Techniques in pharmacology Flashcards
What is electrophysiology?
• Electrophysiology- the study of the electrical properties of cells or tissues
What information does electrophysiology provide?
o Tells us about function (but can also tell us about location)
Why is electrophysiology possible?
o All cells have a membrane potential that is maintained by control by movement of sodium, calcium and potassium
o Using electrophysiology, we can measure the movements of these (and other) ions
What does electrophysiology allow the exploration of?
o How drugs modify the way ion channels activate, open, inactivate and desensitize
o How drugs change neuronal excitability
o How drugs change synaptic transmission and synaptic plasticity
o Location of drug action
o Drug interactions with a mutated protein
What instrument can be used to measure bioelectricity at the amp range?
Fingers
What instrument can be used to measure bioelectricity at the milliamp range?
Wires
What instrument can be used to measure bioelectricity at the microamp range?
Extracellular cell recordings
Microelectrodes
What instrument can be used to measure bioelectricity at the nanoamp range?
Microelectrode/patch
What instrument can be used to measure bioelectricity at the picoamp/attoamp range?
Patch only
What is the advantage of extracellular recordings?
Can be done in vivo
What are the disadvantages of extracellular cell recordings?
- Very hard to control drug administration
- No control over the cell of interest
- No choice of cells
- Very general- not able to pinpoint specific effects
What is the process of extracellular cell recordings?
o As action potential propagates through a cell the electrical current flows in and out of the cell. This changes the voltage in and out of the cell
o If an electrode is inserted into the brain and near a neuron, this extracellular electrode will measure this voltage change
How are single-unit extracellular recordings performed?
o Small electrodes can record activity of one neuron (single-unit)
How are multi-unit extracellular recordings performed?
o Larger electrodes record the activity of several neurons (multi-unit)
Where are extracellular cell recordings mainly used?
o Main use in in vivo anaesthetized or awake behaving animals
How are microelectrode/intracellular cell recordings performed and what does it measure?
o Very fine glass electrode is inserted into a cell of interest
o Metal wire in the liquid filled glass electrode connects to amplifier
o Allows measurement of voltage or current across the membrane compared to a reference electrode
Where are microelectrode/intracellular cell recordings used and why?
o Mainly used in oocytes due to their large size, some neurons (but very few are able to put two electrodes in due to their small size, and hard to control voltage and measure current at the same time with a single electrode)
What are the advantages of using microelectrode/intracellular cell recordings with oocytes?
Advantages:
• Oocytes express proteins well (good for mutation studies)
• Technically easy
What are the disadvantages of using microelectrode/intracellular cell recordings with oocytes?
Disadvantages:
• They are frog oocytes
o Need to consider applicability to mammals
• Difficult to exchange drugs
• Difficult to get good electrical control of neurons as only one electrode
• Noisy
• Makes a hole in the cell
Who developed patch clamping, when and how?
• Patch clamping o 1976- patch clamping arrived Neher and Sackman made first patch-clamp recordings Did this using: • Used better amplifiers • Changed electrode type
How is patch clamping performed and what is the result of this?
o Patch electrode process
Patch electrode pushed up on edge of membrane
Apply small amount of suction so that membrane forms tight seal with glass
Can either
• Pull electrode back to pull out small piece of membrane with just one ion channel of interest
o Record from a single ion channel
• Whole cell- suck on piece of membrane and break it open
o Record from all ion channels/conductances all over the cell
What is the use of patch-clamp, what does it measure and what allows this measurement?
Allows for the voltage-clamp of a cell (set the voltage of a cell)
• Can control voltage-dependent processes
• Amplifier maintains a membrane potential and measures the currents required to maintain it
• This allows measurement of voltage-dependent ion channel responses
• Used to elucidate details of how a drug interacts with a receptor to change its function
What is the advantage of patch-clamp?
o Can control the intracellular milieu of the cells
Manipulate the signalling systems of one cell at a time
o Very low noise and high resolution-can measure the current flow through a single channel
No hole in the membrane around the electrode
o Measure/study activation of one or a few proteins
o Versatile
Permits measurement of several proteins in the same cell
Many different applications
Single cell, cultured cells with mutated proteins expressed, slices, in vivo
What is the disadvantage of patch-clamp recordings?
• Disadvantages- o Quite hard o Can be expensive o Not suitable for all cells o One cell at a time, low throughput o Can lose important components of cell when accessing the inside
What can whole-cell patch clamping study?
• Studies ion channels in: o Isolated cells o Cell culture o Cells in brain slices of in vivo • Can study synaptic transmission
During whole-cell patch clamping, what does the size of post-synaptic current give information on and what does it depend on?
o Size of post-synaptic current informs us about strength of synaptic transmission and depends on: How much neurotransmitter is released Abundance of post-synaptic receptor Location of receptor States of receptor Voltage of post-synaptic cell Ions inside and outside of the cell
What is the current clamp?
• Current clamp: records Vm and tests excitability with a current stimulus
What is the independent variable in current clamp recordings?
o Independent variable- injected current: apply a known constant or time-varying current
Depolarises membrane potential above resting membrane potential and makes excitatory postsynaptic potentials
What is the dependent variable in current clamp recordings?
o Dependent variable- membrane potential: measures the change in membrane potential caused by the applied current
What is the use of the current clamp?
• Controlled injection of current can be used to test how excitable the neuron is and the properties of the neurons
• More natural response to opening ion channel
o This type of experiment mimics the current produced by a synaptic input
What is the voltage clamp?
Voltage clamp: holds membrane potential (Vm) constant so as to record the underlying currents
What is the use of voltage clamps and how do they do this?
• Voltage clamp recordings are used to dissect out the different transmembrane currents and work out the properties of the conductances/ion channels that generate these currents during electrical signal in neurons
o Apply a known membrane voltage and measures the transmembrane current required to maintain that voltage
Control voltage means we can limit the changing variables
What are the components of the voltage clamp? Describe each of their uses and the circuit
o Pair of voltage electrodes and amplifier
First electrode- goes through the membrane and records membrane potential inside cytoplasm of neuron
Second electrode- monitors membrane potential of surrounding fluid
Amplifier- constantly monitors and measures membrane potential
o Current carrying electrodes connected to feedback amplifier
Feedback amplifier makes continuous automatic corrections to ensure that the membrane potential is held essentially constant at the command voltage
• When desired membrane potential is measured by membrane potential amplifier, it sends a signal to the feedback amplifier to stop feeding current
One current carrying electrode used to inject current into cell cytoplasm via command of feedback amplifier
The other one is in the extracellular fluid
o Command device- where the operator commands changes in membrane potential
What is the independent variable in the voltage clamp?
• Independent variable- membrane potential
What is the dependent variable in the voltage clamp?
• Dependent variable- transmembrane current (Im)
In a clamp experiment, what does a downward deflection on the graph indicate?
o Inward current- downward deflection on graph- most likely due to sodium
Positive ions entering, negative ions leaving
In a clamp experiment, what does an upward deflection on the graph indicate?
o Outward current- upward deflection on graph- most likely due to potassium
Positive ions leaving, negative ions entering
What is the purpose of fluorescence-based assays?
Tells us about drug action on expressed receptors and allows high throughput testing
What are the advantages of fluorescence-based assays?
Advantages: o Population response o Very easy to do o Real time readout o Long term recordings o Measure multiple readouts at once o High throughput screening o Optical and non-invasive measures of drug action o Fluorescent dye responds to signaling pathway of interest
What are the disadvantages of fluorescence-based assays?
Disadvantages:
o No access to inside of cell (so need membrane permeable drugs to access intracellular)
o No control of membrane potential
o Fluorescent material could interfere with measurements
o Can’t wash drugs off preparation
o Slower temporal resolution than electrophysiology (milliseconds/seconds)
o May be hard to know exactly what is being measured
What dramatically increased the utilization of fluorescence-based assays in recent years?
Rapid advances in fluorescent materials dramatically increased utilization in recent years.
What can the fluophore in fluorescence-based assays respond to?
o Fluophore can respond to: Membrane voltage Ions cAMP Kinase activity Apoptosis
What does fluorescence imaging allow the exploration of?
Fluorescence imaging allows the exploration of:
o Whether drugs in a library target protein of interest
o Dose-response of drug acting at process of interest
o Drug interaction with mutated protein
o Drug alteration of long term processes
What is the process of fluorescence imaging?
o Cells (with or without expressed proteins) cultured in plates
o Fluorescent material added to plate
o Use plate reader or microscope to excite fluorophore and then measure emitted fluorescence
Fluorescence only emitted when bound to target
o Measure at baseline, in response to drug application or other stimuli
What is the use of immunohistochemistry?
Immunohistochemistry- tells us about location and sometimes state
Allows localization of the antigen within a cell or tissue
Combines histological, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label
What questions does immunohistochemistry allow the exploration of?
Immunohistochemistry allows the exploration of:
o Identification of receptors/enzymes/transporters/lipids etc. in brain region of interest
o Identification of receptors that may be good targets for treating a disease
o Drug-induced change of expression
o Drug-induced movement of receptors in sub-cellular locations
o Drug-induced changes of levels of a cellular event such as apoptosis
What are the four basic steps of immunohistochemistry?
o Tissue preparation o Minimise non-specific labelling o Primary antibody o Secondary antibody o Microscopy to image immunoreactivity
Describe tissue preparation in immunohistochemistry
o Tissue preparation Brain slices or cells onto cover slips Fixing can mask antigens For IHC-P • Deparaffinization and dehydration o Xylene, Xylene 1:1 with 100% ethanol, 100% ethanol down to 50% ethanol • Antigen retrieval o Heat in citrate buffer pH 6.5 around 20 minutes OR enzymatic (trypsin, proteinase K) For IHC-Fr and ICC • Fix slides o 4% PFA for 10 minutes Commonly used Prevents breakdown o Or methanol (ice cold) for 10 minutes o Or acetone (ice cold) for 10 minutes
Describe how non-specific labelling is minimised in immunohistochemistry
o Minimise non-specific labelling
Block 5% serum or BSA for 30 minutes to an hour
• Blocks unspecific binding, usually serum on 2o
Wash in PBS 0.2% Tween 4 times for 5 minutes
Permeabilise the cells if detecting an intracellular target
• 0.2% Triton for 10 minutes (not necessary if fixed in acetone or methanol)
o If antibody is inside of cell->makes hole in cell membrane so antibody can get into the hole and target the antigen
Describe primary antibody incubation in immunohistochemistry
o Primary antibody
Incubate with primary antibody
• 30 minutes to 2 hours RT or overnight at 4oC
Wash in PBS 0.2% Tween 4 times for 5 minutes
• Wash off antibody
Describe secondary antibody incubation in immunohistochemistry
o Secondary antibody
Incubate with conjugated secondary antibody
• 30 minute to 2 hour RT
Interacts with primary antibody
What are two manners in which microscopy is used to image immunoreactivity in immunohistochemistry?
Fluorophore linked secondary antibody
Enzymatic linked secondary antibody
How is the fluorophore linked to a secondary antibody visualised in immunohistochemistry and what are its advantages?
Fluorophore linked secondary antibody
• Viewed with fluorescence or confocal microscope (most cases)
• High structural resolution possible
• Advanced image reconstruction (3D) and signal
• Quantification
• Multiple labelling
o Can use multiple colours to look at different proteins in the same tissue
• Can be used in live cells
How is the enzymatic linked to a secondary antibody visualised in immunohistochemistry and what are its advantages?
Enzymatic linked secondary antibody
• Very high sensitivity as amplified
• Enzyme substrate is processed by enzyme to produce coloured result
• Viewed with light microscopy
• Some reactions that produce colored products used for immune-electron microscopy
What are the disadvantages of immunohistochemistry?
Disadvantages of immunohistochemistry
o Can be false positive or negative results if interactions between antigens/antibodies are not specific enough
o Level of response depends on conditions and is variable between experiments
o Appropriate controls need to be used (in tissue lacking antigen or known to control)
o Tissue needs to be out of the animal/human
o Often sliced into very small sections
What is structure-based drug design?
• Structure-based drug design attempts to use the structure of proteins (or biological targets) as a basis for designing new ligands by applying the principles of molecular recognition
o To inhibit or enhance protein function
o However, ligand may not be specific to protein target and hence can cause potential side effects
What is molecular modelling, what is its use and what does it involve?
• Molecular modelling has become a valuable and essential tool to medicinal chemists in the drug design process
o Molecular modelling describes the generation, manipulation, or representation of 3D structures of molecules and associated physico-chemical properties.
It involves a range of computerized techniques based on theoretical chemistry methods and experimental data to predict molecular and biological properties
What is structural biology and what does it involve?
• Structural biology is the study of molecular structure and dynamics of biological macromolecules, particularly proteins and nucleic acids and how alterations in their structures affect their function
o Structural biology incorporates the principles of molecular biology, biochemistry and biophysics
o Includes:
Recombinant protein expression
Protein purification
Functional analysis
Structural analysis
Why are recombinant proteins produced in pharmacology?
• To investigate how particular proteins regulate biology, researchers usually require a means of producing (manufacturing) functional proteins of interest
o Need a high abundance of protein for study
• Given the size and complexity of proteins, chemical synthesis is not a viable option for this endeavour. Instead, living cells and their cellular machinery are usually harnessed as factories to build and construct proteins based on supplied genetic templates
• Unlike proteins, DNA is simple to construct synthetically or in vitro using well established recombinant DNA techniques. Therefore, DNA templates of specific genes can be constructed as templates for protein expression
• Proteins produced from such DNA templates are called recombinant proteins
What are the basic steps and subsequent uses of making and using recombinant proteins? Describe any considerations that need to be addressed during this process
• Steps: Target gene that expresses protein of interest-> place in plasmid construct-> give plasmid construct to expression system-> produce protein -> functional and structural analysis o Expression Construct design Expression systems choices Conditions o Purification Purification methods Detergent choice Functional analysis o Crystallisation/CryoEM Crystallisation methods (structural analysis) CryoEM conditions Detergent choice Additives/antibody o Data collection Data collection systems Sample mounting/grids o Structure determination Model building
Describe how a construct is designed for recombinant protein production (the necessary elements)
Construct design
• Plasmid/vector design
o Selectable marker- resistance to an antibiotic solely for selection of cells containing vector
o Promoter-allows for polymerase to bind
o Operator- control of expression
o Ribosome binding site-for ribosomes to bind and make protein
o N-terminal/C-terminal tag- to select for target protein amongst other proteins
o Multi-cloning site
o Protein interest
o Transcription terminator
What are the choices of expression systems for expression of recombinant proteins in pharmacology? Describe each in terms of their:
- Speed
- Cost
- Yield
- Post-translational modifications
• Cells o Mammalian cells Low speed High cost Low yield High post-translational modifications o Insect cell Medium-low speed Medium-high cost Medium-low yield Medium-high post translational modifications o Yeast cells Medium-high speed Medium-low cost High typical yield Medium-low post translational modifications o Bacteria cells High speed Low cost Medium-high yield Low post-translational modifications
Describe the use of DNA vectors in pharmacology and what can be done with them
o DNA- electrophysiology/uptake
Introduce a single point mutation and investigate functional effect by using DNA kits
Describe the use of RNA vectors in pharmacology
o RNA-electrophysiology/uptake
Describe the use of protein vectors in pharmacology
o Protein-binding/liposomes/structure
How is overexpression of recombinant proteins controlled? What can be done with overexpressed proteins after induction?
• Overexpression- will express protein in large quantities
o Overexpress protein in E.Coli/yeast/insect/mammalian cells by inducing expression with arabinose/IPTG
Control of expression timing through arabinose/IPTG induction
o Purify protein
o Further study
Crystallography studies (structure)
CryoEM studies (structure)
Binding assay
Isothermal titration calorimetry (ITC)
Reconstitute into liposomes and perform functional studies
• Radiolabelled uptake
• Fluorescence assay
Describe the process of purification for proteins with a His tag
• For His tags o Cell lysis Tris or phosphate buffer (pH 8) 300mM NaCl 10-20 mM imidazole o Ni-NTA resin (column affinity) 30=-60 minutes (Batch or column format) His-tagged protein bound to resin NTA on beads is attached to a nickel • Histidines bind to that nickel o Wash 20-50 mM imidazole o Elute 100-250 mM imidazole • Imidazole will replace histidines at nickel-> helps with elution o Pure 6xHis-tagged protein
What does a protein need to be for structural analysis?
o Pure (homogenenous) o Stable o Soluble o Abundant o Without the tag
How can a protein tag be removed and isolated from the protein of interest?
Tag can be removed with protease digest
Size-exclusion chromatography would then sort proteins by their size
• Bigger ones come first, smaller ones come later
What kind of binding assays can be performed to examine binding of a ligand/substrate to a purified protein and what kinds of results can these assays give?
o Purified protein can be used to examine binding of a ligand/substrate (binding assays)
Determine EC50, Km, K0.5 or IC50 of an inhibitor
o Fluorescence based assays
o Isothermal titration calorimetry (ITC)
o Functional analysis- reconstitution and radiolabelled uptake
What are fluorescence based assays and how are they useful?
o Fluorescence based assays
Uses intrinsic fluorescence of protein or introduced fluorescence
Fluorescence changes depending on whether a ligand or an inhibitor is bound
What is isothermal titration calorimetry and how does it work?
o Isothermal titration calorimetry (ITC)
Measures thermodynamic parameters in solution between a ligand and protein
• Exothermic or endothermic binding
• Allows determinaton binding affinity of drugs
• Reactions release heat-> we can monitor the heat that these reactions release and try to map it
How does reconstitution and radiolabelled uptake work for functional analysis of a purified protein?
o Functional analysis- reconstitution and radiolabelled uptake
Can introduce protein in liposome (membrane of lipids)
• Fake cell where can observe transport of substrates
Why are protein crystallography and cryo-EM useful?
• Protein crystallography and cryo-EM allow us to visualise protein structures at very high resolution (atomic scale)
o Understand conformational changes in proteins
o Develop drugs to specifically bind to proteins
What kind of waves does protein crystallography use?
• Protein crystallography uses X-rays
What is the resolution of protein crystallography limited by?
o Resolution is limited by the wavelength of the electro-magnetic radiation
Describe the theoretical resolution limit of X-ray crystallography
X-ray crystallography (0.1nm or 1 armstrongs)
Describe the theoretical resolution limit of electron microscopy
Electron microscopy (theoretically 0.02A or 0.5A/10000000x)
Describe the theoretical resolution limit of light microscopy
Light microscopy (200nm/2000x)
How does the synchrotron work and what does it produce?
The synchrotron produces light by accelerating electrons almost to the speed of light, 1 million times brighter than the sun
• Electrons accelerate due to magnets around circle
• Every time the electrons turn, they release a light in a straight line
The infrared, UV and X-rays are sent down pipes called beamlines, to work areas where scientists run their experiments
When this bright light is aimed at a very small sample, an image of the samples properties is created on a detector. This image is sent to a computer, which is used to analyse of the sample’s molecular structure
What is used to create the X-rays for protein crystallography?
o Synchrotron for Xray production
What is the cost of the synchrotron?
Cost- 206 million dollars
What is the circumference of the synchrotron?
216 meters
What is the energy of the synchrotron?
3GeV
What is the running cost of the synchrotron?
20 million per year
What is the principle behind crystallisation prior to X-ray protein crystallography and the principle of protein crystallography itself?
When electromagnetic radiation hits electron, 99% of light goes through but the light that is absorbed by the electron/atom will produce an image
• The light that doesn’t go straight through always scatters in the same direction and angle depending on the atom and chemical environment
• So that signal is multiplied, need atoms in same conformation
o This is done by crystallising the proteins
What is the process of crystallisation of the protein prior to protein crystallography and its principles?
o Crystallisation
Crystallisation trials
Vapor diffusion (controlled evaporation)
Crystal contains many molecules in a highly order 3D arrangement
Process
• Reservoir solution with hanging drop containing protein at top
• Reservoir solution evaporates which slowly concentrates the protein and forms protein crystal
What is the disadvantage of using protein crystallography compared to CryoEM?
• Disadvantage: sometimes, protein crystals cannot be made
What are the advantages of using protein crystallography compared to CryoEM?
• Advantage: can see waters, heavy metals that can’t be seen with CryoEM and X-ray crystallography is more powerful than CryoEM
o X-ray crystallography more useful with smaller molecules
Describe how CryoEM works and the principles behind it
CryoEM conditions
• Cryogenic electron microscopy uses electrons
o Uses a beam of accelerated electrons to create an image of the sample
o It is used to investigate the detailed structure of tissues, cells, organelles and macromolecular complexes
• Process:
o Freeze proteins in grid
o Put protein in grid and supply them fast into liquid which freezes protein
Want single layer of protein
o Electrons that come from top hit sample with high speed
o As they hit the sample, the sample disintegrates and gives a picture of the electrons
o Sample analysis
How is a CryoEM sample analysed?
o Sample analysis- Import Motion correction Particle picking Particle extraction 2D classification 3D initial model 3D classification Refinement
What is the cost of CryoEM?
10 million dollars
What are the measurements of a CryoEM machine?
3 metres tall and 1 tonne
What is the energy of a CryoEM machine?
300 kV
What types of proteins is CryoEM most appropriate with?
• CryoEM more useful with larger proteins and membrane proteins
What is the genera process of model building?
• General process:
o Purified protein
o Protein crystals produced by trial conditions
o Diffraction pattern obtained by X-ray
o Electron density map obtained by phases
o 3D model obtained by fitting and model building
What are membrane proteins used for an do they account for a large portion of the protein databank? Describe the exact proportion in both number and percentage and how it compares to their theoretical proportion
- Control movement across cell membrane
- Account for 30% of encoded proteins and major target for pharmaceuticals
- But of the 168600 3D protein structures in the protein data bank (PDB), only 1135 are of unique membrane proteins (0.7%)
Where do most proteins in the protein database come from and why?
o Most proteins in PDB are of prokaryotic origin
They are much easier to express and work with
Why are membrane proteins difficult to purify and how is this issue solved?
• Difficult to work with because they are embedded in lipid bilayer
o If struck from lipid bilayer, would just precipitate and be unable to be purified
o Difficult to remove proteins from the membrane
Need detergent to solubilise membrane proteins
• Need membrane protein with detergent micelles-> makes protein soluble and stable
What is the workflow of drug discovery and preclinical development process?
• Drug discovery and preclinical development process
o Target discovery-> target validation-> lead generation and refinement-> preclinical development
Why are genetically engineered animal models beneficial for drug discovery research?
• Genetically engineered animal models facilitate drug discovery research
• Understanding brain physiology and how different brain regions connect and interact to produce an outcome in normal and disease conditions also requires a whole organism
o This understanding will be useful for designing mechanism-based therapies
• Animal models allow researcher to investigate cellular and behavioural functions of disease-related genes
In the drug discovery/preclinical development workflow, what is the goal of the target discovery step and what genetically engineered models are conducive to that step?
Goal: Find target
Description: Identify genes/proteins linked to specific physiological and pathological pathways
Genetically engineered models:-Knockout
- Conditional knockout
- Knock-in (point mutation)
- Transgenic
In the drug discovery/preclinical development workflow, what is the goal of the target validation step and what genetically engineered models are conducive to that step?
Goal: Eliminate wrong target
Description: Confirm its modulation alleviates the phenotypes associated with a specific pathology
Genetically engineered models: -Knockout
- Conditional knockout
- Knock-in (point mutation)
- Transgenic
In the drug discovery/preclinical development workflow, what is the goal of the lead generation and refinement step and what genetically engineered models are conducive to that step?
Goal: Generate molecules
Description: In vivo stability and tissue distribution of selected compounds-metabolism
Genetically engineered models: Knock-in (reporter)
In the drug discovery/preclinical development workflow, what is the goal of the preclinical development step and what genetically engineered models are conducive to that step?
Goal: Eliminate molecule (toxicity) or Advance molecule (efficacy)
Description: Molecules are tested in vivo to assess safety and efficacy e.g. carcinogenicity/human forms of target protein
Genetically engineered models: For Eliminate molecule: -Knockout -Conditional knockout -Humanised model For Advance molecule: -Knockout -Knock-in -Transgenic -Humanised model
What are the advantages and limitations of using flies, fish and worms as models for drug discovery and preclinical development?
o Flies, fish and worms
Share many of our genes
Basic behaviours possible
Mutations are relatively fast and easy to make
But have fewer neurons and different brain structures
What are the advantages and limitations of using rodents as models for drug discovery and preclinical development?
o Rodents
Share 95% of our genome and many brain structures
Can test more complex behaviours
More difficult to make and more expensive
Many human diseases have well validated rodent models
What are knock-out genetically engineered models?
• Knock-out: remove gene to get an idea of its function
What are knock in genetically engineered models and what is the purpose/advantages of this?
• Knock in: add a mutant gene or a genetic marker
o Introduce a disease-linked mutation (e.g. a mutation identified in a family with a particular condition)
o To localise a protein (in live tissue, if there is good antibody)
o Knock in mice can express a fluorescent marker under a promoter specific for tissue-specific visualisation
What are the consequences of a mutation in the SCN9A gene and how was this found? Is it a genetically valid drug target? How were KOs used to determine this?
• Example- cellular and animal model of a human mutation: target development
o Mutations were found in the SCN9A identified in families who don’t sense pain (but were otherwise pretty normal)
o The SCN9A gene encode the alpha-subunit of the voltage-gated sodium channel Nav1.7
o Expression of the mutant Nav1.7 in a cell line demonstrated the mutation caused a loss of function through electrophysiology
o Mouse where Nav1.7 is knocked out were insensitive to inflammatory pain
o A genetically validated drug target for pain modulation
Nav1.7 KO also used test drug selectivity
Animal models to test if it can control the sensation of pain
Drug development has not been successful yet
Describe the process of homologous recombination and how it can be used in drug discovery/preclinical trials
• Homologous recombination
o Exchange of genetic material between two strands of DNA that have long stretches of similar base sequences
o Can exploit homologous recombination to trick chromosomes into introducing genetic changes into a model organism- commonly knockouts and knockins
How are knock-out/in mice made with homologous recombination?
• Making a knock-out/in mouse with homologous recombination
o Design a target vector
With homologous sequences to target your gene of interest and some selection genes
o Insert the target vector into embryonic stem cells
The vector will recombine with the correct target sometimes
o Inject cells containing the target vector into a new embryo
Select the cells that have correctly incorporated the genetic change and inject them into a normal developing mouse embryo
o Chimeric mouse is born and bred
Chimeric mice contain a mix of its own cells and the heterozygous transgene cells
Select viable KI/KO offspring
What are the limitations of using homologous recombination to create transgenic animals?
• Homologous recombination to create transgenic animals is powerful but has a few limitations:
o Expensive and takes a long time
o Modifications of genes that are essential for development are lethal
o Results are difficult to interpret and control for because the DNA modification is present during embryonic development and wide spread adaptations are likely
Not a very subtle change
What are whole animal KI and KO animal models good for in drug design/pharmacology?
• Whole animal KI and KO animal models are great for:
o Localising specific types of neuron
o Making animal models of genetic disease
o Identifying and validating drug targets
o Humanised models that express human enzymes or express human forms of the target gene can help assess metabolism and cellular drug actions
What are limitations of using whole animal KI and KO animal models in pharmacology and what is a possible solution to these problems?
• Limitations of whole animal KI and KO animal models:
o May be lethal
o Widespread compensatory changes are likely
o However, using tools such as the Cre-Lox system and viral-vectors help to overcome these limitations
The same tools can also be used to express other foreign proteins in subsets of genes to study how specific neuronal populations link to behavioural outcomes
• This is needed to design mechanism-based medicines
• Such as optogenetic and chemogenetic tools
Why are cre-lox models useful in drug design?
• Constitutive (whole body) KI/KO animals may have genetic modifications that are lethal and compensatory mechanisms will have occurred
• Cre-lox systems allow tissue specific gene changes (conditional)
o Cre/lox tissue-specific genetic changes can be used to control the location (conditional) and timing (inducible) of the gene modification
Avoid global developmental adaptations
o Many other variations and uses for the Cre/lox system
How are Cre-Lox mouse models designed/how do they work?
Cre recombinase enzyme+ LoxP target sites on the gene of interest must both be expressed in cells
The genetic change only occurs when a cell expresses both a floxed gene and the cre-recombinase
• Requires two GMO mouse lines (Cre mouse x loxP mouse) OR cre- and/or the floxed gene can be expressed using a viral vector and injected into a mouse
Process-
• Cre will be under control of a tissue-specific promoter in one mouse and the target gene will be flanked by loxP sites in another mouse
• When the two mice are bred:
o The produced mouse will contain both Cre and loxP sites, as well as the target gene
Target gene will be knocked out- will create KO (target cell recombination)
o The produce mouse will contain either only loxP sites or only Cre
The target gene will not be knocked out- expression will remain the same
What are the uses of viral vectors in developing mouse models for drug development, what viral vectors are used and why, and how are they used?
• Viral vectors effectively deliver genetic material into smaller groups of cells
o Viral vectors (AAV and lentivirus vectors) are genetically modified viral particles that:
Don’t replicate
Infect cells locally and express high levels of the desired gene product
Limited immune response
o Must physically put the vectors in the region of interest
Need to inject them
o Promoters help to target specific cell types
Glutamate or serotonin containing neurons
Glial cells
Neurons that express a particular peptide
What is the use of optogenetics and chemogenetics in drug development?
• Opto- and chemo-genetics are two tools that allow scientists to selectively engage specific neuronal circuits
What is optogenetics and how does it work?
• Optogenetics turns on/off neuronal subtypes with light
o Express a non-mammalian ion channel into neurons of interest that opens in response to light
o In combination with fiber-optic and genetic advances, can selectively stimulate/turn off subsets of neurons at near-synaptic precision with low level light of different colours
How was optogenetics developed- series of discoveries that led to its development
o Development
A whole range of light sensitive microbes were described, including an algae that moves in response to light (1985)
• Light sensitivity in the algae is conferred by a single channel called channelrhodopsin-2 (2003)
• ChR2 can change the activity of neurons (2004)
Engineered in to a versatile tool for turning neurons on and off with near synaptic precision- optogenetics
What are chemogenetics and what is it useful for in drug discovery?
• Chemogenetics selectively turns on/off neuronal subtypes with designer drugs
o An engineered GPCR that is selectively activated by a designer-drug
What is DREADDS, how does it work and what is it used for in drug development? What can it be combined with?
o DREADDS
Designer Receptors Exclusively Activated by Designer Drugs can be used to investigate the relationship between behaviours and subsets of neurons
• DREADDs are engineered receptors based on the muscarinic and opiate receptors, but only respond to a novel designer agonists that is otherwise inert
• Systemic injection of the agonist produces long lasting activation
Combine with Cre-lox or viral vectors to manipulate neurons in a cell type specific manner
What are optogenetics and chemogenetics often combined with and why?
• These techniques are used in combination with genetically modified animal models, Cre-lox and viral vectors
o Turn neurons on and off when you want
o Low invasiveness
o High spatiotemporal control
What paper is an example of optogenetics, chemogenetics and Cre/Lox systems combined to discover new drug targets?
• Differential coding of itch and pain by a subpopulation of primary afferent neurons (Sharif et al. 2020)
Summarise the paper Differential coding of itch and pain by a subpopulation of primary afferent neurons (Sharif et al. 2020)
o Present evidence that a subpopulation of peripheral chloroquine-responsive somatosensory afferents can differentially drive itch or pain responses when they are stimulated via metabotropic or fast ionotropic signalling pathways, respectively
o Shows how the same peripheral neuron can discriminate signals of pain and itch
Describe the background that led to the development of the paper Differential coding of itch and pain by a subpopulation of primary afferent neurons (Sharif et al. 2020)
Itch and pain are both unpleasant sensations that can be triggered from the same patch of skin and are both carried by C-fibers
Itch leads to scratching, pain leads to withdrawal behaviours
Animal models for itch has identified the GPCR MrgprA3+ as a receptor that is essential for itch
• MrgprA3+ KO animals lose their itch response
Describe the aim off the paper Differential coding of itch and pain by a subpopulation of primary afferent neurons (Sharif et al. 2020)
o Aim: To explore how the somatosensory system differentiates itch from pain to trigger the appropriate response (sensory modality discrimination)
Describe the method of the paper Differential coding of itch and pain by a subpopulation of primary afferent neurons (Sharif et al. 2020)
o Methods:
MrgprA3-Cre mice
Floxed-ChR2 mice (HR mouse)
• Cre/Lox system
• Optogenetics
AAV9-hSYN-DIO-hM3D (Gq) (AAV-DOI-DREAD)
• Cre/Lox system
• DREAADs
Activate MrgprA3 expressing C-fibers at the skin surface using either optogenetics or DREADDs and measure behaviours
Exploring the possibility of triggering itch by another GPCR
• Excitatory Gq-coupled DREADD channels were expressed in the sensory neurons of MrgprA3-Cre+ mice using an AAV vector
• Intradermal injection of CNO and behaviour monitored
• Control: MrgprA3-Cre- mice or yellow light
Exploring different modes of stimulation
• Expressed ChR2 in the same MrgprA3+ neurons by crossing MrgprA3- Cre+ and floxed-ChR2 mice
• Blue light activation of sensory neurons at the skin and behaviour monitored
• Control: MrgprA3-Cre- mice
Describe the results of the paper Differential coding of itch and pain by a subpopulation of primary afferent neurons (Sharif et al. 2020)
o Results-
DREADD activation resulted in scratching behaviours
GPCR mediated stimulation of MrgprA3 C-afferents induces itch
Optogenetics activation resulted in aversive behaviours such as vocalization, escaping, and attempts to bite the light source, but not scratching
Nocifensive responses are evoked by optical stimualtions of MrgprA3 C-afferents
Describe the conclusions of the paper Differential coding of itch and pain by a subpopulation of primary afferent neurons (Sharif et al. 2020)
o Conclusions-
Fast ionotropic and slower metabotropic stimulation of the same genetically defined populations of neurons can lead to different outcomes at cellular and behavioural levels
• Difference between itch and pain occurs at level of skin
MrgprA3 expressing neurons are multimodal
Suggests a drug target for transdermal analgesics vs anti-itch treatments
Why are animal models needed for development of drugs in neuropsychiatric treatment? What are the limitations of this?
• Animal models are needed as they can be helpful in development of drug compounds for disorders
o Isolate compound and see if it has activity in pre-clinical model to screen whether it could be used to treat condition
o Isolate new target and screen synthetic drug library against target to see if it could be useful for treatment of disorder
• Animal models are there to try and bridge the gap between drug discovery and clinical trials
o Preclinical tests are performed using animal models
o Limitations-
Whilst there is some homology in different targets across different species, human biology is distinct in some ways
When is use of an animal model considered ethical by the animal ethics group?
o Need approval from Animal ethics group to conduct behavioural research using animals
o The “Australian Code of Practice for the Care and Use of Animals for Scientific Purposes” requires animal research must be:
Valid
As humane as possible
Justifiable for scientific or educational benefits
Considerate of the welfare of the animals
Before using an animal model, what are the 3 ethical Rs that need to be considered?
o The 3 Rs
Replacement- techniques that totally or partially replace the use of living animals for scientific purposes must be sought and used wherever possible
Reduction- must use no more than the minimum number of animals necessary to ensure scientific and statistical validity
• Need to do power calculations to specify minimum number of animals
Refinement- must take into account the biological characteristics of the animals to reduce the adverse impact on animal
What is the validity criteria of an animal model? Describe each criteria
• Validity criteria of an animal model
o Face validity
The response observed in the animal model should resemble the behavioural response observed in humans
o Predictive validity:
The animal model should be sensitive to clinically effective pharmacological agents
o Construct validity
How well theoretical and empirical accounts of the human disorder and the disordered behaviour exhibited by the model are brought into alignment
• An animal model with better construct validity will better parallel the behavioural, neurobiological, genetic and molecular features of the human disorder
What are anxiety disorders? Give examples
• Abnormal or inappropriate anxiety reactions o Generalised anxiety disorder o Panic disorder o Acute stress disorder o Phobias
What is the purpose of exploratory models of anxiety and what are examples of such models?
o Mimics the natural conflict between the tendency of a mice or rat to explore a novel environment and to avoid an exposed, open area o Tests Open field Emergence/Light-Dark test Elevated Plus Maze (EPM) Social interaction Predatory odour avoidance
Describe the open field test and the behaviour of mice with an anxiolytic agent in that test
Open field
• Look at amount of time animal spends in the centre vs in the corners
o Mice usually don’t like bright areas in the centre
• Prediction with anxiolytic agent (agent that reduces anxiety)
o Mice will spend more time than baseline in open/light areas than closed/dark ones
Describe the emergence/light-dark test and the behaviour of mice with an anxiolytic agent in that test
Emergence/Light-Dark test
• Measure how much time animal spends in open (light) area vs in closed (dark) area
o Mice will usually spend more time in dark area than light area
• Prediction with anxiolytic agent (agent that reduces anxiety)
o Mice will spend more time than baseline in open/light areas than closed/dark ones
Describe the elevated plus maze test and the behaviour of mice with an anxiolytic agent in that test
Elevated Plus Maze (EPM)
• Have two open arms and two closed arms
o Mice will usually spend more time in closed arms than open arms
• Prediction with anxiolytic agent (agent that reduces anxiety)
o Mice will spend more time than baseline in open/light areas than closed/dark ones
Describe the social interaction animal model test for anxiety disorders
Social interaction
• Look at whether animals explore the other animal-social anxiety
Describe the predatory odour avoidance paradigm for anxiety disorders and the impact of an anxiolytic on the normal response
• Innate fear models- rats/mice have innate fear of predators (like cats)
o Rats were exposed to cat odour during conditioning only
o This predator odour induced an innate anxiogenic response (SAL/SAL vs CONTROL)
Due to innate fear of cat odour
Spend more time hiding away in dark box
o Midazolam during conditioning reversed this anxiety (MDZ/SAL vs SAL/SAL)
• Set-up
o Light-dark box set up
o Put cat odour at end of chamber
Describe the validity of the open field, emergence and elevated plus maze test in terms of face validity and predictive validity
Open field, emergence and elevated plus maze test satisfy:
• Face validity- it is known that mice prefer to spend more time in the dark/closed areas
• Predictive validity- drugs that are known to cause anxiolytic effects will cause animals to explore in the open/light areas more than baseline
Describe the predatory odour avoidance test in terms of face validity, predictive validity and construct validity
Predatory odour avoidance model satisfies
• Face validity- mice/rats have an innate fear of cats/predators
• Predictive validity- anxiolytic agent in humans can reduce fear response to cat odour
• Construct validity- Regions of the brain attributed to anxiety/fear mediation light up in response to cat odour
What is PTSD, in which populations is it most prevalent and how is it currently treated?
• Arises due to exposure to a traumatic event
• Increased rates in military, police, firefighters and ambulance officers
• Now is included in a new category of disorders, the Trauma- and Stressor-Related Disorder in DSM-V
o Not classified as an anxiety or mood disorder
• Exposure therapy only effective in 56% of patients
o Extinction of response over time- if paired in the drug, can make
• Crisis in PTSD drug development
What are the two mice models for PTSD and how appropriate are they?
• Repeated shock mouse model for PTSD:
o Mice will be exposed to a series of shocks that are paired to context
o Placed back into context with the absence of shock- measure their response
o Testing for a more holistic mice model (testing for more than 1 PTSD symptom at a time) might be better (one shock paradigm induced PTSD-related)
• Shock paradigm induced PTSD-related mouse model:
o Mice exposed to one intense shock paired to context
o Placed back in context with the absence of shock- measure their response
• May have greater translational potential using a model that encompasses many different aspects of what is occurring in humans
Compare PTSD symptoms in humans and the relevant shock paradigm induced PTSD-related effect in mice
PTSD symptoms in humans on the left
Shock paradigm induced PTSD-related effect in mice on the right
Anxiety=Increase in anxiety-related behaviour
Long-lasting exaggerated stress response to trauma-related stimuli=Increase of conditioned fear to context even 55 days after the original shock-context pairing
Fear potentiated startle= Increase in fear potentiated startle
Nightmares=Cannot be assessed directly in mice
—–However, some evidence to show that there is altered REM sleep in one-shock model
Hippocampal volume and grey matter loss=Increase in hippocampal volume loss
What are the symptoms of depression?
• Symptoms
o Depressed mood (intense sadness and helplessness)
o Apathy (indifference, lack of motivation)
o Anhedonia (inability to experience pleasure)
o Difficulty to think or concentrate, paying attention or make decisions
o Suicidal thoughts
What are animal models of depression?
• Animal models of depression
o Behavioural despair (forced swim test, tail suspension test)
Forced swim test-
Tail suspension test
o Learned helplessness (chronic unpredictable stress)
o Chronic mild stress
o Novelty-induced hypophagia
What is the forced swim test model for depression? Describe how it works
Forced swim test-
• Mice placed in cylinder with water-> they will typically start to swim
• Measure the time at which they start to become immobile (immobility time)
o Immobility is a sign that animal has given up
Describe the validity of the forced swim test model for depression with examples
• Forced swim test satisfies:
o Predictive validity- Desipramine (antidepressant) decreased immobility in the forced swim test
o Construct validity- P2RX7 (gain of function variant/SNP), a gene coding for a purinergic ligand-gated ion channel, is associated with major depressive disorder
Decreased immobility of P2X7 knockout mice in the forced swim test
P2X7 triggers cytokine release (pro-inflammatory) which may contribute to depressive phenotype
What is the tail suspension model for depression? Describe how it works
Tail suspension test
• Mice hung by the tail-> they will typically try to fight their way out
• Measure the time at which they start to become immobile
Compare the human symptoms of schizophrenia to its corresponding animal model
Human symptoms on the top/left
Animal behavioural model on the bottom/right
Positive symptoms: hallucinations, delusions and bizarre behaviours
—Thought to be influenced by dopamine
==
DA agonists (cocaine, amphetamine)
-NMDA antagonists (PCP, MK-801)
—-Can give rise to hyperactivity and stereotypy
—-The repeated administration of these drugs can lead to cognitive deficits-> cognitive impairment is an aspect of schizophrenia
Negative symptoms:
Blunted affect and apathy= Animal models of anxiety
anhedonia= Reduced consumption of sucrose
social withdrawal= Social interaction
alogia (poverty of speech)= No test available
Cognitive symptoms:
decreased attention= Prepulse inhibition of startle (PPI)
decreased memory= Y maze and Morris Water Maze
Describe the prepulse inhibition of startle model of schizophrenia and what drugs it is affected by
• Prepulse inhibition of startle
o Sensory gating and attentional dysfunction
o PPI- ppre-exposure to a weak acoustic stimulus usually reduces the startle response to a loud stimulus
Schizophrenia patients show deficits in PPI that are normalised by antipsychotic drugs and nicotine
• Schizophrenia patients startle more
Describe the predictive validity of the prepulse inhibition of startle animal model. Mention a drug predictive validation and a genetic one.
o Predictive validity-
Risperidone (anti-psychotic drug) increases PPI
• However, THC exposure reduces the neurobehavioral effects of risperidone
Nrg1 HET mouse of schizophrenia
• Hrg1 hyperactivity reversed by clozapine
• Nrg1 HET mice display enhanced sensitivity to drugs that trigger psychosis in humans e.g. methamphetamine and THC
How does THC exposure reduce the neurobehavioural effects of risperidone on prepulse inhibition? What is the impact of this on drugs targeting schizophrenia?
o THC seems to be reducing brain accumulation of risperidone due to overexpression of P-gp protein which is a drug efflux pump that drags risperidone out of the brain more effectively
Henceforth, non-P-gp substrate antipsychotic drugs as treatment off choice in first-episode psychosis patients with a cannabis use history
• THC exposure did not influence the non-P-gp substrate clozapine
• Clozapine treatment of choice in treatment-resistant patients
• Better APDs will be those that aren’t P-gp substrates such as blonanserin
Describe the face validity of the nrg1 HET mouse model of schizophrenia
nrg1 HET mouse of schizophrenia
• Nrg1 heterozygous mice display hyperactivity and decreased PPI
o Hyperactivity may be due to increased dopaminergic activity
• Cognitive deficits- nrg1 HET mice display impaired learning in the Morris Water Maze
Describe the construct validity of the nrg1 HET mouse model of schizophrenia
Nrg1 HET mice have:
• Increased c-Fos expression in the nucleus accumbens
• Altered D2, 5-HT2A and NMDA receptor expression
• Endocannabinoid dysregulation in cognitive and stress-related brain regions in the Nrg1 mouse model of schizophrenia
o Schizophrenia patients have increased hippocampal 2-AG and cognitive deficits
• Nrg1 HET mice have increased C4 expression
o C4 is upregulated in schizophrenia brain and is part of the MHC, the standout risk gene for schizophrenia
What is a limitation on basing schizophrenic drug targets on nrg1 HET mice models?
However, only subset of schizophrenic patients have Nrg1 mutation (not even sure if it plays a role in schizophrenia) so drug design based on that model may not be appropriate for entire schizophrenic population
