8/24: Research Methods

brain damage

→ the best way to identify and study which parts of the brain are responsible for a certain function

• Is this the best research method for neurosci T _ T ???

computerized tomography

CT → imaging technique that uses x-ray beams (high energy light/radiation)

• Patients lie in a cylindrical x-ray source to get images of their body at all angles, 360°

• Following scanning, x-ray detections are computerized and reconstructed into a 3D model of the patient’s body

• CT scans are cheap and fast, but not the best for soft tissue (like the brain)

• Sometimes contrast agents (fluids like iodine) are administered to improve resolution

magnetic resonance imaging

MRI→ imaging technique that uses magnetic fields/radio waves (low energy light)

• Patients lie in a cylindrical radio frequency coil (magnet) to get images of their body at all angles, 360°

• As magnetism is administered, the hydrogen atom protons in a patient’s body absorb the energy, orient themselves in line with the magnetic fields, and return emissions

• Following scanning, radio wave detections are computerized and triangulated to form an estimate of the relative density of hydrogen atoms throughout the scanned

• MRI scans have good spatial resolution for imaging tissues (hydrogen atoms are especially prevalent in lipids … myelin is a kind of fat and there’s plenty of that in the brain)

• Safer than CT; orientation of an atom is inconsequential to internal biochemistry

diffusion tensor imaging

DTI → MRI technique that measures the direction and speed of the diffusion of water molecules in the brain

• Alternative to normal MRI (whose magnets are optimized for lipids)

• Used to identify axon tracts

• Colours indicate the direction of water molecule diffusion

functional magnetic resonance imaging

fMRI → imaging technique that uses a rapid series of MRI scans measuring the movement of magnetic field distortions over time

• Same process as MRI, but with measuring blood oxygen levels in a locale

• As magnetism is administered, blood oxygenation in a given area changes;

• An area’s rapid ↑ in blood flow correlates positively with ↑ neural activity

• fMRI scans have good spatial resolution and temporal resolution, providing great structural and functional information

• Popular for being non-invasive

fMRI upcoming modification

→ to measure fluctuations in neurotransmitter signaling

• Would involve using “enzyme-activated magnetic resonance contrast agents” to distort magnetic fields differently when they bind to neurotransmitters

positron emission tomography

PET → imaging technique that involves injections of radioactive compounds

• Radioactive sugar molecules are commonly used to detect changes in energy use

  • 2-DG → similar to glucose (consumed by active cells), however, it’s not metabolized as easily (endures)

• Scanning where and how radioactive sugar molecules move over time

• PET substances can also measure changes in the expression levels of neurotransmitter receptors across weeks

• Main disadvantages of PET scans are operating costs and safety precautions (radioactive molecules decay from the moment they’re produced, so they have to be made on-site and as close to the procedure as possible)

• More recently superseded by fMRI

electroencephalogram

EEG → measures electrical activity in the brain

• Macroelectrodes attached to the scalp to record summed population-level activity of millions of neurons

• Can be used a diagnostic tool since specific patterns of EEG activity are associated with different states of consciousness (awareness), subconsciousness (sleep stages), and cerebral atrophy (neural loss)

experimental ablation

→ lesion study; involves the procedural removal or destruction of a portion of the brain

• Inferential findings: Functions that no longer work after the procedure are probably controlled by the lesioned brain area

radiofrequency lesion

→ small lesions made by passing an electric current through a metal wire insulated everywhere but the tip (whose heat will burn surrounding cells)

• Shape and size of lesion is determined by the duration and intensity of the current

• Shortcoming is that passerby axons are burnt in the process

excitotoxic lesion

→ small lesions made by injecting a glutamate receptor agonist

• Localized drugs cause so much excitation (Ca2+ influx) such that the affected neurons undergo apoptosis

• This method spares passerby axons

ex.

• Kainic acid

sham lesion

→ not an actual lesion; made by mimicking the procedures for an actual experimental ablation, except the damaging part

reversible lesion

→ temporary “lesions” made by injecting drugs that block or reduce neural activity in a given region

• Localized drugs cause so much excitation (Ca2+ influx) such that the affected neurons undergo apoptosis

• This method spares passerby axons

ex.

• Voltage-gated sodium channel blockers → stops action potentials

• GABA receptor agonists → hyperpolarizes cell bodies

microelectrodes

→ neural activity implants made from thin metal wires with a fine tip that record single-unit recordings (the electrical activity of individual neurons; chronic or acute)

• Can be used in behaving animals to record action potentials

• Wires are connected to a “socket” in the animal’s head so that it can be “plugged” into a recording system at any time

electrical stimulation

→ method of manipulating neural activity that involves passing an electrical current through a wire inserted into the brain

• Affects all receptor components in the area (axons of neurons)

• Overstimulation (rapid stimulation frequencies) actually produces the same behavioural effects as lesioning the area (deliberate damage)

chemical stimulation

→ method of manipulating neural activity that involves administering drugs into a particular brain region

• Usually pushed through a guide cannula (hollow tube)

• Anesthetics → can completely shut down neural activity, entirely or locally

• Receptor agonists/antagonists are alternatives since they don’t affect passerby axons

optogenetics

→ refers to the use of light to control light-sensitive neurons

• Neurons are made light-sensitive through the introduction of foreign DNA, which provides instructions to make light-sensitive proteins called opsins

  • Opsins in eye are metabotropic receptors with 30ms~ delay

  • Similarly, opsins optogenetically manipulated are ion channels that open and close instantly in response to light

  • First opsins (ex. ChR2) were discovered in bacteria and algae

• Excitatory opsins → light delivery generate action potentials

• Inhibitory opsins → light delivery prevent action potentials

channelrhodposin-II

ChR2 → excitatory photosensitive ion channel permeable to sodium (Na+) ions

• Responds to blue light, depolarizing neurons

• This opsin evolved from single-cell organisms

IC++

→ inhibitory photosensitive ion channel permeable to chloride (Cl-) ions

• Responds to blue light, hyperpolarizing neurons

• This opsin was designed by humans

virus

→ small infectious agent that replicates by injecting its DNA into normal cells of a host organism

• Replication-deficient → when a virus has its DNA removed

• Microbiologists can insert foreign DNA into replication-deficient viruses

  • This lab-made foreign DNA encodes proteins for a cell to express, such as fluorescent proteins or optogenetic proteins

viral-mediated gene delivery

→ using viruses to deliver expressive DNA into neurons

1. Modified viruses (replication-deficient viruses filled with foreign DNA) are injected into the brain. Surrounding cellular components become infected.

2. When viral DNA accesses the nucleus of a cell, that cell will start to transcribe the viral DNA to make its associated proteins.

fluorescent calcium imaging

→ imaging technique that involves implanting a fiber optic cable into the brain to measure neural activity triggered by calcium-sensitive proteins

• Green fluorescent protein (GFP) → bioluminescent protein discovered in jellyfish

• GCaMP→ modified GFP to bind to calcium (which is involved in action potentials) and to fluoresce much brighter when it does

  • GOOGLE SAYS: GFP + calmodulin (CaM) + M13, a peptide sequence from myosin light-chain kinase

tracing methods

→ reveal the input and output structures involved in a particular brain region involved with a particular function

• Retrograde

• Anterograde

retrograde labeling

→ determining neural connections by tracing afferent axons; inputs

• Asks “what brain areas send their axons here?”

• Retrograde tracer:

  • Taken up by axon terminals and transported back to the cell body

• Retrograde tracer → chemicals taken up by axon terminals and transported back to the cell body

  • ex. Fluorogold

anterograde labeling

→ determining neural connections by tracing efferent axons; output

• Asks “where do these axons go?”

• Anterograde tracer → chemicals taken up by cell bodies and transported back to axon terminals

  • ex. PHA-L

stereotaxic brain surgery

→ surgical intervention that uses a stereotaxic apparatus to administer electrical or chemical stimulation

• Injections: drugs, viruses, tracers, etc.

• Implantations: cannula, electrodes, fiber optic cables, wires, etc.

• Lesions: radiofrequency, excitotoxic, reversible, etc.

• Bregma → junction where pieces of the skull fuse together; often used as a reference point for stereotaxic surgery

microdialysis

→ old-fashioned approach to measuring fluctuations in neurotransmitters in behaving animals

• Dialysis → refers to the use of a semipermeable membrane to either deliver or measure the concentration of molecules in some solution or area

• Microdialysis probe → small insertable metal tube that holds dialysis tubing

• Sampling rate → frequency of sampling the concentration of molecules over the dialysis membrane

  • Since equilibration takes time, sampling is usually once every 10 minutes, but it could be more frequent

man-made fluorescent receptors

→ modern approach to measuring fluctuations in neurotransmitters in behaving animals

• Executed via viral-mediated gene delivery → method to deliver protein receptors that fluoresce when bound to neurotransmitters