Methods in Modern Neurosciences Flashcards
Tell me about Sharp Electrode Recordings
Records the activity of a small group or population of neurons in living animals & humans.
Uses a thin glass or metal electrode to puncture the cell membrane.
Can study activity of specific neurons while the subject is performing a task, also used to study neuronal activity during neurological diseases such as epilepsy.
Juu Sola lab used it to investigate the effect of temperature on the activity of neurons.
What are the disadvantages of Sharp Electrode Recording
Can’t change the solution inside or outside the cell. Limited Possibility for controlling the membrane potential. Can’t measure the activity of a single ion channel/receptor.
Tell me about whole-cell patch clamping
Pipette is pressed against the membrane, suction creates a gigaseal (>gigaohm of resistance). Further suction ruptures the membrane providing a direct electrical connection between the intracellular space and the electrode in the pipette.
Allows recording of voltage and ligand-gated ion channels.
Tell me about outside-out patch clamping.
A small piece of membrane is pulled away from the cell via suction. Allows the study of ion channels/receptors on the outer surface
Tell me about inside-out patch clamping.
A small piece of membrane is pulled away from the cell but remains attached to it. Allows the study of ion channels/receptors on the inner surface.
Tell me about on-cell patch clamping.
A loose seal is maintained which allows the recording of electrical signals without disrupting physiological functions. This allows you to change the conditions and measure the response in electrical activity.
Tell me about lucifer yellow.
Lucifer Yellow is a dye that can be introduced into neurons through patch clamping. It will diffuse across the entire neuron (30mins-2hours) allowing the study of morphology alongside electrophysiology.
What are some disadvantages of using lucifer yellow through patch clamping
You can’t label to many cells. Limited ability to stain specific cell types (takes a long time, there are lots of neurons). Limited ability to stain live cells (neurons tend to die quite quickly). Can’t label cellular components .
What are the key differences between patch clamping and sharp electrode recording?
SER doesn’t allow precise control over the extracellular conditions. SER isn’t as detailed as patch clamping. SER is for populations of neurons while patch clamping can be used for individual cells or small clusters of cells.
Tell me about Green Fluorescent Protein
Beta-Barrel with an active centre. Stimulated by blue light to release green light.
Tell me about simple fluorescent microscopes.
Uses emission and excitation filters specific to the wavelengths passing through them. Dichroic mirror ensures only the wavelengths of light targeted form the image. Often used to view neurons GFP has been expressed in.
Tell me about GCaMP
It’s a GFP-based calcium indicator. Involves M13 and Calmodulin (CaM) fused to GFP which is stable in a dim conformation. When calcium is present (eg during neuronal activity) M13 and CaM interact changing the GFP conformation to bright.
What are the benefits to using a confocal microscope?
Contains a pinhole gap between the lenses ensuring only light from the focal plane is collected. Allows for much greater spatial resolution.
What issues are faced in measuring neuronal activity in live animals?
Animals are often sedated using Na+ channel blockers, this affects neuronal activity. Animals are stressed which releases neuron modulators. Animals don’t perform behaviours they normally would.
How can live animals be used to provide more accurate data.
Use a VR environment to simulate what would happen in the wild. Use free moving animals (can require complicated setups for studying)
