Techniques

Question 1

Golgi stain

Answer 1

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

Question 2

Nissl stain

Answer 2

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

Question 3

DAPI stain

Answer 3

Fluorescent dye that binds to DNA
Labels the nuclei of neurons and glial cells
Good for visualizing cell density and brain boundaries

Question 4

Immunohistochemistry

Answer 4

Targeting proteins with antibody staining
Can use indirect or fluorescence to visualize
Good for visualizing specific cell types (via specific proteins)

Question 5

In situ hybridization

Answer 5

Binding antisense probe to mRNA sequences
Can use enzymatic or fluorescent detection
Good for visualizing specific cell types (via gene expression)

Question 6

Anterograde tracing

Answer 6

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

Question 7

Retrograde tracing

Answer 7

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

Question 8

Lectin peptides

Answer 8

Anterograde tracer
Bind to sugars and label glycoproteins
Ex. PHA-L

Question 9

Choleratoxin B

Answer 9

Retrograde tracer
Peptide
Can be identified via fluorescent tagging or antibody detection

Question 10

Fluorogold

Answer 10

Retrograde tracer
Synthetic molecule, green fluorescence

Question 11

Wheat Germ Agglutinin

Answer 11

Anterograde tracer
Can cross synapses and label the post-synaptic cell

Question 12

Retrobeads

Answer 12

Retrograde tracer
Plastic, fluorescent beads that enter synapses via reuptake machinery

Question 13

Common reporters include…

Answer 13

Green Fluorescent Protein: absorbs blue and UV light -> emits green light
LacZ: colorimetric enzyme

Question 14

Cre-LoxP system

Answer 14

Cre: recombinase enzyme
Cre responds to LoxP sequences in DNA
Used to delete genes and allow reporter expression

Question 15

Cre-ER system

Answer 15

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)

Question 16

Anterograde viral tracers

Answer 16

1. Adeno-associated virus (AAV): most common; cannot replicate; delivered via vector

Question 17

Retrograde viral tracers

Answer 17

1. Rabies virus: monosynaptic (can only jump one synapse); allows visualization of one neuron connection
2. Retro-AAV

Question 18

MRI

Answer 18

Signaling from hydrogen nuclei varies in strength based on the surroundings (essentially detecting water content)
Differentiating between gray matter, white matter, and CSF

Question 19

Diffusion tensor MRI

Answer 19

Using the diffusion of water molecules to generate contrast
Measuring macroscopic axonal organization and connectivity of the human brain

Question 20

Electrode imaging

Answer 20

Tetrodes can detect several neurons at a time (via APs)
Impacted by the electrode proximity
Good for measuring neuronal activity

Question 21

Calcium imaging

Answer 21

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

Question 22

Limitations of calcium imaging include…

Answer 22

Indirect measurement
Only done on the exterior of the brain
NOTE: microendoscopy via GRIN lenses allows for deep imaging at single cell resolution

Question 23

Fiber photometry

Answer 23

Detecting average signal for a population of neurons (low resolution)
Can be done in freely moving animals
Good for measuring neuronal activity

Question 24

fMRI

Answer 24

Measuring human brain activity via blood flow

Question 25

Limitations of fMRI

Answer 25

1. Delayed response
2. Low resolution
3. Signals are noisy and small

Question 26

Silencing approaches include…

Answer 26

1. Lesions: chemical, surgical, electrical
2. Drugs: muscimol (GABAa agonist), Glutamate antagonist

Question 27

Activation approaches include…

Answer 27

Electrical stimulation

Question 28

Optogenetics

Answer 28

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

Question 29

Chemogenetics

Answer 29

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