Modern methods in neuroscience 2 Flashcards
What is GCaMP?
A GFP based calcium indicator
They are genetically encoded calcium indicators
What’s the principle behind GCaMP?
- Fusion of GFP and two calcium binding proteins
- In presence of calcium these two proteins interact
- GFP becomes much brighter
What happens to GFP in GCaMP if there’s no Ca?
GFP is dim
Why does the GCaMP work?
When a neuron is active its calcium levels rise and neurons become brighter
What do high resolution microscopes create and state two types
Generate movies
Wide field and confocal
Describe wide field microscopy
Collects light from, below and above the focal plane
Describe confocal microscopy and its benefit
Rejects light coming not from focal plane and decreases the area of excitation
Allows for massive increase in spatial resolution – has a higher resolution
How is a neuron depolarised by light?
guanylyl cyclase produces cGMP which stimulates Na+ channels to open
The channel opens rapidly in response to blue light, and in neurons the inward flow of cations is enough to produce depolarisation beyond threshold for action potentials.
How is a neuron hyperpolarisaed by light?
light - conformational change in retinal - activates opsin - activates transducin - acvitates Phosphodiesterase to break down cGMP
Na+ channels close
What is Halorhodopsin?
light-gated ion pump, specific for chloride ions
Propose an experimental paradigm for understanding the morphology of a particular neuron
Virtual reality
- Mouse is placed in a ball
- Turn of the ball leads to turn of field of view
- Head is fixed
- Objective is placed on top
Free moving mice
- Tiny fluorescent microscope is placed directly in the skull
- Image delivered via light guide
- When a neuron is active its calcium levels rise and neurons become brighter
problems with using high resolution microscopy and GFP to visualise animal brain excitation
Animal is sedated using Na+ channel blockers
or
Animal is stressed
and
Animal does not perform behaviour that it usually does
