Research Methods in Pharmacology Flashcards
What are neurobiological techniques?
*Research in neuropsychopharmacology can span the entire spectrum of neuroscience research
Define in vivo
analyses performed in a living organism
Define in vitro
in glass, analyses performed in test tube
Define ex vivo
analyses performed in live tissue removed from an organism
Define in situ
analyses performed in context to living tissue but typically after dissection
Describe stereotaxic surgery
*Apparatus for specific positioning of instruments/syringe into the brain
*Stereotaxic atlas gives coordinates to target specific regions
*Can be used to give very precise injections, microsurgeries, lesions
Describe micro dialysis
*Technique for collecting fluid from a live and awake animal – analysis of neurotransmitter levels, signalling molecules, drugs, etc in vivo
Describe chromatography
*Chromatographic techniques separate molecules based on their size, charge, relative polarity, or specific interactions
*Compounds can then be detected, quantified, or further analysed
What are analytes?
*Proteins – column chromatography (size exclusion, cation/anion exchange, affinity)
*Proteins/peptides – high-performance liquid chromatography (HPLC) often coupled to mass spectroscopy (HPLC-MS)
*Neurotransmitters – HPLC, gas chromatography (GC)
*Drugs – HPLC, GC, thin layer chromatography (TLC)
Describe electrophysiological recordings
*A variety of techniques for measuring electrical activity in organisms, tissues, or cells
*Useful in vivo, ex vivo, or in vitro
*Classic studies by Hodgkin and Huxley on squid giant axon
Describe in vivo recordings
*Implantable electrodes connected to an external recorder
*Can measure activity in specific regions during normal behaviour
*Typically measure field activity (relatively large number of neurons)
Describe intracellular recording
*Measures activity across the membrane of a single cell
*Whole-cell (sharp electrode)
*Patch-clamp (smaller areas)
*Can be used to isolate single ion channels for measurement
Describe molecular methods
*A variety of techniques have been used over the years to locate neurotransmitters and receptors in the brain
*Early techniques (sometimes used today) used radioactive isotope labelled drugs or antibodies to visualize drug binding
*Autoradiography
*Radioimmunoassay
*These have been largely supplanted by immunofluorescent and transgenic techniques
What are immunological techniques?
*Antibodies are proteins produced by the adaptive immune system
*Antibodies have specific binding sites for recognition of foreign antigens
*Antibody binding is high affinity and is responsible for targeting foreign bodies for destruction
What is innate immunity?
*Immunity can be exploited experimentally to produce antibodies against biologically relevant proteins
*Typically an isolated protein is injected into a rabbit (mouse, goat, donkey, chicken), an immune response generated, and antibodies are isolated from the rabbit serum
Describe immunofluorescence
*Antibodies can be conjugated to fluorescent molecules and used to identify specific proteins in cells of the CNS.
What is immunohistochemistry?
the detection of proteins in tissue
What is immunocytochemistry?
the detection of proteins in cells (typically cells cultured in vitro)
Describe confocal microscopy
*Specialized technique used in fluorescence microscopy to filter out-of-plane light
*Allows imaging of an optical ‘slice’ of tissue
*Can be used to generate 3D images of biological structures
Describe 2 photon microscopy
*Uses infrared lasers (700-900 nm) to image living tissue
*IR light can penetrate ~10 mm through brain tissue
Describe immunoassays and ELISA
*Enzyme-linked immunosorbant assay
*Detects an immobilized antigen using an enzyme-linked antibody
*Higher sensitivity
Describe Western blotting
*Detects an immobilized antigen using an enzyme or fluorophore linked antibody
*Higher throughput
*Evaluating gene expression
*RNA expression (transcripts) can be measured as a proxy for protein expression
*RNA is translated into proteins but protein is the functional unit of biological systems
*Numerous processes can modify or alter translation so RNA ≠ protein
What is FISH?
*Fluorescence in situ hybridization (FISH) binds a fluorescent probe in tissue sections to show which cells may be expressing a gene of interest
What is qPCR?
Quantitative polymerase chain reaction (qPCR) uses the PCR process to identify specific transcripts from tissue homogenates
Describe cDNA micro assays
*cDNA Microarrays can identify thousands-millions of transcripts in a single sample
*Usually comparative between two samples (e.g. control vs drug treated)
Describe imaging the brain
*Both structural and functional imaging technologies can be used to evaluate the human brain
What are structural techniques?
Structural techniques can reveal differences in gross anatomy in various pathological conditions
What are functional techniques?
Functional techniques can reveal changes in brain function with between pathological conditions or during specific tasks
Describe MRI as an imaging technique
*Magnetic resonance imaging uses the phenomenon of nuclear magnetic resonance to generate structural images of living tissue
*Imaging modes can be varied for normal anatomy (T1), pathology (T2), or grey/white matter differences (PD)
Describe MRI imaging modes
Imaging modes can be varied for normal anatomy (T1), pathology (T2), or grey/white matter differences (PD)
Describe PET
- PET Uses positron-emitting radiotracers to analyze brain receptors or metabolism
- Radioligands for specific receptors can be used to reveal binding in different brain regions
- Tissue uptake of 2-deoxy-2-[18F]fluoro-D- glucose (FDG) is proportional to glucose uptake and hence activity
Describe connectivity measures
- fMRI is used to measure functional connectivity by correlating changes in activity during tasks between regions (fcMRI)
- Effective connectivity is established by information flow between regions
- Structural connectivity is established using DTI
Describe single photon emission computed tomagraphy
- Uses gamma-emitting radiotracers to analyze brain blood flow
- Tissue uptake of [99mTc] hexamethylpropyleneamine oxime (99mTc- HMPAO) is proportional to blood flow and hence activity
Describe the role of genetic engineering
- Transgenic mice are a commonly used technique for assessing genetic contributors to disease or for determining functions of specific genes
- Common models use genetic knockouts (KO mice), knock-ins, or selective or conditional knockouts
Describe optogenetics
- Specific bacterial proteins function as light-gated ion channels
- Expression of opsins can be used to non- invasively stimulate or inhibit neuronal function in vivo
- Can be combined with specific genetic techniques to target different neuronal populations
- Can be used with fibre optics to alter activity involved in behaviour in live, free-moving animals
What are the benefits of optogenetics
Optogenetics offers several improvements over conventional electrical stimulation including a less invasive procedure, and cell- or region- specific modulation of activity.
Describe behavioural pharmacology
- Animal models are used to mimic human behaviours and pathologies
- Use of animals presents challenges for psychopharmacology as there is no such thing as a depressed or schizophrenic mouse
- Behavioural tests are designed to provide measurable proxies for human behaviours
Describe construct validity
- Similarity between the methods by which the models is
induced and the etiology of disease - Genetic and environmental factors
Describe face validity
- Ability of the model to recreate key features of a
disease - Anatomical, behavioural, and neurochemical features of disease
Describe predictive validity
- Utility of the model to predict drug effects
- Assumes similarities in effect are based on shared mechanism
Describe the Morris water maze
- Morris water maze tests spatial
memory - Animals are tested for their ability to recall the location of a hidden submerged platform in a pool
- Decreased time to find the target in repeated trials indicates spatial memory
Describe radial 8 arm maze
- Mazes are useful for measuring
memory in animals - Many possible configurations
- The radial 8-arm maze tests spatial memory
Describe novel object recognition tests
- Novel object recognition tests can
evaluate episodic memory - Animal is habituated with two objects
- In a repeat trial one object is replaced
*Time spent investigating the novel object is indicating of episodic memory
Describe anxiety tests
- Anxiety tests are common measures for depression
- Open field test places animal in a novel open box environment
- Mice have both strong exploratory drive and fear of open spaces
- Movement tracked over a defined time period
- Decreased time spent in open vs peripheral areas is indicative of anxiety
- Amenable to automated analysis
Describe the elevated plus maze
- Plus maze consists of two open
arms and two closed arms - Entry into open arms is indicative of novelty-seeking behaviour
- Depressed / anxious animals spend more time in closed arms
Describe fear conditioning
Animals are presented with a signal(light or tone) followed by an unescapable foot shock
- Later trials measure the fear response (freezing) to the conditioned stimulus
- Fear-conditioned startle is an alternative in which the conditioned stimulus if followed by a startling noise
Describe reward-operant conditioning
- Animals are readily trained to obtain rewards (food or drugs) in response to operant behaviours (lever press)
- Useful for developing models of substance abuse (operant self- administration)
Describe conditioned place preference
- Pavlovian conditioning paradigm
- Drug injection (unconditioned stimulus) paired with a neutral environmental stimulus to create a conditioned stimulus
- 2-3 chamber conditioning box
- Three phase CPP protocol
- Pre-conditioning (habituation)
- Open exploration, 15 min each for 3 days
- Conditioning
- Alternate injections of drug and vehicle in alternate chambers
- Post-conditioning (test)
- Open exploration, time spent in each chamber measured
