modern methods 1+2 Flashcards
lesion experiments
where whole parts of the brain are removed, and compare behavioural output in control animals and animals with lesions
2 types of lesions
directed - done on model animals
naturally occurring - after strokes and other neurodegenerative diseases
whole brain imaging techniques
fMRI, non invasive used in visual processing studies
methodological approach to study a new neuronal type
first - cluster neutrons into different types by describing their morphology
second - map connections of neurons
describe activity
what does a sharp electrode recording measure
measures the electrophysiology of a cell
what is a sharp electrode
an electrode with a very thin tip filled with electrolyte
inject the current into the cell and record the changes In membrane potential and can stimulate and calculate the number of spikes
how do you know if the electrode is not inside the neuron
will measure a potential of 0mv
disadvantages of sharp electrode recordings
no solution change inside or outside of the cell
limited possibility for controlling the cell membrane
cannot measure single channels - as measure the potentials generated by the entire membrane
what is the patch clamp method
electrode has a larger tip diameter. use glass electrode filled with electrolyte similar to intra(KCL) or extra(NaCl) solution.
touch the cell and apply negative pressure. part of the membrane will form a tight junction with the electrode and will be able to record the currents of individual channels
what is an inside out patch clamp ?
pull part of the membrane
what is a whole cell configuration patch clamp ?
brake the membrane and record currents of the whole cell
what is an outside out configuration
when in a whole cell configuration, pull the membrane giving and outside-out configuration
what is the difference between inside out and outside out configurations?
inside out - can change the intracellular solution and see the cytoplasmic domain of the specified channel
outside out - can change the extracellular solution, and see how this affects outer part of the channel
how can we measure morphology and physiology in one experiment
making a whole cell and add fluorescent dye in the pipette solution
why will the dye only label one neuron
as not soluble in water so can’t spread unless there are gap junctions
problems with dye and labelling
cannot label many cells
limited ability to label specific cell type
limited ability to label cellular compartments
limited ability for live labelling
what can be used instead of dye
Green fluorescent protein (GFP)
what is GFP
fluorescent protein
stimulated by blue light
emits green light
wavelength of blue light
450-495 nm
wavelength
495-570nm
what do you need to image GFP
a fluorescent microscope
what are the important modules of a simple fluorescent microscope
objective lens and tube lens
eye/camera at the top
form of excitation light (blue)
excitation filter - rejects anything that isn’t blue light
emission filter - so for GFP rejects everything not green
dichroic mirror - passes light with a certain wavelength and rejects light with another wavelength
how can GFP be used to understand the function o a neuron
live imaging
genetically encoded calcium indicators - proteins based on GFP, stabilised in a dim conformation
two different proteins - calmodulin and M13
functions of calmodulin and M13 in GFP imaging
interact with each other in presence of calcium causing entire molecular to be stablished in a bright conformation
can observe activity of calcium - neuron active = calcium levels rise and neuron becomes brighter
what is wide field microscopy
collects light from below and above the focal plane
what is confocal microscopy
rejects light not coming from focal plane and decreases the area of excitation
what is infa-red light used for
can go deeper into the tissue than blue light and allows to increase amount of tissue observed
problems of high resolution microscopy on animals
animal sedated using Na+ channel blockers which decrease excitability of many neurons
animal is stressed
animal does not perform normal behaviour
solutions of problems in high resolution microscopy
virtual reality
freely moving animal
what is channelrhodopsin
non selective channel stimulated by blue light, depolarises the neuron
what is halorhodopsin
chloride channel activated when stimulated with yellow light
leads to hyper polarisation of the membrane
benefits of confocal microscopy
can control depth of field, elimination or reduction of background information away from the focal plane
