NEURO: Neurons and Glia Flashcards
What important step is needed in order to slice/section the brain?
It needs to be stored and firmed up by paraformaldehyde.
Without it, the brain is a similar softness to raw chicken (if we put a bit of pressure on it, it deforms, so we can’t get clean slices).
What are two ways in which we can slice brains?
The two ways are:
- using a microtome: you embed the brain in wax in a particular orientation, then you mount it in the microtome and slice it
- using a cryostat: you freeze the brain, then slice it in the cryostat
What does the Nissl stain label?
The Nissl stain labels RNA.
What does the Golgi stain label?
The silver chromate labels the cell body of some neurons.
It’s not too effective.
In what ways can we target opsins to specific cells?
- viral delivery
- Cre/Lox technology
Describe the viral delivery of opsins to specific cells.
The DNA sequencing of the GFP genetic code is cleaved out, and that DNA is packaged into a virus.
The virus infects the neurons, inserting a GFP genetic code into the DNA of the neuron.
DNA transcription and translation of the GFP DNA occurs to produce a fluorescent protein.
The fluorescent protein DNA code is inserted into the viral DNA code along with a promoter, and it is this promoter that determines which cell types the virus can infect.
List the different types of cell promoters, and what types of cells they will induce the infection of.
The CMV/CAG promoter infects all neurons and glia.
The GFAP promoter infects glia only.
The hSYN promoter infects neurons only.
The Cam KII infects excitatory neurons only.
Describe the Cre/Lox technology in targeting specific cells.
Cre recombinase is an enzyme that recognises loxP sites. Different genetic modifications take place depending on the loxP location/orientation.
List the different modifications that occur based on the loxP orientation.
- if we have 2 loxP sites facing each other, the gene gets flipped by the enzyme (now coding for a different protein)
- if the loxP sites are facing the same way, it results in the complete removal of the gene between them [this is the one that we are interested in as it is what happens when we introduce fluorescent proteins in neurons]
- if the loxP sites are on different strands, it cuts and swaps them
Describe the soma of the neuron.
Soma is greek for body, so this refers to the cell body.
The different parts that make up a cell body are:
- a K+-rich cytosol
- the nucleus (responsible for DNA replication and transcription)
- the endoplasmic reticulum (involved in RNA translation)
- the Golgi apparatus (involved in protein folding)
- the mitochondria (known as ‘the powerhouse of the cell’)
List the kind of membrane proteins that are found on the different membrane.
There are many types of membrane proteins, such as:
- ligand-gated ion channel proteins
- G-protein coupled receptors
- voltage-gated ion channel
Ligand-gated ion channels and G-protein coupled receptors are found mainly on the dendritic membrane.
Voltage-gated ion channels are mainly found on the axonal membrane.
Describe the structure of the cytoskeleton of the prototypical neuron.
The cytoskeleton is made of different parts:
MICROTUBULES:
- they are relatively large (20 nm in diameter)
- they run the length of the neurites
- they are composed of tubulin
- (they do not extend into the axon)
MICROFILAMENTS:
- they are 5 nm in diameter (the same as the neural membrane)
- they are numerous in neurites
- they are composed of actin filaments
NEUROFILAMENTS:
- they are 10 nm in diameter (they are called intermediate filaments in other cell types)
- they are composed of 5 proteins (NFL, NFM, NFH, internexin, peripherin)
- their protein combination is dependant upon the neuronal cell type and their developmental stage
Describe the axons of the prototypical neuron
The initial segment of our axon from the soma is the axon hillock. It is where the EPSP and IPSP are summed, and an action potential is fired/inhibited.
Some axons are 1 mm in length, while others can get up to 1 metre in length.
The axons branch to form collaterals.
What are the two types of synaptic boutons that we can have?
Terminal boutons: where they synapse at the end of an axon
A bouton en passant: when the synapse is along the length of an axon
How are vesicles transported up and down the axon?
The axon contains no ribosomes - materials are enclosed in vesicles and transported by kinesin.
