Techniques Flashcards
Golgi stain
First technique to visualize neurons
Fixing tissue in potassium dichromate then silver nitrate leads to black precipitate forming on stoma and processes
Labels less than 1% of neurons
Nissl stain
Positively charged dye binds to negatively charged molecules in cell
Labels RNA in the ER
Labels most cells
Good for visualizing cell boundaries and densities, identifying regions
DAPI stain
Fluorescent dye that binds to DNA
Labels the nuclei of neurons and glial cells
Good for visualizing cell density and brain boundaries
Immunohistochemistry
Targeting proteins with antibody staining
Can use indirect or fluorescence to visualize
Good for visualizing specific cell types (via specific proteins)
In situ hybridization
Binding antisense probe to mRNA sequences
Can use enzymatic or fluorescent detection
Good for visualizing specific cell types (via gene expression)
Anterograde tracing
Labeling axonal projections from the cell
Tracer is injected into cell body -> transported along axon
Tracers include Lectin peptides and WGA
Good for demonstrating brain connections
Retrograde tracing
Identifying input neuron cell bodies for a specific area
Tracer is injected into synaptic cleft -> take up by axons and sent to cell body
Tracers include CTB, Fluorogold, and Retrobeads
Good for demonstrating brain connections
Lectin peptides
Anterograde tracer
Bind to sugars and label glycoproteins
Ex. PHA-L
Choleratoxin B
Retrograde tracer
Peptide
Can be identified via fluorescent tagging or antibody detection
Fluorogold
Retrograde tracer
Synthetic molecule, green fluorescence
Wheat Germ Agglutinin
Anterograde tracer
Can cross synapses and label the post-synaptic cell
Retrobeads
Retrograde tracer
Plastic, fluorescent beads that enter synapses via reuptake machinery
Common reporters include…
Green Fluorescent Protein: absorbs blue and UV light -> emits green light
LacZ: colorimetric enzyme
Cre-LoxP system
Cre: recombinase enzyme
Cre responds to LoxP sequences in DNA
Used to delete genes and allow reporter expression
Cre-ER system
Cre can be controlled via drug (tamoxifen)
Cre binds to ER (estrogen receptor)
ER is a transmembrane protein -> exports Cre out of the nucleus and into the cytoplasm (inactive)
Tamoxifen can be administered to bind ER, causing it to release Cre
Cre will return to the nucleus (active)
Anterograde viral tracers
- Adeno-associated virus (AAV): most common; cannot replicate; delivered via vector
Retrograde viral tracers
- Rabies virus: monosynaptic (can only jump one synapse); allows visualization of one neuron connection
- Retro-AAV
MRI
Signaling from hydrogen nuclei varies in strength based on the surroundings (essentially detecting water content)
Differentiating between gray matter, white matter, and CSF
Diffusion tensor MRI
Using the diffusion of water molecules to generate contrast
Measuring macroscopic axonal organization and connectivity of the human brain
Electrode imaging
Tetrodes can detect several neurons at a time (via APs)
Impacted by the electrode proximity
Good for measuring neuronal activity
Calcium imaging
Ca flow signals APs in neurons
Coupling fluorescent sensors to Ca activity (GCamP is inserted via vector -> Ca sensitive fluorescent protein)
Good for measuring neuronal activity
Limitations of calcium imaging include…
Indirect measurement
Only done on the exterior of the brain
NOTE: microendoscopy via GRIN lenses allows for deep imaging at single cell resolution
Fiber photometry
Detecting average signal for a population of neurons (low resolution)
Can be done in freely moving animals
Good for measuring neuronal activity
fMRI
Measuring human brain activity via blood flow
Limitations of fMRI
- Delayed response
- Low resolution
- Signals are noisy and small
Silencing approaches include…
- Lesions: chemical, surgical, electrical
- Drugs: muscimol (GABAa agonist), Glutamate antagonist
Activation approaches include…
Electrical stimulation
Optogenetics
Use of light to activate and de-activate neurons via light-sensitive ion channels and pumps
ChannelRhodosin (ChR2): activating neuron with light exposure
HaloRhodopsin (eNpHR): silencing neuron with light exposure via K+ pump
Chemogenetics
Using drugs and synthetic channels to manipulate neurons
DREADDS: designer receptors exclusively activated by designer drugs; delivered via vectors
hM3D9: activating neuron via depolarization upon binding
hM4Di: silencing neuron via hyperpolarization upon binding
Highly specific method of manipulation
