Visualization and identification of neurons and networks Flashcards
Two types of visualization (broad classes)?
Static visualization
Dynamic visualization
static visualization
- Good for cell counting
- done on fixed tissue/cells
- can be done on a cell level (using Nissl staining/DAPI), mRNA (ISH), or protein (IHC) level
Dynamic visualization
- Allows for measurement and identification of neuronal responses w/ detailed morphology (can see morphology, signal propagation, and connectivity)
- Provides better understanding of neuronal communication in networks
- Done on living tissue/preparation
First (modern) neuroimaging technique
Labeling cells w/ GFP and imaging w/ fluorescence microscopy
What is DIC microscopy?
Differential interference contrast microscopy
- Allows us to enhance contrast in unstained, transparent tissue
- Separated single polarized light into two polarized, mutually coherent parts
- based on concept of interferometry to gain info about optical path length of sample
Labeling using patch clamp
- it’s possible to inject different substances to “fill” the cell to distinguish between different parts of the neurons and look at what parts are active
- Substance injected depends on type of connectivity of interest (either functional or structural)
- ex: biocytin, fluorescence dyes, calcium dyes
Biocytin
- used to look at functional connectivity
- shows how morphology and firing patterns together identify the cell
- visualization is possible only post-experiment
Fluorescence dyes (w/ E-phys)
- used to look at structural connectivity
- shows morphology and firing pattern by adding colors according to change in fluorescence of the structure
- no temporal resolution, only live morphology
Calcium dye (for visualization)
- Takes advantage of intracellular calcium flux to directly visualize Ca2+ signaling in living neurons
- AMPAR and NMDAR let calcium into spines
- Makes it possible to look at activity in dendritic spines
- The dye is a fluorescence dye that changes its fluorescence w/ increases in [Ca2+]
- Gives us temporal information, but little information about connectivity
Advantages of Ca2+ dye and voltage sensitive dyes?
- Do not require patching of the neuron, because the structures and substrates for the dyes occur naturally in the cell
- Make it possible to study many things both in vivo and in vitro
On what is optogenetics based?
Based on opsins, allowing us to see neuronal morphology, dynamic changes, and connections
What are opsins?
opsins = light-sensitive channels found in algae and bacteria; can be introduced to a neuron and activate it when an activation light is flashed on it
-Sit on membrane and undergoes a conformational change from all-trans to 13-cis when light hits it
Types of opsins
Channelrhodopsin
- Pumps Na+, Ca2+, K+, and H+ into cell (depolarizes)
Halorhodopsin
-Cl- channel (hyperpolarizes)
Bacteriorhodopsin
- Pumps protons out of the cell
Activation/emission spectra of opsins
Channelrhodopsin
- Activate in blue light (325-600 nm w/ peak at 425 nm)
- see in yellow light
Halorhodopsin
- Activate (to silence/inhibit neurons) in orange light (450-725 nm w/ peak at 625 nm)
- See in green
Ways of opsin expression
- Non-specific expression
- Artificial channel induced in virus→infect brain with virus → channel expression in all neurons - Specific expression
- AAV vector recognizes specific gene sites (using Cre-Lox system)→ induced w/ channelrhodopsin within lox sites and introduced to neurons→ binds specific cells that recognize it (cre expressing)
(or, can use a cre line w/floxed line where the cre line is injected w/ AAV containing light activated opsins within lox sites and the floxed line is injected w/ an AAV vector containing cre-recombinase under a specific promoter)
