of experimental research in neuroscience
Historical introduction
- Example → studies into Autism Spectrum Disorder (ASD)
- Correlation and causation
- Observational versus experimental research
Autism spectrum disorder (ASD) → difficult to study
- Defined on behavioral symptoms → broad clinical definition
- Lack of known causal factors
- Poor access to brain tissue
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 can be obtained
4. Developmental aspects can be studied (life span)
Observational 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)
,Lecture 2 – Molenhuis
Genes and behavior
Genetic association studies (‘forward genetics)
Behavioral trait → which genotypes?
- Family studies (e.g., Mendelian disorders)
- Population genetics (e.g., case-control, behavioral difference)
Gene manipulation experiments (‘reverse genetics’)
Effects on behavioral traits? ← genotype
- Genetic manipulation (e.g., gene knock-out, CRISPR/Cas9)
- Behavioral readouts
- Conditional gene manipulation (e.g., spatio-temporal control)
Twin studies → can be used dissect genetic and environmental influences
- Monozygotic twins share ~100% of their genes, while dizygotic twins share only ~50%
- Allows to assesses the genetic and environmental influence on a trait
Heritability always depends on the environmental context in which it was measured → Flynn-effect
Heritability = 2 x (rMZ – rDZ)
- Autism: > 80%
- Reading disability: 40%
Linkage study (1990s) → searching genetic locus for aggressive behavior in family
- Defect in MAOA gene
Sanger sequencing (1977) → a technique
used to read the full implicated DNA
sequence
Family studies using Whole Exome
Sequencing (2010-now) → using ‘next-
generation sequencing’ to read all exomes
at once
- 180000 exons
- 30 million base pairs
Used to identify mutations in families
- Rare inherited mutations
- ‘de novo’ mutations
Mendelian disorders → are due in families to mutations with large effect
Behavior → is influenced in populations by many variants with small effects
Genome-wide association study (GWAS) → used 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 for burrowing behavior
- Multiple loci explaining difference in tunnel length – 3 cm each
- Locus explaining probability to make escape tunnel
Mice with human-like mutation monoamine oxidase (MAO-A) display enhanced aggression.
Genetic disorders → animal models
- Animal models focus on genes disrupted by rare mutations
- Study in fixed genetic and environmental background
Fmr1 KO mice
- No difference in general brain morphology
- Increased testicular weight and abnormal exploratory behaviors
- Impaired Morris Water Maze Performance
- Lack of preference for a conspecific mouse
Generation of genetic knockout animals by
homologous recombination of a targeting
vector (since 1988)
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
Oxytocin receptor knockout causes reduced preference for social novelty in male prairie voles
Genes are often expressed in multiple tissues and brain regions → conditional gene KO mouse
models can be achieved using the Cre-loxP system
- Tissue-specific KO model → 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
- Temporal KO model → gene of interest is temporally inactivated at a given time-point in
embryonic, post-natal or adult animals following injection with Tamoxifen
Behavior and Neuroscience
Lecture 1 – Molenhuis & Buwalda
Introduction Into BN Research Skills
Learning goals at the end of the course
- Describe research techniques commonly used in neuroscience
- Explain the possibilities of experimental research in neuroscience
Historical introduction
- Example → studies into Autism Spectrum Disorder (ASD)
- Correlation and causation
- Observational versus experimental research
Autism spectrum disorder (ASD) → difficult to study
- Defined on behavioral symptoms → broad clinical definition
- Lack of known causal factors
- Poor access to brain tissue
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 can be obtained
4. Developmental aspects can be studied (life span)
Observational 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)
,Lecture 2 – Molenhuis
Genes and behavior
Genetic association studies (‘forward genetics)
Behavioral trait → which genotypes?
- Family studies (e.g., Mendelian disorders)
- Population genetics (e.g., case-control, behavioral difference)
Gene manipulation experiments (‘reverse genetics’)
Effects on behavioral traits? ← genotype
- Genetic manipulation (e.g., gene knock-out, CRISPR/Cas9)
- Behavioral readouts
- Conditional gene manipulation (e.g., spatio-temporal control)
Twin studies → can be used dissect genetic and environmental influences
- Monozygotic twins share ~100% of their genes, while dizygotic twins share only ~50%
- Allows to assesses the genetic and environmental influence on a trait
Heritability always depends on the environmental context in which it was measured → Flynn-effect
Heritability = 2 x (rMZ – rDZ)
- Autism: > 80%
- Reading disability: 40%
Linkage study (1990s) → searching genetic locus for aggressive behavior in family
- Defect in MAOA gene
Sanger sequencing (1977) → a technique
used to read the full implicated DNA
sequence
Family studies using Whole Exome
Sequencing (2010-now) → using ‘next-
generation sequencing’ to read all exomes
at once
- 180000 exons
- 30 million base pairs
Used to identify mutations in families
- Rare inherited mutations
- ‘de novo’ mutations
Mendelian disorders → are due in families to mutations with large effect
Behavior → is influenced in populations by many variants with small effects
Genome-wide association study (GWAS) → used 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 for burrowing behavior
- Multiple loci explaining difference in tunnel length – 3 cm each
- Locus explaining probability to make escape tunnel
Mice with human-like mutation monoamine oxidase (MAO-A) display enhanced aggression.
Genetic disorders → animal models
- Animal models focus on genes disrupted by rare mutations
- Study in fixed genetic and environmental background
Fmr1 KO mice
- No difference in general brain morphology
- Increased testicular weight and abnormal exploratory behaviors
- Impaired Morris Water Maze Performance
- Lack of preference for a conspecific mouse
Generation of genetic knockout animals by
homologous recombination of a targeting
vector (since 1988)
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
Oxytocin receptor knockout causes reduced preference for social novelty in male prairie voles
Genes are often expressed in multiple tissues and brain regions → conditional gene KO mouse
models can be achieved using the Cre-loxP system
- Tissue-specific KO model → 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
- Temporal KO model → gene of interest is temporally inactivated at a given time-point in
embryonic, post-natal or adult animals following injection with Tamoxifen
