Neuroscience Techniques

Question 1

What are the types of preparations of the neural system we can do?

Answer 1

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

Question 2

How do we practically measure activity in humans?

Answer 2

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

Question 3

What techniques can be used to measure neural activity?

Answer 3

Intracellular
Extracellular
Patch clamp
Two-electrode voltage clamp

Question 4

What is the intracellular technique?

Answer 4

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

Question 5

What is the use of dye in these techniques?

Answer 5

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

Question 6

What is the extracellular technique?

Answer 6

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)

Question 7

What is the patch clamp technique?

Answer 7

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

Question 8

What is the two-electrode voltage clamp?

Answer 8

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

Question 9

What chemicals can block channels?

Answer 9

Tetrodotoxin blocks Na+ channels

Tetraethyl-ammonium blocks K+ channels

Question 10

What is an oocyte and why are they used?

Answer 10

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

Question 11

What are optogenetics?

Answer 11

Switching neurones on/off

E.g. we can control neurones with light

Question 12

How do we prepare brain tissue for light microscopy?

Answer 12

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

Question 13

What are the 4 staining methods?

Answer 13

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

Question 14

What is Nissl staining?

Answer 14

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)

Question 15

What is Golgi staining?

Answer 15

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)

Question 16

What theroy was correct about neurones?

Answer 16

Cajal’s neurone doctrine - neurones were like other cells in the body and were the elementary unit of the brain

Question 17

What is the progression of the electron microscope?

Answer 17

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)

Question 18

How does the transmission electron microscope work?

Answer 18

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

Question 19

What is immunohistochemistry?

Answer 19

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

Question 20

What technique can be used in immunohistochemistry?

Answer 20

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

Question 21

Describe the excitation/emission of dyes?

Answer 21

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

Question 22

How can immunohistochemistry be used in disease?

Answer 22

We can see which motoneurones are surviving

Question 23

What is axoplasmic transport?

Answer 23

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)

Question 24

What are the types of axoplasmic transport?

Answer 24

Anterograde transport - movement of substances from the soma to the axon terminals

Retrograde transport - movement of substance from the axon terminals to the soma

Question 25

What can we use axoplasmic transport for?

Answer 25

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

Question 26

What can be used when we want to trace back through more than one synapse?

Answer 26

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

Question 27

What is double labelling using transneuronal tracers?

Answer 27

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

Question 28

How can we see axonal trajectories?

Answer 28

We can use MRI and computer manipulation

These are stored in a Biobank