Inspired by what you\'ve learned in the first week, you decide to use HRP (horse
ID: 143651 • Letter: I
Question
Inspired by what you've learned in the first week, you decide to use HRP (horseradish peroxidase) to map the inputs to a region of the occipital cortex of a rat brain. Accordingly, you carefully inject a small volume of HRP in the cortex of a freshly dissected brain and then place the brain in a vial of formaldehyde (a technique called drop-fixation). One week later you carefully slice the fixed tissue and look at it under a microscope. Other than a small signal at the injection location, you are unable to find any labeled cells. Is it correct to conclude that this means no other cells send projections to the occipital cortex?Explanation / Answer
The occipital cortex is the visual processing center of the cells that receives signals from the eyes.
The horse raddish peroxidase is an enzyme that breaks down hydrogen peroxide. Hence, HRP labeling is detected using a chromogenic substrate and hydrogen peroxide. When HRP is injected into the occipital cortex, it should diffuse to different regions of the occipital lobe. However, the amount of HRP transported depend on the fixation protocol. Formaldehyde fixes tissues by crosslinking proteins, mostly basic amino acid.
When the HRP is injected, it will start diffusing to the other regions of the occipital lobe.. However, as it is fixed immediately with formaldehyde, the formaldehyde will irreversible crosslink the proteins. This will result in loss of enzyme activity. The relationship of the enzyme to other tissue components may be altered. This limits the access of the enzyme to other sites. As a result, HRP staining will progressively decrease away from the site of injection. As fixation was performed for over a week, any diffused HRP is no longer detected due to denaturation. Fixation should be done for shorter time in order to preserve HRP activity. Formaldehyde and paraformaldehyde severely reduce HRP enzymatic activity as compared to glutaraldehyde.
As the concentration of HRP was highest at the injection site, some labeling of HRP was still exhibited at the site. The enzyme activity was not completely abolished by the fixative.
Hence, it is incorrect that no cells send projections to the occipital cortex. as the problem lies with the fixative.