PSCYH 275 - Research Methods

lectures from Dr. Kathleen Munley

What is neuropsychology?

The scientific discipline focused on studying relationships between brain functions and corresponding behaviour, especially in humans

What are 3 common approaches to neuropsychology?

1. Non-invasive imaging

2. Complex neuroanatomical measurements

3. Sophisticated behavioural analyses

What is behavioural neuroscience?

The scientific discipline focused on studying the biological bases of behavior in human and non-human animals

What is ethology?

The objective study of non-human animal behavior, especially under natural conditions

What are Neuroanatomical studies?

Studies that visualize, map, and analyze the structure of the nervous system

• Typically involve post-mortem investigations of brain tissue from humans and non-human animals

• Commonly use histology

What is histology?

The study of tissues and organs through sectioning, staining, and microscopy

• Allows visualization of individual cells and structures in neuroanatomical studies

What are histological stains?

Dyes used to colour tissue for better visualization and disgnosis

What are Chromogenic stains?

Coloured stains that absorb wavelengths of visible light and can be viewed by the human eye

What are some common chromogenic stains?

• Golgi stain: randomly stains ~5% of neurons in a slide

• Myelin stain: stains myelin surrounding axons

• Nissl stain: stains cell bodies of neurons

What are fluorescent stains?

Coloured stains that absorb wavelengths of UV or blue light and must be re-emitted as visible light to be viewed by the human eye

What are some common fluorescent stains/approaches?

• Autoradiography

• Immunocytochemistry (ICC)

• Multiphoton microscopy (MPM)

What is autoradiography?

A fluorescent technique that uses radioactively-labeled substances to stain for proteins

What is Immunocytochemistry (ICC)?

A fluorescent technique that uses labeled antibodies attached to a chromogenic or fluorescent dye to identify proteins

What is multiphoton microscopy (MPM)?

A fluorescence imaging technique that enables 3D imaging of live cells and tissues (up to ~1mm deep)

What is in situ hybridization (ISH)?

A molecular technique that uses labeled complementary DNA, RNA, or modified nucleic acid strand (probe) to detect and localize DNA or RNA sequences in cells or tissue sections

What is hybridization chain reaction (HCR)?

An in situ hybridization that uses multiple fluorescently labeled probes for simultaneous detection of multiple RNA or DNA sequences of interest

What are some common microscopes used to visualize neurons?

• Optical (light) microscopes

• Scanning electron microscopes (SEM)

• Transmission electron microscopes (TEM)

What are optical/light microscopes?

Imaging instruments that use visible light and glass for visualizing tissue

• Bounces light off a tissue sample and funnels it through lenses that bend and magnify the image produced

What are scanning electron microscopes (SEM)?

Imaging instruments that use focused beam of electrons to create high-resolution 3D images of a sample’s surface topography

What are transmission electron microscopes (TEM)?

Imaging instruments that use transmitted beam of electrons to visualize thin slices of tissue at high resolution

What is neuropsychological testing?

Uses specifically designed tasks to measure brain functions known to be linked to a particular brain structure or neural pathway

• Typically applied to people who have sustained a brain injury

• Eg. Patients with temporal lobe damage often have memory disturbances

What are 3 types of neuropsychological tests that can be used to examine three distinct forms of memory:

1. Verbal learning and memory tests

2. Visual and visuospatial memory tests

3. Working memory and short-term memory tests

What are 3 examples of neuropsychological tests of memory?

1. Corsi block-tapping test - Examiner taps out a sequence of blocks for the participant to remember

2. Mirror-drawing task - Task is to trace between two outlines while only looking at your hand in a mirror

3. Test of recent memory - Task is to identify which picture you saw most recently

What does behavioural analysis of non-human animals look like?

Most research continues to be conducted on animal models to determine neural circuitry underlying specific behaviors

• Historically, majority of behavioral neuroscience studies conducted on rodents (mice, rats)

• Field has been evolving over the past few decades to include non-traditional animal models (other mammals, fishes, birds, reptiles, invertebrates)

What are Morris swimming tasks?

Assessments used to test visuospatial learning in rats

What are the three different Morris swimming tasks?

1. Place-learning task: the rat must find a platform using external cues

2. Matching-to-place learning - platform is in the same location each trial, but in a different location each day

3. Landmark-learning task - platform is identified by a cue on the wall

What is a skilled reaching task?

A movement series displayed by rats trained to reach through a narrow vertical slot to obtain a food pellet:

1. Aim the hand

2. Reach over the food

3. Grasp the food

4. Withdraw the hand and move food to the mouth

What is an automated touchscreen platform?

Used for cognitive and motivational testing of rodents

• Removes the variation and stress that humans introduce when testing animals

• Can be programmed to deliver tests that are highly similar to the touchscreen tasks used in human cognitive testing

What are two reasons for brain/behaviour manipulations?

1. To develop and test hypotheses about how the brain affects behavior

2. Develop animal models of neurological and psychiatric disorders

How are case studies used to study brain lesions?

They investigate behaviour of individuals with existing brain damage

• Assess changes in behavior since damage and compare differences in abilities with control individuals

• Examples: Broca’s and Wernicke’s areas (aphasia), Phineas Gage, split brain patients

What experimental approaches are used to study brain lesions?

Ablation and lesioning

What is ablation?

The surgical removal of brain tissue

• Imprecise and often used for large areas of the brain

• Performed via aspiration

What is lesioning?

The surgical damaging of brain tissue

• Precise and sometimes reversible

• Performed via electrical current, heat, chemicals, or via microscalpel

What are stereotaxic instruments?

Devices that allow precise positioning of all brain regions relative to each other and landmarks on the skull using probe

• Use stereotaxic atlas (map of the brain) to determine appropriate probe location

• Probes are typically fine-wire electrodes

• Can also use microelectrodes to monitor and/or stimulate a single neuron

What is compensation?

The neuroplastic ability to modify behavior after damage to make up for lost functionality

What are some temporary and reversible lesion techniques?

1. Regional cooling

2. Local administration of GABA agonist

3. Transcranial Magnetic Stimulation (TMS)

What is deep brain stimulation?

A neurosurgical technique in which electrodes are implanted to stimulate targeted areas of the brain

• Used to treat essential tremor, dystonia, obsessive-compulsive disorder, epilepsy

What is transcranial magnetic stimulation (TMS)?

A non-invasive technique that uses magnetic pulses to stimulate the brain

What are some research approaches involving drug manipulations?

• Cannulation - a neurosurgical procedure in which plastic tube is implanted into specific brain regions for direct application of drugs

• Brain microdialysis: an in vivo sampling technique used to monitor chemical composition of brain’s extracellular fluid in real time

What is optogenetics?

A biological technique that combines light and genetic engineering to control activity of neurons with high temporal and spatial precision

How does optogenetics work?

• Light-sensitive protein channels inserted in specific types of neurons

• Exposure to light selectively activates only that type of cell

What is chemogenetics?

A neuroscience technique that uses genetically engineered receptors and small molecule ligands to remotely control specific cell populations

• Commonly involves DREADDs

What are DREADDs?

• Designer Receptors Activated Exclusively by Designer Drugs

• Engineered G-coupled protein receptors that remotely and reversibly control neuronal activity, behavior, and cellular signaling

What is CRISPR/Cas9?

• Clustered Regularly Interspaced Short Palindromic Repeats

• Gene editing technology that enables researchers to edit DNA in living organisms

• Adopted from naturally occurring genome editing system that bacteria use for immune defense

What is electrophysiology?

A branch of physiology that studies the electrical properties and activities of biological cells

• Electrical measures of brain activity are important for studying brain function

What are the 4 major electrophysiological techniques?

1. Single-cell recordings

2. Electroencephalography (EEG)

3. Event-related potentials (ERPs)

4. Magnetoencephalography (MEG)

What is patch clamping?

An electrophysiology technique for measuring ionic currents and membrane potentials in neurons (single cell recordings)

• Uses fine electrodes to measure single- neurons action potentials

• Electrodes are either placed next to cells (extracellular recording) or inside cells (intracellular recording)

What is electroencephalography (EEG)?

A non-invasive measurement of summed graded potentials of all neurons in the brain (or a brain region) on a visual trace

• High temporal resolution → records events as lose as 1 ms apart

• Poor spatial resolution → imprecise approach unless electrodes are applied directly to the brain surface

What are event-related potentials (ERPs)?

Precise, non-invasive electrophysiological measures of brain activity derived from EEG

• Represent graded potentials on dendrites that a sensory, cognitive, or motor stimulus triggers

• Complex electroencephalographic waveforms are related in time to a specific sensory event

• Stimulus is presented repeatedly to counter noise effects and recorded responses are averaged

What is magnetoencephalography (MEG)?

A neuroimaging technique for mapping brain activity by recording magnetic fields produced by electrical currents from neurons

• Magnetic fields recorded by detectors on the skull

• Allows 3D visualization of active cell groups

What is anatomical brain imaging?

a technique that produces detailed, high resolution 3D images of brain structures

What are the 4 major anatomical brain imaging techniques?

1. Computed tomography (CT)

2. Magnetic resonance imaging (MRI)

3. Diffusion Tensor Imaging (DTI)

4. Magnetic Resonance Spectroscopy (MRS)

What is Computed tomography (CT)?

An imaging approach that uses X-rays to create detailed images of the body

• Produces flat static images that can be combined into 3D composites via computer

• Dye in the bloodstream allows imaging of soft tissues

What is magnetic resonance imaging (MRI)?

A medical imaging technique that uses magnetic field and computer-generated radio waves to create detailed images of organs and tissues

• Measures radiation emitted from hydrogen atoms

What are me MRI images based on?

The density of hydrogen atoms in different brain regions

• Areas with high water content stand out from sites with lower water content

What is diffusion tensor imaging (DTI)?

An advanced MRI modality that measures directional movement of water molecules to image nerve-fiber pathways in the brain

• Enables non-invasive visualization of neural pathways

• Used to detect, track, and treat neurological conditions and abnormalities in neural pathways

What is magnetic resonance spectroscopy (MRS)?

A non-invasive diagnostic technique that measures biochemical composition (metabolites) of tissues

• MRI variant that uses hydrogen proton signal to determine concentration of brain metabolites

• Useful in detecting persisting abnormalities in brain metabolism in disorders

What is functional brain imaging?

The use of neuroimaging technology to measure brain function

• Infers changes in brain activity by measuring blood flow or levels of blood’s constituents (Eg. oxygen, glucose, iron)

What are the 3 major functional brain imaging techniques?

1. Functional magnetic resonance imaging (fMRI)

2. Functional near-infrared spectroscopy (fNIRS)

3. Positron emission tomography (PET)

What is functional magnetic resonance imaging (fMRI)?

A neuroimaging technique that maps brain activity by measuring changes in blood flow and oxygenation (BOLD signal) compared to neuronal activation

How does fMRI work?

Changes in oxygen content of the blood alter magnetic properties of water in the blood

• Allows monitoring of changes in blood oxygenation as activity (and resource requirements) change

What is functional Near-Infrared Spectroscopy (fNIRS)?

An imaging technique that measures neural activity by detecting blood oxygenation changes using near-infrared light

How does fNIRS work?

Oxygen-rich hemoglobin and oxygen-poor hemoglobin differ in absorption spectra

• Allows brain to be reconstructed by gathering light transmitted through it

What is positron emission tomography (PET)?

An imaging technique that detects changes in blood flow by measuring changes in uptake of compounds (e.g., oxygen, glucose)

How does PET work?

• Radioactive molecules are injected into the blood

• Scanner picks up emitted positrons to determine relative activity

• Used to analyze metabolic activity of neurons

What are 3 important considerations when deciding what method should be chosen for your research?

1. Temporal resolution → How quickly are measurements or images obtained?

2. Spatial resolution → How accurate is localization in the brain?

3. Degree of invasiveness → Is it internal or external? Does it affect tissues long-term?

What are the experimental goals of all mentioned research methods?

What two areas of study set parameters for using research tools?

1. Neuroethics

2. Ethical treatment of non-human animals

What is neuroethics?

The discipline that examines the ethical, legal, and social implications of neuroscience and neurotechnology

• Key areas: brain data, cognitive enhancement, free will

What are the APA’s five general ethical principles?

1. Beneficence and nonmaleficence

2. Fidelity and responsibility

3. Integrity

4. Justice

5. Respect for people’s rights and dignity

How are ethical standards for research governed?

• Most governments regulate the use of animals in research, and universities and other research organizations have additional rules governing animal use

• All accredited North American universities that receive government grant support are required to provide adequate treatment for all vertebrate animals

What are 5 key ethical standards for research?

1. Institutional review board (IRB)

2. Informed consent

3. Deception

4. Research misconduct

5. Animal research

What is research misconduct?

Fabrication, falsification, or plagiarism (FFP) in proposing, performing, reviewing, or reporting research

What is fabrication?

Making up data and/or results and recording or reporting them

What is falsification?

Manipulating research materials, equipment, or processes or changing or omitting data or results, such that research is not accurately represented to the scientific community

What is plagiarism?

The appropriation of another person’s ideas, processes, or results without giving appropriate credit

What is informed consent?

An individual’s voluntary agreement to participate in a study with the understanding of potential adverse effects

• Necessary in research with humans

What is deception?

Misinforming or failing to disclose to participants the details of the study

• Many restrictive guidelines on appropriate deception

• Informed consent required if there is a risk of physical pain or severe emotional distress

What is speciesism?

The systematic discrimination, prejudice, or bias against non-human animals based solely on their species

What do ethics look like in research with non-human animals?

• Some feel risky experiments are more ethical when performed on animals

• Animal research guidelines are tightening over time

What is gene therapy?

A medical technique that treats or prevents disease by correcting, replacing, or inactivating faulty genes in a patient’s cells

What are 5 ethical concerns of gene editing?

1. Somatic vs. germline editing

2. Safety and unintended effects

3. “Designer babies” and eugenics

4. Consent and autonomy

5. Equity and access