April 9, 2021, by mbacs2

Immunohistochemistry in Neuroscience

By Emma Gow, 3^rd Year Neuroscience BSc

Neuroscience is a complex and ever-growing field of science, requiring constant research to understand how the brain functions. We will never completely understand every aspect of the brain, but there is a great effort from the scientific community to expand our knowledge of such a complex but fascinating subject.

A large portion of the research within the School of Life Sciences is dedicated to enhancing the knowledge around the brain and its functions, including developing new techniques to observe the molecular systems within the brain. The brain is so densely packed with a large variety of different cells and chemical compounds, from various forms of neuronal cells to a vast array of different neurotransmitters, which makes it very important to have a definitive method of identifying each one. As more discoveries emerge, this becomes increasingly necessary to improve the accuracy of new studies.

One recent subject of interest is the compound 5-hydroxymethylcytosine (5hmC).  As well as being an intermediate in the DNA demethylation process [1], 5hmC is thought to also work as a stable epigenetic marker to regulate gene expression [2]. DNA demethylation leads to the activation of associated genes, so the presence of this intermediate cytosine form could be considered a marker for gene activation. 5hmC has been found to be abundant in neuronal cells in the brain, and thus could play an important part in gene expression within the central nervous system [3]. Due to its potentially crucial role in the nervous system, 5hmC has undergone much research to determine its exact functions, but also to find an effective way to analyse it within the brain.

In the School of Life Sciences, studies by Marcus Willis and Rebecca Trueman have outlined a definitive method to visualise 5hmC in the neuronal cells of rodents by using immunohistochemistry [3]. Immunohistochemistry is an important methodology that is utilised in many biological sciences as a way to image specific molecules or cells within the body using antibodies. In the protocol, Willis and Trueman used primary antibodies that bind to 5hmC on prepared brain segments, then secondary antibodies with a fluorescent marker attached. These secondary antibodies are bred against the animal the primary antibodies were developed in, causing them to bind to the primary antibodies attached to any 5hmC present. The brain segments are then able to be imaged using a specialised fluorescence microscope, allowing the concentrations of 5hmC to be visualised due to the fluorescent marker on the secondary antibodies.

Despite immunohistochemistry being relatively less quantitative than other methods of imaging, it is very specific and can be adapted to measure fluorescence levels to determine a quantitative concentration of the substance [3]. This methodology is utilised in a large variety of studies, including animal models of central nervous system disorders. For example, 5hmC is currently being investigated for its potential importance in the development of Alzheimer’s Disease [4]. High levels of 5hmC have been found to aid in the survival and continued function of neurons [4], so 5hmC could be implemented as a drug target to help relieve some symptoms for those with Alzheimer’s Disease. Protocols such as those outlined by researchers in the School of Life Sciences are critical to investigating and developing these types of novel treatments.

Image by Simon Litherland from University of Nottingham Image Bank

References

[1]	L. D. Moore, T. Le and G. Fan , “DNA Methylation and Its Basic Function,” Neuropsychopharmacology, vol. 38, no. 1, pp. 23-38, 2013.
[2]	Y. Zhang, Z. Zhang, L. Li, K. Xu, Z. Ma, H.-M. Chow, K. Herrup and J. Li, “Selective loss of 5hmC promotes neurodegeneration in the mouse model of Alzheimer’s disease,” The FASEB Journal, vol. 34, no. 12, pp. 16364-16382, 2020.
[3]	M. D. Willis and R. C. Trueman, “Immunohistochemical Detection of Modified Cytosine Bases in Rodent Brain In: DNA Modifications,” in Methods in Molecular Biology, vol 2198, New York, NY, Humana, 2020, pp. 183-191.
[4]	D. Cao, D. Jiang, D. Zhou, H. Yu and J. Li, “A Comparative Study on 5hmC Targeting Regulation of Neurons in AD Mice by Several Natural Compounds,” BioMed Research International, vol. 2020, p. 5016706, 2020.

 

 

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