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Flashcards in lecture 22 Deck (10):
1

What information about the brain should be collected and interrelated?

- 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)

2

What do the tissues of the nervous system look like?

- 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

3

What techniques are used to view neurons?

- 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)

4

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

- 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

5

Who is Ernst Abbe?

- 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

6

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

- 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

7

What are new technologies in microscopy?

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

8

What is the problem with light with light microscopes?

- 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

9

What are the super resolution methods?

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

10

What is temporal resolution?

- 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