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Psych 478 > Research Methods in Pharmacology > Flashcards

Research Methods in Pharmacology Flashcards

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1
Q

What are neurobiological techniques?

A

*Research in neuropsychopharmacology can span the entire spectrum of neuroscience research

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2
Q

Define in vivo

A

analyses performed in a living organism

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3
Q

Define in vitro

A

in glass, analyses performed in test tube

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4
Q

Define ex vivo

A

analyses performed in live tissue removed from an organism

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5
Q

Define in situ

A

analyses performed in context to living tissue but typically after dissection

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6
Q

Describe stereotaxic surgery

A

*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

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7
Q

Describe micro dialysis

A

*Technique for collecting fluid from a live and awake animal – analysis of neurotransmitter levels, signalling molecules, drugs, etc in vivo

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8
Q

Describe chromatography

A

*Chromatographic techniques separate molecules based on their size, charge, relative polarity, or specific interactions

*Compounds can then be detected, quantified, or further analysed

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9
Q

What are analytes?

A

*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)

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10
Q

Describe electrophysiological recordings

A

*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

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11
Q

Describe in vivo recordings

A

*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)

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12
Q

Describe intracellular recording

A

*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

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13
Q

Describe molecular methods

A

*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

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14
Q

What are immunological techniques?

A

*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

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15
Q

What is innate immunity?

A

*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

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16
Q

Describe immunofluorescence

A

*Antibodies can be conjugated to fluorescent molecules and used to identify specific proteins in cells of the CNS.

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17
Q

What is immunohistochemistry?

A

the detection of proteins in tissue

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18
Q

What is immunocytochemistry?

A

the detection of proteins in cells (typically cells cultured in vitro)

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19
Q

Describe confocal microscopy

A

*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

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20
Q

Describe 2 photon microscopy

A

*Uses infrared lasers (700-900 nm) to image living tissue

*IR light can penetrate ~10 mm through brain tissue

21
Q

Describe immunoassays and ELISA

A

*Enzyme-linked immunosorbant assay

*Detects an immobilized antigen using an enzyme-linked antibody

*Higher sensitivity

22
Q

Describe Western blotting

A

*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

23
Q

What is FISH?

A

*Fluorescence in situ hybridization (FISH) binds a fluorescent probe in tissue sections to show which cells may be expressing a gene of interest

24
Q

What is qPCR?

A

Quantitative polymerase chain reaction (qPCR) uses the PCR process to identify specific transcripts from tissue homogenates

25
Q

Describe cDNA micro assays

A

*cDNA Microarrays can identify thousands-millions of transcripts in a single sample

*Usually comparative between two samples (e.g. control vs drug treated)

26
Q

Describe imaging the brain

A

*Both structural and functional imaging technologies can be used to evaluate the human brain

27
Q

What are structural techniques?

A

Structural techniques can reveal differences in gross anatomy in various pathological conditions

28
Q

What are functional techniques?

A

Functional techniques can reveal changes in brain function with between pathological conditions or during specific tasks

29
Q

Describe MRI as an imaging technique

A

*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)

30
Q

Describe MRI imaging modes

A

Imaging modes can be varied for normal anatomy (T1), pathology (T2), or grey/white matter differences (PD)

31
Q

Describe PET

A
  • 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
32
Q

Describe connectivity measures

A
  • 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
32
Q

Describe single photon emission computed tomagraphy

A
  • 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
33
Q

Describe the role of genetic engineering

A
  • 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
34
Q

Describe optogenetics

A
  • 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
35
Q

What are the benefits of optogenetics

A

Optogenetics offers several improvements over conventional electrical stimulation including a less invasive procedure, and cell- or region- specific modulation of activity.

36
Q

Describe behavioural pharmacology

A
  • 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
37
Q

Describe construct validity

A
  • Similarity between the methods by which the models is
    induced and the etiology of disease
  • Genetic and environmental factors
38
Q

Describe face validity

A
  • Ability of the model to recreate key features of a
    disease
  • Anatomical, behavioural, and neurochemical features of disease
39
Q

Describe predictive validity

A
  • Utility of the model to predict drug effects
  • Assumes similarities in effect are based on shared mechanism
40
Q

Describe the Morris water maze

A
  • 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
41
Q

Describe radial 8 arm maze

A
  • Mazes are useful for measuring
    memory in animals
  • Many possible configurations
  • The radial 8-arm maze tests spatial memory
42
Q

Describe novel object recognition tests

A
  • 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

43
Q

Describe anxiety tests

A
  • 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
44
Q

Describe the elevated plus maze

A
  • 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
45
Q

Describe fear conditioning

A

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
46
Q

Describe reward-operant conditioning

A
  • 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)
47
Q

Describe conditioned place preference

A
  • 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

Psych 478 (28 decks)

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