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https://drive.google.com/open?id=0B-NHmA3zMW-PeEstclFIZmxRQmc

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Media from Chiodo, A (2017).

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Dentate gyrus, CA3, and CA1 hippocampal region differences in DNA methylation based on the measured Bdnf gene. Novel rats were used to show differences in gene expression.

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DNA methylation differences of the Bdnf gene in three subregions of the hippocampus including the dentate gyrus, CA3 and CA1. Methylation is shown in grey and unmethylation is shown in black.

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DNA methylation controls hippocampal memory overtime but has no affect on the initial encoding of memory.

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4 hour delay between injections

Zebularine injection shows decline in DNA methylation compared to control

Significance: destabilization of hippocampal memory in Zebularine injected mouse due to removal of gene expression silencing

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15 minute delay between injections

No significant difference between control and Zebularine injection observed

Significance: spatial memory encoding intact and unaffected by the demethylating agent addition

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IMAGE: Roth et al. 2015. Figure 3b (modified)
Mean Pearson correlations of sessions 1 and 2 (comparison 1) and sessions 3 and 4 (comparison 2). Zebularine results are shown in black and controls are shown in white.

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The chosen one: Bdnf gene and DNA methylation

Bdnf gene: associated with learning and memory, contextual fear learning and neural plasticity

DNA methylation has been found to directly regulate hippocampal Bdnf activity (Roth et al. 2015) making it the perfect gene for the researchers to examine changes in DNA methylation

Novel rats exhibited increased Bbnf DNA methylation in the CA3 sub-region of the hippocampus

Increased DNA methylation was not seen in the CA1 or Dentate gyrus of the novel rats

Main conclusion: The CA3 sub-region of the hippocampus is responsible for modulating changes in gene expression caused by DNA methylation.

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Previous studies have looked at DNA methylation/demethylation in the HPC (hippocampus) and its role in neurogenesis and memory formation

No studies have looked at how chemical modification of DNA can impact memory learning and formation

Main Goal: to determine the contribution of DNA methylation in spatial memory formation and maintenance of this memory in the HPC circuit (including the dentate gyrus, CA1 and CA3)

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#3.  A final suggestion or alternative for Roth et al.’s team of researchers in their 2015 paper is to assess the level of activating ACETYLATION in their less-stressed rats in comparison to those that were much more stressed. Acetyl tags have the opposite effect of methyl tags since they increase the amount of DNA transcription. To further solidify their hypothesis that this particular spatial learning and memory pathway in the hippocampus was dependent on epigenetics, Roth et al.’s team of researchers could have looked at various other epigenetic tags other than methylation. Since the less-stressed rats were shown to have greater levels of neuronal plasticity, scientists would have subsequently seen greater amounts of acetylation present in their hippocampi and visibly less in the more-stressed rats. Both methylation and acetylation were measured in Weaver et al.’s (2004) study that assessed epigenetic tags in mice that were either highly cared for as pups in comparison to those who were neglected. If there would be no difference in the levels of acetylation, the entire CA1/CA3 spatial learning pathway may have other underlying genetic influences possibly found somewhere in the genome that have nothing to do with epigenetics at all… (ENJOY THE FINAL VIDEO BELOW!)

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http://learn.genetics.utah.edu/content/epigenetics/rats/images/Pup.jpg

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#2. Roth et al’s (2015) experiment only assessed destabilization in neurons and potential for plasticity and therefore did not actually measure if zebualrin rats learned better than their wild type counter parts. This is a critical hole in the experiment that could have added easily been tested using one of the most well known and effective behaviour tests for spatial representation and memory formation -  a Morris Water Maze (SEE VIDEO to the LEFT!). Rats with more methylation present were shown to have less plasticity of their neurons responsible for this particular spatial learning CA1/CA3 pathway in the hippocampus. If placed in a Morris Water Maze along with their wild-type counterparts, the highly methylated rates would have spent more time in the water trying to locate and remember where the hidden platform was. Once the platform would be removed, these rats would also have difficulty remembering what symbol/shape it was located under and would thus show NO PREFERENCE to the specific compartment that contained the hidden platform. If there would be no difference in the Morris Water Maze task for both groups of rats, then Roth et al.’s (2015) hypothesis and results could be refuted.

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www.criver.com/products-services/basic-research/find-a-model/long-evans-rat

www.lovethispic.com/uploaded_images/42225-Chocolate-Sprinkle-Donut.jpg

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In order to address their experimental shortcomings, there are several directions Roth et al (2015) can take in the near future:

#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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http://nl.bu.edu/wp-content/uploads/2011/07/animat.png

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Media from Chiodo, A (2017).

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Agnihotri, N. T., Hawkins, R. D., Kandel, E. R., and Kentros, C. (2004). The long-term stability of new hippocampal place fields requires new protein synthesis. Proceedings of the National Academy of Science USA, 101(10), p. 3656-3661. doi: 10.1073/pnas.0400385101

Bilang-Bleuel, A., Ulbricht, S., Chandramohan, Y., De Carli, S., Droste, S. K., and Reul J. M. (2005). Psychological stress increases histone H3 phosphorylation in adult dentate gyrus granule neurons: involvement in a glucocorticoid receptor-dependent behavioural response. European Journal of Neuroscience, 22(7), p. 1691-1700. doi: 10.1111/j.1460-9568.2005.04358.x

Chandramohan, Y., Droste, S. K., and Reul, J. M. (2007). Novelty stress induces phospho-acetylation of histone H3 in rat dentate gyrus granule neurons through coincident signalling via the N-methyl-D-aspartate receptor and the glucocorticoid receptor: relevance for c-fos induction. Journal of Neurochemistry, 101(3), p. 815-828. doi: 10.1111/j.1471-4159.2006.04396.x

Feng, J., Zhou, Y., Campbell, S. L., Le, T., Li, E., Sweatt, J. D., Silva, A. J., and Fan, G. (2010). Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nature, Neuroscience, 13(4), p. 423-430. doi: 10.1038/nn.2514

Lubin, F. D., Roth, T. L., and Sweatt, J. D. (2008). Epigenetic regulation of BDNF gene transcription in the consolidation of fear memory. Journal of Neuroscience, 28(42), p. 10576-10586. doi: 10.1523/JNEUROSCI.1786-08.2008

Miller, C. A., and Sweatt, J. D. (2007). Covalent modification of DNA regulates memory formation. Neuron, 53(6), p. 856-869. doi: 10.1016.j.neuron.2007.02.022

Oliveria, A. M. M., Hemstedt, T. J., and Bading, H. (2012). Rescue of aging-associated decline in Dnmt3a2 expression restores cognitive abilities. Nature Neuroscience, 15(8), p. 1111-1113. doi: 10.1038/nn.3151

Penner, M. R., Roth, T. L., Chawla, M. K., Hoang, L. T., Roth, E. D., Lubin, F. D., Sweatt, J. D., Worley, P. F., and Barnes, C. A. (2011). Age-related changes in Arc transcription and DNA methylation within the hippocampus. Neurobiology of Aging, 32(12), p. 2198-2210. doi: 10.1016.jneurobiolaging.2012.01.009

Rice, J. C., and Allis, D. C. (2001).Histone methylation versus histone acetylation: new insights into epigenetic regulation. Current Opinion in Cell Biology, 13, p. 263-273.

Roth, E. D., Roth, T. L., Money, K. M., SenGupta, S., Eason, D., E. and Sweatt, J. D. (2015). DNA methylation regulates neurophysiological spatial representation in memory formation. Neuroepigenetics, 2, p. 1-8. doi: 10.1016/j.nepig.2015.03.001

Roth, T. L., Zoladz, P. R., Sweatt, J. D., and Diamond, D. M. (2011). Epigenetic modification of hippocampal Bdnf DNA in adult rats in an animal model of post-traumatic stress disorder. Journal of Psychiatric Research, 45(7), p. 919-926. doi: 10.1016.j.jpsychires.2011.01.013

Sweatt, J. D. (2013). The Emerging Field of Neuroepigenetics. Neuron, 80 (3), doi: 10.1016 j.neuron.2013.10.023

Vaissière, T., Sawan, C., and Herceg, Z. (2008). Epigenetic interplay between histone modifications and DNA methylation in gene silencing. Science Direct, 659(1-2), p. 40-48.
doi: 10.1016 j.mrrev.2008.02.004

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.

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Two major limitations found within the Roth et al. (2015) paper include the following:

• Roth et al.'s (2015) paper cannot truly solidify epigenetic mechanisms unless methylation alterations proven to be passed down to rat offspring & with cross fostering of pups

• The researcher's data only analyzed destabilization in neurons and potential for plasticity; they did not actually measure if zebualrin rats learned better than their wild type counter parts. This could have added a Morris Water Maze test to test for spatial representation in memory formation (SEE VIDEO!)

• In order to fully prove this mechanism is dependent on epigentitics, researchers would have to test whether such alterations in methylation patterns are passed down to future generations as well as cross fostering ; possible novel experiment ? Weaver et al., (2004) were successful their epigenetic experiments that used CROSS-FOSTERING FIRST!

•Only methylation patterns (inhibitors) were evaluated; scientists  could potentially look at ACETYLATION patterns(activators) and their effects in the very same stressful/non-stressful conditions they placed the mice in just as was done in Weaver et al.'s (2004) study as well as reviewed by Peixoto and Abel (2013)

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The following results were found to be consistent with the Roth et al. (2015) paper:

•Memory was found to involve complex interactions of  external, non-genetic factors such as contextual learning elements and stress that are both capable of inducing epigenetic modifications and altering gene expression

(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).

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•These results support the initial hypothesis that DNA methylation epigenetics plays a key role in regulating neurophysiological spatial memory formation (Miller and Sweatt 2007; Lubin et al. 2008; Feng et al. 2010).

•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)

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#1: DNA Methylation patterns can be altered based on external stressors
•Rats exposed to only novel environments had visibly more methylation present in their hippocampus

•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

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Image from Chu, A (2017).

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Fig. 2: Visual Depiction of the 5 Day Experiment in Experiment 2

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Fig. 2: Visual Depiction of the 5 Day Experiment in Experiment 2

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This figure visually explains the second experiment done in this paper. In contrast to the previous experiment, this spanned 5 days (2 days for training, 1 day of rest and 2 days with the introduction of the drug treatment).

Rats were injected with the drug treatment one hour prior to the 1st session and immediately after the 3rd session.

In one testing day, the day was split into two parts with 3 sessions in each, for a total of 6 sessions/day.

Sessions 1 and 2 took place in Env. A, while sessions 3-6 took place in Env. B. Rats were allowed to rest for 15 minutes in between each session, with the exception of sessions 3-4, where there was a 4 hour break between the morning and afternoon.

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Experiment 2: HPC Single Unit Recording 

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Experiment 2: HPC Single Unit Recording 

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Drug Treatments
Rats were randomly given either the control treatment or the drug treatment.

Control treatment: 10% dimethyl sulfoxide (DMSO)

Drug treatment: zebularine - 600 ng/µL in 10% DMSO

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)

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Fig. 1: Visual Depiction of the 8 Day Experiment in Experiment 1

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Fig. 1: Visual Depiction of the 8 Day Experiment in Experiment 1

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This figure provides a visual depiction of experiment 1. It shows that the experiment occurred over 8 days (3 days for training and 5 days for testing) and where the rats performed the experiment during each day (either environment A or B)

On a testing day, rats completed 20 laps for the foraging task in the familiar environment, took a 10 minute break and then completed another 20 laps.

Day 8 shows how the two groups differ - the control group had the 10th session in environment A, while the experimental group had that session in environment B.

Image from Roth et al. (2015) - Figure 1a

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Experiment 1: Assessment of DNA Methylation Following Exposure to a Novel Environment - Methods

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Experiment 1: Assessment of DNA Methylation Following Exposure to a Novel Environment - Methods

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Biochemical Analyses
Rat brains were removed 90 minutes after completing the last testing session and had biochemical analyses completed to observe the methylation status of the rat's brains.

DNA was extracted from HPC regions such as the dentate gyrus, CA1 and CA3.

They assessed the methylation via methyl-specific real time PCR.

They wanted to detect methylated/unmethylated DNA in the target neurotrophin, BDNF - specifically in exons 1,4 and 6.

The methylation of these exons were observed via direct bisulfide sequencing, where anti-body tags are added to the methyl groups.

To determine if changes to BDNF was consistent with the observed methylation changes, they also extracted RNA from the HPC regions.

They performed reverse transcription and used statistical analyses to detect the differences.

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Subjects: adult Long-Evans rats
Task: foraging task for experiments 1 and 2

Rats were trained to run clockwise on a circular track while foraging for chocolate sprinkles

Tracks were customizable and could change the colour and texture to produce novel environments

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What is Epigenetics?

“epi” – means above

Epigenetics is above the level of genes as it is defined to be changes in gene expression that control cell differentiation (Sweatt et al. 2013)

Neurogenomics focuses on changes taking place at the level of the genome, where as epigenetic research attempts to look above this and considers outside environmental factors being capable of causing phenotypic change.

Epigenetics does not cause a change in the sequence of the DNA, but is rather defined as the addition of chemical tags onto histones or directly onto DNA itself (Vaissière et al. 2008)

Histone acetylation and DNA methylation are two prominent epigenetic modifiers, which ensure the transmission of the altered gene expression across many cell generations, allowing epigenetic changes to be classed as heritable (Weinhold et al.2006)

DNA methylation silences gene expression (Rice et al. 2001)

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A Little Intro to Epigenetics!

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Epigenetics_ Why Inheritance Is Weirder Than We Thought