lecture 22 Flashcards

1
Q

What information about the brain should be collected and interrelated?

A
  • 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)
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2
Q

What do the tissues of the nervous system look like?

A
  • 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
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3
Q

What techniques are used to view neurons?

A
  • 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)
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4
Q

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

A
  • 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
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5
Q

Who is Ernst Abbe?

A
  • 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
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6
Q

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

A
  • 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
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7
Q

What are new technologies in microscopy?

A
  • even higher resolution
  • can zoom into the cell without harming it
  • can therefore look at the natural function of the cell in real time
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8
Q

What is the problem with light with light microscopes?

A
  • 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
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9
Q

What are the super resolution methods?

A

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
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10
Q

What is temporal resolution?

A
  • 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
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