Neurons and Circuits

Question 1

What are the different types of neurons?

Answer 1

1) Unipolar: cell, then dendrites, then axon
i. e. invertebrate neuron
2) Bipolar: dendrites and axon on either side of cell body
i. e. retinal cell
3) Pseudo unipolar cell: cell body on the axon, dendrites on one side and axon continues to other
i. e. ganglion cell of dorsal root
4) Multipolar cell
- dendrites on cell body, one axon extending from cell body, different types
i. e. spinal chord motor neurons, pyramidal of hippocampus, purkinje cell of cerebellum

Question 2

What is the main function of a neuron?

Answer 2

Inputs, Integrative, Conductive, Output

Question 3

What is a neuroenodrine cell?

Answer 3

Contacts a capillary that releases a peptide/hormone into the bloodstream

Question 4

What are the methods of visusalization we talked about in class?

Answer 4

Golgi
Dye Filling
Immunohistochemistry
Genetically Encoded FPs
Electron Microscopy
Brainbow
GRASP
Rabies Virus

Question 5

What is the Golgi Stain?

- Benefits/Deficits

Answer 5

Ramon y Cahal 
Silver nitrate impregnation
Small subset of neurons stained: unknown why which ones
Dead Tissue 
Easy technique 

PROS: easy, good general visualization
CONS: only get a random subset, not super specific

Question 6

What happens to the dendritic spines in life? How about those of schizophrenic patients?

Answer 6

The prefrontal cortex develops over the first few decades and synapes are ‘pruned’, you are given more than you need to start with.
Schizophrenic patients have less dendritic spines in prefrontal cortex, they have OVERPRUNED synapses

Question 7

What is Dye Filling?

Answer 7

Glass pipette with electrode, pierce membrane and fill die (30 minutes diffuse)
Electrode measures electrophysiological activity.
Live or Dead tissue

PROS: select exact cells and isolate. See functionality and morphology
CONS: Difficult, time consuming

Question 8

What is autism associated with?

Answer 8

Fewer dendritic spines

Question 9

What is Immunohistochemistry?

Answer 9

Targets/identifies any specific protein that has a specific type of cell/function IF you have the antibody
Antibodies can be combined to examine multiple cell types in the same tissue
Dead Tissue: detergent makes holes in membranes
Cheap & Easy

Identifying Specific Molecules, exploiting specificity of protiens

You can generate an antibody against ANY protein

1) Make an antibody
2) Attach a fluorescent tag that emits light at a wavelength
3) See where that protein is in the cell

All cells have the same DNA, they just express only certain genes

Question 10

what is doublecortin?

Answer 10

a tag for immature cells: used to study newborn cells, where they put they axons/dendrites to make connections
-cells only express this for the 1st part of their lives

Question 11

what is Calretinin?

Answer 11

calcium binding protein used to tag a subset of inhibitory neurons askbecca

Question 12

What is Genetically encoded FP?

Answer 12

BASICALLY
certain promotors are only active in specific cell types
- by articially expressing genes under the control of a cell specific promor, you can get cell specific gene expression
- can be exploited to visualize specific stuff

By manipulating the genome, you can exploit the activity of that promotor and make it express things.

i. e. make it express a fluorescent protein downstream where it would have expressed the other protein, and will make neuron light up **
* **

Question 13

what is the most common genetically encoded FP? How is it excited?

Answer 13

GFP!
isolated from jellyfish, now you can get it in any colour not just green
Excitation spectra peak at 490nm, shines light at 520nm

excited by blue and emits green light
(longer wavelengths are emitted back to you)

Question 14

what is RFP?

Answer 14

a different FP, can be used with GFP because they don’t have the same wavelengths of exitation

Question 15

How does genetically encoded fluorescent proteins WORK? pros/cons

Answer 15

• transgenic animal lines or virus conduction
• costly to set up, but can be cheap and efficent (breed transgenic animals), and viruses are cheap
• can be used to visualize cells in living tissue

Pro:you can label your genetically modified cell! if you exrepss it along with other genes, it acts as a tag “reporter” to tell you which have been modified

con: gene expression is variable, you may use a given promotor but it just isn’t the same

Question 16

what is the CAG promotor? why is this useful?

Answer 16

active in all cells (expresses GFP) in all cells

• synthetic promotor
• useful for tumor/transplant cells when you want everything, not as good for imaging neurons because it stains everything.

Cytomegalovirus, Actin, Globulin

Question 17

What is the Thy1 promotor?

Answer 17

active (expressed) in a fraction of all types of neurons (golgi like labelling

• good for imaging neurons, stains a SPECIFIC subset of cells
• gives a wide perspective
• labels tons of cell types so you can look at lots of different regions

Question 18

what is the L7 promotor?

Answer 18

active in Cerebelllar purkinje neurons and nowhere else!

Question 19

why is picking your promotor so important?

Answer 19

you can look at different cells in the same brain region depending on the promotor you use!

Question 20

How do you express FPs in neurons with viruses?

Answer 20

injected into specific brain region

• different virus for different cell types (dividing/non dividing etc.)
• much more common to use virus to insert into cell of interest
• requires surgery
• good if you care about one regions, but bad if you want a big picture because virus only spreads so far

need

1) the right virus
2) the right promotor

Question 21

what is a disadvantage of traditional genetically encoded FP expression?

Answer 21

brain tissue has to be cut to viusalize neurons so that light doesn’t scatter and it can penetrate)
- this means you must cut into sections then do immunohistochemistry!

Question 22

what is CLARITY? Why is it needed?

Answer 22

clearing/ridding brain of fats enabling the imaging of FPs in large blocks of tissue
“brain clearing method”
Everything is bound together in a polymerizing step, and then the membranes are washed away the polymer holds it all together: no need to cut the brain!

• brain is filled with mostly lipids and fats, and the inside is mostly aqueous: microscopic lenses going though liquids scatter/diffracts, but after treatment with clarity, its almost only aqueous stuff left!
• allows light and antibodies to penetrate
• allows tracking axons as they travel long distances and characterize networks

Question 23

How do you visualize FPs with confocal microscopy?

What is the benefit to this?

Answer 23

Confocal: pinhole that eliminates things that are out of focus, allowing you to visualize a single focal plane
Benefit: you don’t have things that are blurring your vision. In a normal microscope, you have light being reflected from blow and above plane of focus (its out of focus but its still visible and can distract you)

Question 24

How can you visualize GFP neurons in vivo?

What can you see when doing thing?

Answer 24

Using 2 photon microscopes using laser (that can go through very think skull deep into tissue
- shows appearing/disappearing spines

Clarity is good for only DEAD tissue

Question 25

What is electron microscopy?

Answer 25

combines immunohistochemistry (sometimes) with microscopy 
Its converted into a precipitate signal inside the cell (electron dense at the site of the antibody) 
-can show you the structure and whatnot of neurons on its own too

Tissue imbedded in resin, cut into TINY sections, imaged on an EM

PROS: best resolution we have today, shows detailed synapse to infer function

CONS: requires microscope and personnel, time consuming (12+ years to map C elegans nervous system)

Question 26

What is a circuit connnectivity and what is Brainbow?

Answer 26

Circuit connectivity
-identifying these circuits is essential to understand how they work and how they can be manipulated.
Many methods lable too few/many/nonspecific neurons and can’t map a connectome (Except EM but it takes Years)

BRAINBOW: Multiple coloured FPs which randomly combine and create colors in different neurons
- goal is to trace dendrites and axons of all or many to see how they connect/form circuits

good in theory but not in practice

Question 27

what is the connectome?

Answer 27

wiring/synaptic connectivity of all neurons.

If you know the connectome for a circuit you may be able to infer its function

Question 28

What is GRASP?

Answer 28

Identify synaptically connected neurons
(GFP reconstitution across synaptic partners)
- GFP is taken apart, fragmented and it does NOT fluoresce. You target it half in presynaptic cell and half in the post synapic cells. Where they MEET GFP will reconstitute and fluoresce again!

Also use a fluorescent tagger on each to know if they’re in the right place (same promotor drives fragment and ‘reporter’

post synaptic cell: dTomato
pre synaptic cell: mCerulean

Question 29

How do you map circuit connectivity with the rabies virus?

Answer 29

Rabies infects neurons and travels RETROGRADE to infect upstream/presynaptic neuron

Manipulating this turns it into a useable tool

Rabies virus trav
Pros: can show connectivity
cons: can only travel one neuron at a time retrograde

Question 30

which is the one true way to view functional synapses?

Answer 30

to stimulate one neuron and watch for connection in the other!

Question 31

how would you infer direction of flow in genetically encoded FP?

Answer 31

loot at the anatomy! where the dendrites are in comparison to axons

Question 32

what is the standard number of antibodies for immunohistochemistry?

Answer 32

3 antibodies! limitation!!!