Lab 4 - THE CELLULAR STRUCTURE OF CNS TISSUE and PERIPHERAL NERVES Flashcards
What do Nissl stains show?
Nissl stains show charged structures (Nissl bodies) in the soma of neurons and glia. The Nissl stain is most intense in nucleoli and in the rough endoplasmic reticulum of neurons.
Shows rER in neurons and it specifically stains RNA
blue dots = cells (RNA is inside cells), blue spines = dendrite (can have rER), flattened blue cells = endothelial (lifeblood vessels)
What molecular components are stained with nissl stain?
DNA and RNA in the nucleus and the nucleolus, RNA in the ribosomes of the rER
Why do you think the grey matter is folded?
As our brains expanded, the subsequent pressure was mitigated by folding. This folding (gyrification) increases the surface area of the cortex and so increases
the space for more neurons.
increases surface area
What type of axons would be found in the white matter underlying the gray matter?
Myelinated/unmyelinated axons
Density of cells
close to the edge of the cortex = sparse in terms of cells
further in/deeper shows more density of cells
Glial cell most common in white matter
oligodendrocyte
2 other glial cell types that you would find in the white matter
microglia
astrocytes
Neuron with nissl stain and using LM
stained cytoplasm (can see small stained processes leaving the soma, most obvious at the upper aspect of soma; nucleus in soma large/pale).
astrocyte with nissl stain and using LM
nucleus only (speckled), there does not seem to be any cytoplasm.
endothelial cell with nissl stain and using LM
flattened endothelial nuclei, some of these are curved in a crescent shape.
Golgi staining of CNS tissue
method for staining nervous tissue
The Golgi stain selects cells at random and impregnates and stains the entire cell so that the entire dendritic tree and the axon and its branches can be followed. Commonly however, the axon and many of the dendrites will leave the section containing the cell body. So that we get as much of the cell in one section we cut thick sections, around 2-300μm, so you need to focus down through the section to see the cells clearly.
(NOTE: Glial cells can be stained, and sometimes parts of the vasculature are also stained - they have the appearance of solid black-stained smooth processes but we will not look at them here.) The Golgi method is particularly useful for determining the arrangement of dendritic and axonal processes, on individual neurons, and how these change during life
Look at the different sized cells in the different layer. Are there any differences in the dendrites leaving the cells in the different layers (Golgi stains)
The dendrites of the deeper cells go right to the surface of the cortical layer and have more branches off the main apical dendrite than the more superficial cells. They also have more basal dendrites leaving the cell body and these branch more than those of the superficial cells.
This does depend on the staining but the question is designed to get the students to think about why the dendrites might be different. A different type of axon and different pattern of axonal branching occurs at the different layers of neurons and on different neurons and it is this synaptic contact that dictates the pattern of the dendritic tree.
Write down what differences you can see between the neurons of the superficial and deeper layers of the cortex. Looking at one particular depth (i.e. in one layer) of the cortex move within a layer from the medial to lateral aspects of the section. Do the cells change in any way, size, shape or density of them?
A distinct layer of labelled cells can be seen in the more superficial part of the cortex. The cell soma are small compared to cells that are located more deeply. The staining seems more sparse in the deeper layers but that is due to the cells being larger. As you move from the the medial to the lateral portions of the cerebral cortex you will see different patterns of staining indicated by changes in the density and the shape size of the cells. The changes in cell density and cell soma size you see are representative of those seen in a Nissl stained section.
lateral to medial = sparse to dense
List the neuronal features visible in Golgi stained section that you could not see in the Nissl stained section
Why were you not able to see these structures in the Nissl stained section?
Lots of dendrites, some leaving around the soma and a large one leaving at the apex of the soma, they are branching, also spines on the dendrites. Can see a finer process leaving the cell body, does not change thickness like the dendrites and is smooth. Only see a few of the cells but see the processes leaving the cell body and can see they are all in different planes within the section.
dendritic spines
Why were you not able to see these structures in the Nissl stained section?
The dendrites are not visible, as they only have a small number of organelles such as mitochondria, rough endoplasmic reticulum, the main element stained in the cell soma with the Nissl stain. If there is less for the stain to bind to there will be less staining and will not be visible. Also, as the dendrites get smaller, they are too small to be seen with the conventional bright field light microscope that you use in this class. The Golgi stain fills the cell process with a dense precipitate so even small branches can be seen. The axons have even fewer organelles, cytoskeleton and a few mitochondria so would not be visible in bright field light microscope with a Nissl stain. The Nissl-stained section is also thinner so there is not as much tissue in the Z axis.
for density use what stain…
nissl stain because it stains all the cells
for branches use what stain…
for branches look at Golgi stain because it stains the morphology
List the features of the dendritic spines you could measure/record in this Golgi stained tissue?
The number of spines, the shape and size/length of the spines
Are you seeing all the spines on this length of dendrite and if not, why not?
You will not see spines that are behind the branch or are standing out at right angles to the surface of the branch as they will be obscured by the density of the dendrite.. Remember that some of the spines that appear to be short may be coming from the mid-point of the branch and extending out laterally until they are seen. You might see a fat spine that appears to have no neck, this may be a normal spine that is coming from behind the dendrite and it is just the head of the spine you are seeing.
You could ask the students if they can think of another way of looking at spines. Confocal images if the spine has been stained or filled with a fluorescent dye or three dimensional reconstruction using stacks of serial sections viewed in the electron microscope.
real diameter equation
measure diameter (mm) /magnification = real diameter (mm)
real diameter (mm) to microns
mm x1000 = microns
