lecture 2/3 - modern methods Flashcards
briefly describe the patch clamp technique
part of a membrane is sucked into a pipette
membrane voltage is measured
describe the 4 types of patch clamp
cell-attached = single channel activity, study ion function without disrupting the cell
whole cell = total cell currents, study whole cell activity(eg. APs)
inside out = intracellular regulation to study secondary messengers, you can change the solution outside the cell
outside in = extracellular ligand effects, to test drug interactions, you can change the solution outside the cell
describe the sharp electrode technique
sharp glass micropipette poked through membrane
current injected and APs measured
what are the key differences between sharp electrode and patch clamp?
SE- pierces membrane
measures Vm
cant measure single ion channels
more invasive
measure APs
PC- forms seal
measures ion currents
measures single channels and whole cell
less invasive
measures ion channel pharmacology
what is a lesion?
a damage or change in tissue
can be induced (animal models) or from injury
what is fMRI used for?
measuring brain activity by detecting changes in blood flow
what are the advantages of fMRI?
non invasive
hight spacial resolution
what is GFP stimulated by?
blue light
what 2 things can GFPs tell us about a neuron?
morphology (shape)
function
how can GFPs be used to understand morphology of neurons?
they can be genetically expressed in neurons, allows for in vivo tracking
then can use a fluorescent confocal microscope to detect these certain GFPs
How does a fluorescent confocal microscope detect GFPs
it reflects certain wavelengths and passes others which allows for GFPs of different wavelengths to be detected
what are the benefits of a confocal microscope
high resolution
enhanced depth of field
reduced background noise
how can GFPs be used to understand the function of neurons?
GFP can be fused to 2 Ca2+ binding proteins
when Ca2+ is present , the 2 proteins interact and the GFP becomes much brighter
when a neuron is active, Ca2+ levels are higher, therefore brighter GFP
what are some issues involved with trapping mice and using GFPs to test neuron function
sedation and stress will influence their neuron activity
how are the trapping issues with mice solved when using GFP to track neuron function
virtual reality
freely moving mice with microscope added in skull
what are the advantages and disadvantages of using GFP to track neuron function
+ = measure large numbers of neurons at the same time
- = cant measure individual action potentials
what is the difference between confocal and widefield microscopy
confocal- Uses a laser and pinhole to focus light on a specific optical plane, rejecting out-of-focus light.
Produces sharper, high-resolution images, especially for thick or 3D samples.
Ideal for detailed structural imaging and z-stack (3D reconstruction).
Widefield - Illuminates the entire sample at once.
Captures both in-focus and out-of-focus light, leading to potential image blur.
Best for thin samples or quick imaging.
what is channel rhodopsin and what happens when its stimulated?
It is a light-gated ion channel that, when activated by blue light, allows cation influx (Na⁺, K⁺, Ca²⁺), leading to neuronal depolarization and activation.
what are the main uses for channelrhodopsin?
can activate/block the channel to stimulate/block neuronal behaviour
what is halorhodopsin?
a light-activated chloride pump used to inhibit neuronal activity. When exposed to yellow light, it transports Cl⁻ ions into the cell, causing hyperpolarization and preventing the neuron from firing.
what is the relationship between channelrhodopsin and halorhodopsin?
they counteract eachother
how are channelrhodopsin and halorhodopsin used together?
to study the function of individual neurons
Give the process of understanding morphology of a neuron
1) define the region of interest, choose between individual neuron or subset
2) label with GFP
3) prepare tissue, either live or fixed
4)use confocal microscope for 3D reconstruction
