Zebularine is a drug that has been known to inhibit DNA methylation, making it a demethylating agent.
Surgical Treatment
Before this experiment began, the rats had microdrive arrays inserted into their brains, right above the dorsal lateral HPC to record from the neurons.
 
IMAGE: https://www.intechopen.com (zebularine molecule)

•Essentially more stressed and thus their memory was negatively impacted compared to the rats placed in familiar environments  (less methylation present)

#2: Pharmacological addition of a demethylation agent improves hippocampal plasticity
•Rats injected with ZEBULARINE (demethlyation agent) had significantly more destabilization in their hippocampal neurons MORE PLASTICITY = BETTER LEARNING & MEMORY!

•When methyl groups removed, over time observed a greater capacity for long term spatial memory and learning 

•Highly suggestive that long term place memory may require molecular memory processes that are influenced by epigenetic mechanisms like DNA methylation.

•This is the first study to demonstrate that a novel spatial experience alters DNA methylation patterns that vary by different regions of the brain (i.e. hippocampus)

(Bilang-Bleuel et al. 2005; Chandramohan et al. 2007; Lubin et al. 2008; Penner et al. 2011; Roth et al. 2011)

•Short-term vs. long-term results coincided well with previous reports. Injections of anisomycin (protein synthesis blocker) revealed that protein synthesis was necessary for long term (6 hrs. between sessions) place field stability but not necessary for short term stability (1 hr. between sessions) (Agnihotri et al. 2004).

•This work provides support for the speculative notion that the decline in spatial memory associated with aging and neurological diseases, such as Alzheimers, could reflect dysregulation of hippocampal circuit function due to alterations in DNA methylation (Oliveira et al. 2012).

Weaver, I. C. G., Cervoni, N., Champagne, F. A., D’Alessio, A. C., Sharma, S., Seckl, J. R., and Meaney, M. J. (2004). Epigenetic programming by maternal behaviour. Nature Neuroscience, 7, p. 847-854. doi: 10.1038/nn1276

Weinhold, B. (2006). Epigenetics: The Science of Change. Environmental Health Perspectives, 114(3), A160–A167.

 

#1. Firstly, in order to fully prove this particular mechanism on spatial learning and memory in the hippocampus is fully dependent on epigentitics, researchers would have to test whether such alterations in methylation patterns are passed down to future generations. Rats with visibly more methylation detected (as a result of constantly being exposed to novel environments) should have been forced to breed with one another. The same would have to be done for wild-type rats exposed to familiar environments and were thus less stressed. Once the groups of offspring were of a suitable age for testing, neuronal cells could be extracted from their hippocampi and subsequently measured for levels of methylation via BISULFITES SEQUENCING (antibodies easily and specifically precipitate out methyl groups.) The offspring of the stressed rats, who would also have been exposed to the stressful environment, should yield greater levels of methylation than their wild-type counterparts. CROSS-FOSTERING should then be done by switching the rat pups to opposite families in order to see if this had any impact on their hippocampal methylation levels. If the amount of methylation changes due to different environmental exposure, it can be successfully concluded that the particular hippocampal pathway studied by researchers is, in fact, influenced by epigenetic tags and NOT regular inherited changes to the genome. If there is no visible change in the levels of methylation, then Roth et al.’s (2015) hypothesis can be refuted!

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