L7 - techniques Flashcards
electrophysiology
measuring electrical activity of biological tissue
potential difference
measured between electrode inside and outside of cell
what causes -ve intracellular charge
high K+ concentration inside cell
K+ leaves cell via K+ leak channels
3 methods of preparation for measuring electrical changes to neurons
1 - dissociated neuron cultures
2 - acute brain slices
3 - whole animal
dissociated neuron cultures method
plate fragments of neurons from embryos and add growth medium so they grow into networks
advantages of dissociated neuron cultures
easily accessible for recording
cells well isolated
easy to carry out
disadvantages of dissociated neuron cultures
only good for embryos
not normal physiological environment for neurons
acute brain slices methods
record from neurons in thin slices of brain tissue
advantages of brain slices
local circuits remain intact
carry out at any developmental stage
easy to identify specific regions
disadvantages of acute brain slices method
long range inputs/outputs get cut
not normal physiological environment
whole animal method
animal under anaesthesia
implant electrodes
recordings made when animal is awake
advantages of whole animal recordings
all circuits remain intact
allows correlation of electrical activity wth behaviour
disadvantages of whole animal recordings
difficult to carry out
2 ways of intracellular recording membrane potential of a neuron
sharp electrode
patch clamp recording
sharp electrode method
electrode made of glass
filled with solution similar to that of the cell
very high resistance - narrow tip
poke a hole in side of cell
allows access to record membrane potential relative to outside the cell
patch clamp recording
more modern/popular method lower resistance pipette - wider tip put on surface of membrane apply negative pressure (suction) bursts membrane under pipette cytoplasm continuous with inside of pipette allows access for recording
equipment required for intracellular recordings
whole set up = rig placed on anti-vibration table microscope manipulators big metal cage
how do you isolate pipette from external vibrations
rig placed on anti-vibration table
what is the microscope for
visualising individual neurons within the brain slice
what are the manipulators for
very delicately place the glass pipette onto the surface of the cell
what is the big metal cage for
surrounds the rig
isolates rig from ambient electrical noise
amplifer
takes small analogue/electrical signal and makes it larger
converted to digital signal before being sent to computer
why is saline solution used
similar to csf
cell attached recording
pipette attached to membrane via small suction
doesnt break through surface
low resistance seal
whole cell recording
more suction/negative pressure applied
membrane bursts underneath pipette
allows electrical access to inside of cell
inside out patch
suction from whole cell recording pulls small bit of membrane from underneath pipette and separates from cell
allows recording of individual ion channels
Ohm’s law
V= I x R voltage = resistance x current
low resistance occurs when
lots of ion channels are open
how do you make current clamp recordings
inject different currents to stimulate the cell to give the resistance of the membrane
why do you apply different drugs in current clamp recordings
drugs alter the ion channels of membranes
causes changes in resistance of membrane
describe the EPSP of a CA1 pyramidal neuron
fast rising
slow decaying
what are voltage clamp techniques for
they fix the voltage so it cant change
allows studying of underlying currents
the amplifier injects -ve current to maintain cell at -80mV…
the cell cant depolarise
opposes +ve ion flow via glutamate receptors
EPSC stands for
excitatory post synaptic current
what is an epsc
the flow of ions that causes an epsp
as +ve ions move into the cell what happens to the extracellular space
becomes more -ve
advantages of measuring extracellular space
does not disrupt intracellular content
can record spikes in vivo
can record many cells at once
what causes the size of the extracellular recording spike to vary
the distance of the recording from the electrodes
electrodes pick up spikes closest to them
tetrode
bundle of 4 electrodes
what does functional imaging measure
neuronal activity
benefits of functional imaging
measures a larger anatomical area that electrodes
gCaMP
calcium indicator protein
fluorescent when bound to Ca2+
limitations of gCaMP
difficult to distinguish fluorescence between 6 small spikes or 1 large Ca2+ signal
