3. Cellular Neuroscience Neurons, their organelles, and glia

What glial cells myelinate the axons in the central nervous system?

oligodendrocytes

What glial cells myelinate the axons in the peripheral nervous system?

schawnn cells

What are the 3 functions of myelin?

1. Provide multilayered, membranous sheath

2. Insulates axon as plastic on electrical wire

3. Allows electrical impulses to travel quickly down axon

Synapses require additional metabolic support by _____ which make close contact with synapses.

Astrocytes

Transmission electron microscopy has electron a resolution limit of ___ A

2.5

What kind of things do electron microscopes observe?

fine details, smaller organs and vesicles

Light microscopy has electron a wavelength of ___ A

350-700 nm

Transmitted light microscopy uses white(___ wavelength) light

mixed

In transmitted light microscopy, contrast derives from interaction of light through specimen: ___.

Diffraction

T/F Contrast of most cells is low without help

True

Collection of diffracted light may be enhanced by __ in microscope

optics

Visible light + cells = _____

change in amplitude or phase

The goal of _____ is to separate out phase-shifted light from unaffected light

phase microscopy

____ uses specific wavelength of light for illumination.

Fluorescence microscopy

T/F Fluorescence microscopy collects higher energy, smaller wavelength, light

False - lower energy, longer wavelength

Fluorescence Microscopy relies on special properties of ___.

Fluorosphores

Fluorescence Microscopy is used to to look at what? (4)

(1) Visualize cell morphology and (2) organelles (3) identify cell types and (4) organelles by markers

Excitation of flurorphores VS emission of flurorphores

Excitation- absorb light at a wavelength

Emission- emit light at a longer wavelength

Exictation and emission spectra of fluorphores are different and can be separated by _____.

optic filters

The difference between the excitation and emission of wavelengths is called the _____

Stokes Shif

What are the 3 advantages of fluorescence microscopy?

1. Low background

2. High signal

3. Can visualize specific cells, cell types, organelles, presence of proteins/macromolecules

Can you look at individual macromolecules by fluorescence microscopy?

No - light microscopy

What is an example of fluorescence microscopy?

Dye filled neuron

What are 2 kinds of organelle specific fluorescent dyes

1. DAPI Stain

2. MitoTracker Stain

What does DAPI stain bind to and what does it help us visualize?

Binds to dna, used to visualize the nucleus

Where does MitoTracker dye accumulate

Mitochondria

What kind of technique uses dye stained antibodies.

Immunofluorescence

Antibodies recognize specific dyed molecules that end up dying the whole antibody. These molecules are called ___.

Antigens

What is added to an antibody to dye it?

A fluorophore

What are the 2 types of immunofluorescence?

1. Direct

2. Indirect

What is the process of direct immunofluorescence?

A single primary antibody is directly conjugated to a fluorophore

What is the process of indirect immunofluroescence?

An unlabeled primary antibody binds to a target protein. A fluorophore-conjugated secondary antibody then binds to the primary antibody.

When is direct immunofluorescence needed?

It is needed when high specificity and low background are needed

When is indirect immunofluorescence needed?

When trying to detect low abundance proteins

Does direct or indirect immunofluorescence have higher amplification?

Indirect because multiple secondary antibodies bind to one primary.

What is immunofluorescence used for?

Identifying specific cells, specific organelles, and subcellular structures

With immunofluorescence astrocytes are labeled with what protein

GFAP

With immunofluorescence neurons are labeled with what protein

MAP2

Is DNA(nuclei), dyed blue also observed with immunofluorescence?

No, it’s dyed by fluorescent dye.

With immunofluorescence golgi apparatus are labeled with what protein

GM130

With immunofluorescence neurons are labeled with what protein?

MAP2

With immunofluorescence postsynaptic density are labeled with what protein?

PSD-95

With immunofluorescence presynaptic terminals are labeled with what protein?

synapsin

When you combine per and post synaptic markers(dye) you can identify what?

a synapse

Our ability to manipulate and introduce modified genes into organisms allows researchers to introduce fluorescence into living specimens, this is called _____.

Florescent tagging

What dye is used to look at the specimen: Aequoria Victoria (jellyfish)

GFP

What dye is used to look at the specimen: Discosoma (coral)

dsRed

What are the 2 steps to fluorescent tagging?

1. Create a ____ of a fluorescent protein and promoter, targeting sequence, and/or protein of interest.

2. Introduce ____ into cultured cells or whole organisms

fusion gene

modified gene

Using cell type specific ____ to label neuron subpopulations in mice

promoters

What are the 5 advantages/uses of fluorescent tagging?

1. Many colors of fluorescent proteins (FPs) exist now, from blue to far red

2. High levels of brightness and specificity

3. Observing cell/organelle morphology, protein distribution in living cells

4. Labeling cells based on genetic identity (using cell type specific promoters to drive FP expression)

5. Modified FPs can report on enzyme activity, protein-protein interactions, calcium dynamics, and much more.

Modified fluorescent proteins can report on what 4 things?

1. Enzyme activity

2. Protein-protein interactions

3. Calcium dynamics