Neuroscience Techniques Flashcards
What are the types of preparations of the neural system we can do?
Single cell - Isolated neurone preparations (no synaptic connections)
We can look at resting/action potential in a single cell, we can see types of ion channels that are used
Brain or spinal cord slices - neurons in vitro with local synaptic connections intact
Here we need to bathe it in artificial cerebral spinal fluid to keep them alive in the recordings we need to make
Larger in vitro preparations – the brainstem spinal cord preparation and longer connections are maintained here
We can stimulate the brain stem and be able to record information from a neurone much further down
How do we practically measure activity in humans?
Measuring activity of single neurones - can be done around the knee or arm for example
Microneurography: can look at problems with conduction velocity or unusual levels of activity
Electroencephalography (EEG): Electrodes are placed on the scalp, we are measuring the input of thousands of neurones
Uses: we can record activity and then drive an action by using your EEG activity
What techniques can be used to measure neural activity?
Intracellular
Extracellular
Patch clamp
Two-electrode voltage clamp
What is the intracellular technique?
Sharp electrode (30-150 M Ohms), placed inside the membrane and voltage difference is measured to the outside of the cell
Used on single cells, brain slices and in vivo
Impaling the neurone can be challenging
Useful for measuring resting membrane potential, action potentials, firing rate (in bursts) and synaptic potentials
What is the use of dye in these techniques?
We can fill the electrode with dye, which will diffuse throughout the cytoplasm of the neurone
The brain slice can be processed and the dye can be visualised using light microscopy
The neurones can be drawn allowing for correlation of electrophysiological and neuroanatomical properties
What is the extracellular technique?
Uses a low resistance electrode recording voltage from outside the cell; with small, inverted signals
Used on brain slices, in vivo and intact nerves
Useful for recording action potential firing rates without impaling the neurone, it measures field potentials (synaptic potentials) from large population of neurones
The size of the action potential recorded will be weaker and the graph will go down (instead of up in depolarisation)
What is the patch clamp technique?
Uses a glass low resistance patch electrode that is placed against the membrane of a neuron, and gentle suction is applied.
A very high resistance ‘seal’ is formed between the glass and the membrane (‘gigaohm seal’)
Can produce microscopic currents (voltage and current can be measured)
Used on single cells and brain slices but difficult to learn
Useful to learn more about the properties of neurones in disease states compared to normal
What is the two-electrode voltage clamp?
Measuring conductance through voltage gated ion channels whilst controlling (clamping) the voltage across the membrane
The recording electrode measures the membrane potential (Vm) and is connected to the voltage clamp amplifier
When the Vm is different from the desired potential, the voltage clamp amplifier injects current into the axon through the second (current-passing) electrode
The current passed through the axon, and thus across the membrane, is recorded
This is used on single cells and is relatively easy
What chemicals can block channels?
Tetrodotoxin blocks Na+ channels
Tetraethyl-ammonium blocks K+ channels
What is an oocyte and why are they used?
Ocytes - a cell in an ovary which may undergo meiotic division to form an ovum
They can be injected with mRNA, synthesising foreign ion channel proteins
The properties of these ion channels in question can be studied in isolation
What are optogenetics?
Switching neurones on/off
E.g. we can control neurones with light
How do we prepare brain tissue for light microscopy?
Tissue needs to be sliced thin: in order to resolve individual cells
The tissue is fixed using formaldehyde
This fixes proteins, prevents autolysis and decomposition
It is then sectioned using a microtome (cut into 10) - 200 µm thick
Stain is then used selectively
What are the 4 staining methods?
Nissl – used to detect neurones and glia in the brain
Golgi – used to detect neurones in the brain
Nauta silver stain – used to detect degenerating axons
DAPI – used to detect DNA in living and fixed neurones
What is Nissl staining?
Stains the nuclei and clumps of material (rough endoplasmic reticulum)
Useful for: selectively labelling neurones and gives an indication of the arrangement of cells (cytoarchitecture) of neurones
We can see how many are packed into an area
However, it only labels around the nucleus (no neurites - dendrites)
What is Golgi staining?
Stains the cell body and the neurites of neurones
Golgi suggested neurites of different cells were fused together to form a continuous reticulum (he was wrong)
However, it only stains a portion of neurones in the tissue slice (we don’t know why)
What theroy was correct about neurones?
Cajal’s neurone doctrine - neurones were like other cells in the body and were the elementary unit of the brain
What is the progression of the electron microscope?
The limit of the light microscope is around 0.1 μm
However, the synapse is around 0.02 μm = not possible to tell if the neurones are connected or not
The electron microscope has a resolution of around 0.1 nm
This revealed the existence of synapses and the ultrastructure of neurones (e.g. mitochondria etc)
How does the transmission electron microscope work?
A stream of electrons is accelerated toward the specimen using a positive electrical potential in a vacuum
The electron stream is confined and focused using metal apertures and magnetic lenses into a thin, focused, monochromatic beam
The interactions of electrons with the tissue are detected and transformed into an image
What is immunohistochemistry?
In order to see proteins inside neurones we manufacture very specific antibodies to a protein (e.g. Ca2+ binding protein, neurotransmitter etc.) and seeing these antibodies in sections of the brain tissue
We chemically tag antibodies with a visible marker (to amplify the signal)
Sometimes this requires a second antibody attached to it
It allows discrete labelling of cells that contain the candidate protein, and is useful for localising cells using a particular neurotransmitter
What technique can be used in immunohistochemistry?
Fluorescence techniques
We can combine fluorescent tags, which allows staining of multiple proteins in one section
A UV lamp is needed to visualise the fluorescence
Describe the excitation/emission of dyes?
They get excited at one wavelength and emits at another
‘Red’ dyes emits at around 570 nm
‘Green’ dyes emit at round 515 nm
Example: From the ‘brainbow’ retinal ganglion cells - dendrites all point in one direction = very clear function
How can immunohistochemistry be used in disease?
We can see which motoneurones are surviving
What is axoplasmic transport?
Movement of material down the axon
Neurones use microtubules to transport substances from the cell body to the axon terminal
There are no ribosomes in axon terminal so no protein synthesis e.g. peptide neurotransmitters
Fast axoplasmic transport (1000 mm per day)
Slow axoplasmic transport (1-10 mm per day)
What are the types of axoplasmic transport?
Anterograde transport - movement of substances from the soma to the axon terminals
Retrograde transport - movement of substance from the axon terminals to the soma
What can we use axoplasmic transport for?
Horseradish peroxide (HRP), can be injected into a living brain to be taken up by axon terminals to be retrogradely transported to the cell body Staining technique will then be used to visualise the HRP in slices of the brain
What can be used when we want to trace back through more than one synapse?
Instead of retrograde labelling use:
Transneuronal labelling
We can look at selective pathways all the way from the start i.e. All the way back up to the brain, to see which circuit is controlling a group of neurones
What is double labelling using transneuronal tracers?
Inject one tracer into one organ and another of a different colour into a different area
Examine tissue for single or double labelled neurones
We can see if there are neurones controlled by multiple regions or are some masking others
How can we see axonal trajectories?
We can use MRI and computer manipulation
These are stored in a Biobank