Animal research methods Flashcards
What are the ways to study animal brains?
- neuronal tracing
- microelectrode EEG
- electrical stimulation
- pharmacological manipulation
- optogenetics
- calcium imaging
- excitotoxic lesions
- radio lesions
- microdialysis
- immunohistochemistry
- cFos immunohistochemistry
What is experimental ablation?
- lesion study
- the removal or destruction of a portion of the brain
- functions that can no longer be performed following the surgery are probably controlled by that brain region
What are radiofrequency lesions?
- small lesions made by passing radiofrequency current through a metal wire that is insulated everywhere but the tip
- burns cells around the tip of the wire
- size and shape of lesion, determined by the duration and intensity of the current
What is a downside to radiofrequency lesions?
- axons just passing through will also be burned
What is an excitotoxic lesion?
- injection of a glutamate receptor agonist
- cause so much excitation (and calcium influx) that the affected neurons undergo apoptosis
- axons passing through are spared
- permanent lesion
What is a sham lesion?
- placebo procedure
- duplicates all steps of producing a brain lesion except for the step that causes extensive brain damage
What is a reversible lesion?
- temporary brain “lesion”
- injecting drugs that block or reduce neural activity in area
- permanent
What are common drugs to use for a reversible lesion?
- voltage-gated sodium channel blockers (stops all action potentials, affects axons passing through)
- GABA receptor agonists (hyperpolarize cell bodies, does not affect axons passing through)
What is the most direct measurements of neural activity?
- made with metal wires placed in the brain
- macroelectrodes
What are macroelectrodes?
- thin metal wires with a fine tip
- record the electrical activity of individual neurons (single-unit recordings)
- used in behaving animals to record every action potential from a given neuron
- record from hundreds of single neurons simultaneously
- implanted in brain during stereotaxic surgery
- wires connected to socket in animal’s head so that they can be ‘plugged in’ to a recording system at any time
- permanently attached set of electrodes, with a connecting socket cemented to the skull
What are chronic electrical recordings?
- made over an extended period of time
What are acute electrical recordings?
- made over a relatively short period of time
- often during surgery when the animal is anesthetized
What can we know when we manipulate neural activity?
- how the activity of specific receptors or cell populations influence behaviour
What are ways to manipulate neural activity?
- electrical stimulation
- chemical stimulation
- optogenetics
- viral-mediated gene delivery (record + manipulate)
What is electrical stimulation?
- passing an electrical current through a wire inserted into the brain
- affect everything in the area (even passing)
- fast stimulation frequencies counterintuitively produce the same behavioural effects as lesioning the brain area
- stop action potentials
What is chemical stimulation?
- drugs
- administered through a guide cannula (hollow tube) implanted in a particular brain region
- anesthetics to shut down all neural activity
- receptor agonist/antagonist can be used (don’t affect passing)
What are optogenetics?
- use light to depolarize and hyperpolarize neurons with millisecond precision
- turn up or down the activity of specific cells or receptors
- use of light to control neurons that have been made sensitive to light through the introduction of foreign DNA
How do optogenetics work?
- foreign DNA provides instructions to make light-sensitive proteins
- opsins we use to manipulate neural activity (optogenetic techniques) are often ion channels that open and close instantly in response to light
- light sensitive protein (ion channel that opens in response to light)
- take gene for protein
- insert DNA into specific neurons in the brain
- neurons communicate by firing
- can cause neurons to fire by flashing light
What are opsins?
- proteins that are activated by light
What are types of photosensitive ion channels that evolved in bacteria and algae?
- ChR2
- IC++ (designed by humans)
What is ChR2?
- excitatory opsin
- permeable to sodium ions
- when activated with blue light, it depolarizes neurons, causing them to spike
What is IC++?
- inhibitory light-gated ion channels
- pass chloride and hyperpolarize neurons when activated by blue light
What do excitatory opsins do?
- pulse light or leave it on to generate action potentials
- depolarize neuron, causing them to spike
What do inhibitory opsins do?
- continuous light delivery can prevent action potentials
- like halorhodopsin
What is viral-mediated gene delivery?
- remove DNA from virus
- renders the virus “replication-deficient”
- add foreign DNA to a virus
- DNA that encodes proteins we want a cell to express
- modified virus is injected, infect all cells in area
- Once a virus gets its DNA into the nucleus of a cell, that cell will start to transcribe the viral DNA and make the associated proteins
- section of DNA that encodes a fluorescent protein
- Fluorescent proteins are used to identify which cells got infected
- study function of neurons, use viruses to deliver DNA to them
- make neurons express proteins that will change their resting membrane potential or generate action potentials
How does viral-mediated gene delivery work?
- light sensitive protein (light gated ion channel)
- take gene for protein
- insert DNA into hollowed out virus
- insert virus into brain
What is a virus?
- type of DNA delivery system
- replicate by injecting viral DNA into a host organism
- contains instructions on how to make more virus
- small infectious agent that replicates by injecting its DNA into normal cells
What is virus DNA?
- instructions for how to make more virus
What is GCaMP?
- protein
- modified the fluorescent protein GFP
- causing it to bind calcium and fluoresce much brighter when it does
What does measuring GCaMP do?
- little calcium influx always occurs during action potentials
- monitoring GCaMP fluorescence is good way to measure neural activity (in cells made to express GCaMP protein)
What are efferents?
- outputs
What are afferents?
- inputs
How do we trace neural connections?
- retrograde labeling
- anterograde labeling
What is retrograde labeling?
- tracing afferent axons
- What brain areas send their axons here?
- label the cells that innervate (project to) a given region
- retrograde tracers (chemicals like fluorogold)
- fluorogold taken up by axon terminals and transported back to the cell body
What is anterograde labeling?
- tracing efferent axons
- Where do the axons from these cells go?
- label where axons from a particular location go to
- anterograde tracers (chemicals like PHA-L)
- PHA-L is taken up by cell bodies and transported down to axon terminals
What is stereotaxic surgery?
- surgical intervention that uses a stereotaxic apparatus
- put something into a very specific part of the brain
- inject drugs, viruses, or tracers (dyes) in brain
- permanently implant things, like cannula, electrodes, or fiber optic cable
What is a bregma?
- junction where pieces of skull fuse together
- used as a reference point for stereotaxic brain surgery
Why is stereotaxic surgery commonly used for one-time injections of drug or virus?
- Lesion a brain area
- Lesion a specific type of cell in a particular brain area
- To change gene expression
Why else is stereotaxic surgery used?
- Implant a guide cannula (allow for later infusions of drugs)
- Implant microelectrodes
- Implant fiber optic cables
How do we measure fluctuations in neurotransmitter levels?
- microdialysis
- man-made fluorescent reporter proteins
What is microdialysis?
- old-fashioned approach
- measuring changes in neurotransmitter levels in a brain region
What is dialysis?
- use of a semipermeable membrane to either deliver molecules to or measure the amount of molecules in some solution (or brain area)
- takes time for the concentration of molecules to equilibrate across dialysis membrane
- fastest sampling rate possible is once per minute
- typically, once every 10 minutes
What is a microdialysis probe?
- small metal tube that holds dialysis tubing, which can be placed in an animal’s head
What are man-made fluorescent reporter proteins?
- design proteins, like receptors that become fluorescent when bound to neurotransmitter
- use viral-mediated gene delivery to get neurons to express man-made fluorescent sensors
- visualize neurotransmitter release in a living brain
How would a man made fluorescent glutamate receptor work?
- receptors fluorescence whenever glutamate binds to them
- can see all places there’s glutamate release in brain in moment in time
What is the short description of neuronal tracing?
- Fluorescent molecule is injected to visualize afferents/efferents – identify inputs and outputs to a cell population
What is the purpose of neuronal tracing?
- Structure
What are the pros of neuronal tracing?
- Can create a circuit diagram of brain
What are the cons of neuronal tracing?
- Molecules don’t fill whole neuron
What is the short description of microelectrode EEG?
- Electrode in brain records electrical changes – observe activity changes with behaviour
What is the purpose of microelectrode EEG?
- Correlation
What are the pros of microelectrode EEG?
- High temporal resolution
What are the cons of microelectrode EEG?
- Can’t tell what you’re recording from
What is the short description of electrical stimulation?
- Electrode in brain delivers electrical current, stimulates nearby neurons – observe behavioural changes
What is the purpose of electrical stimulation?
- Causation
What are the pros of electrical stimulation?
- High temporal precision
What are the cons of electrical stimulation?
- Can’t tell what you’re stimulating
What is the short description of pharmacological manipulation?
- Drug delivered into brain region through guide cannula – alter cell activity in region
What is the purpose of pharmacological manipulation?
- Causation
What are the pros of pharmacological manipulation?
- Animals can be their own controls
What are the cons of pharmacological manipulation?
- Drug diffusion makes it not very spatially specific
What is the short description of optogenetics?
- Light sensitive ion channel expressed in cell population; shining light changes cell activity
What is the purpose of optogenetics?
- Causation
What are the pros of optogenetics?
- Temporal specificity; Can target specific cell populations
What are the cons of optogenetics?
- Nonphysiological
What is the short description of calcium imaging?
- Fluorescent molecule binds to calcium; can measure fluorescence as proxy of neural activity
What is the purpose of calcium imaging?
- Correlation
What are the pros of calcium imaging?
- Temporal specificity; Can target specific cell populations
What are the cons of calcium imaging?
- Can be difficult to handle so much data
What is the short description of excitotoxic lesions?
- Ionotropic glutamate receptor agonist causes excitotoxicity, killing cells in region
What is the purpose of excitotoxic lesions?
- Causation
What are the pros of excitotoxic lesions?
- Only affects cell bodies in region
What are the cons of excitotoxic lesions?
- Need an additional sham lesion group for control
What is the short description of radio lesions?
- Radio wave delivered through metal wire burns targeted brain area
What is the purpose of radio lesions?
- Causation
What are the pros of radio lesions?
- The neurons are dead for SURE
What are the cons of radio lesions?
- Destroys passing axons
What is the short description of microdialysis?
- Sample extracellular fluid through a dialysis membrane – quantify molecule concentration during behaviour
What is the purpose of microdialysis?
- Correlation
What are the pros of microdialysis?
- ID molecules present during behaviour
What are the cons of microdialysis?
- Poor temporal resolution (10+ mins)
What is the short description of immunohistochemistry?
- Localize proteins in brain tissue – fluorescent antibodies washed onto brain slice bind to protein of interest
What is the purpose of immunohistochemistry?
- Structure
What are the pros of immunohistochemistry?
- Can localize any protein you have the antibody for
What are the cons of immunohistochemistry?
- Cannot localize small molecules (neurotransmitters)
What is the short description of cFos immunohistochemistry?
- Recently active cells make cFos
- Localize cFos in brain tissue as a correlate of recent activity
What is the purpose of cFos immunohistochemistry?
- Correlation
What are the pros of cFos immunohistochemistry?
- ID regions active during behaviour
What are the cons of cFos immunohistochemistry?
- Poor temporal resolution (post mortem)
