lecture 22

Question 1

What information about the brain should be collected and interrelated?

Answer 1

• morphology (shape, projections, branching patterns, soma shape)
• location (clusters, nuclei, layers, tissue)
• connectivity (inputs and outputs: size, location, type)
• output neurochemistry (primary and secondary neurotransmitters)
• input neurochemistry (receptors subtypes, 2nd messenger systems/interactions)
• electrical behaviour (distribution of channels and pumps)
• homologies in other brains
• ontogeny (history of gene expression, migration, environmental interaction)
• functional data (effects of lesions, results of modelling, experiments)

Question 2

What do the tissues of the nervous system look like?

Answer 2

• different types of neurons
• e.g. in cerebral cortex there are clearly two types of cells: pyramidal cells and stellate cells
• varieties of each
• mapped out presence of pyramidal/stellate cells in different regions of the cortex
• subtle differences in types and distributions of variations of cells in the cerebral cortex
• Brodman’s cortical map

Question 3

What techniques are used to view neurons?

Answer 3

• eye: can resolve strucutres around about to the thickness of a hair, 100µm
• light microscope: 1mm to ~100nm
• electron microscope: ~50µm to ~10^-10m (atom)

Question 4

How do we resolve images of nervous tissue through a microscope?

Answer 4

• getting a fairly removed thing that we are looking at compared to what we started with
• convert brain tissue to a form that you can look at –> usually involves killing the tissue
• fix tissue sample as a covelently crossed linked molecule, make stable
• make tissue have a refractive index as much like glass as possible, use various clearing compounds
• cut thinly
• clearing compounds often allow it to be infiltrated with wax or plastic to allow it to be
• so what you have now is not so much the brain you started with, but a crosslinked protein embedded in plastic or wax, thinly sliced
• to get the image you shine light through it
• light is scattered by the specimen
• and interference of that light makes in image
• need to collect as much light as possible
• hence microscopes tend to have large objective lenses that are incredibly close to the glass slide

Question 5

Who is Ernst Abbe?

Answer 5

• over 100 years ago
• worked theory bhind microscopes
• formula for spatial resolution
• governed by wavelength of light you use / refractive index of the material and certain angle sin theta (how much of light you cancollect)
• there is a limit to what you can resolve with a light microscope
• therefore can’t use a light microscope to look at e.g. respiratory centres on the inner surface of mitochondrial membrane

Question 6

What do we use to look with finer resolution than the light microscope?

Answer 6

• e.g. electron microscope
• piece of plastic that has heavy metal deposits based on where it bound to the protein that was crosslinked by the fixative
• sort of looking at a strange shadow
• beautiful, high resolution image
• still looking at a dead thing: snapshot
• how do we look at something in a temporal sense?
• e.g. synaptic contacts (red fluorescent molecule) on a neuron whose cytoskeletal proteins are stained green (GFP)
• therefore can look at number, size etc of neurons, shape of neuron
• excite a molecule it gives of visible light
• great to have fluorescent molecules, but can insert a protein as a gene (GFP), transgenic animals, can control the conditions under which it is expressed
• very useful biological tool

Question 7

What are new technologies in microscopy?

Answer 7

• even higher resolution
• can zoom into the cell without harming it
• can therefore look at the natural function of the cell in real time

Question 8

What is the problem with light with light microscopes?

Answer 8

• can focus light up to a point
• refraction
• the lens defracts the light, so you can’t focus the beam to anything smaller than the wavelength
• so if you’re hoping to look at something that is less than the wavelength of light, you don’t have adequate resolution
• 300-400nm vs 3nm of a molecule
• impossible to look at individual molecules

Question 9

What are the super resolution methods?

Answer 9

STED

• STimulated Emission Depletion
• use another beam of light to blah blah
• end up with a much smaller spot of fluorescense
• more detail

PALM
- Photo-Activated Localisation Microscopy

STORM
- Stochastic Optical Reconstruction Microscopy

• both taking advantage of the fact that the path of the molecule is switchable
• switch off the spot
• determine the centre
• this is the location of the molecule
• trick is you switch on the molecules separately, in small groups
• but looking at a computer generated map of locations of molecules
• far removed from the tissue

Question 10

What is temporal resolution?

Answer 10

• function
• cell membrane level
• neurons are excitatory cells: membrane
• changes that happen across of neurons occur in miliseconds
• how quickly things change
• record electrical activity with very high temporal resolution - 0.1 miliseconds

patch clamp electrode tip

• glass electrode made into a fine fine point
• so fine that you have to use an electron microscope to see it
• leaves a tiny hole in the end of the tube
• nm wide
• can be applied to the surface of cell membrane
• would cover approximately 1 ion channel
• measuring single channel conductances
• fundamental unit of excitability in the nervous system
• picoAmps