Reflection on The Research on Epigenetics Ability to Encode Memory

The article stated that when it comes to long-term memory formation, it is accepted that it is formed by the strength of the synapse (space between two neurons). Neurons are the cells in our brain which retrieve and send information throughout our body. There is a theory that long-term memory is actually encoded by modifications in gene expression or, epigenetics. This modification is not due to heredity but to the gene itself being turned on or off. This phenomenon can be mediated by RNA.

Researchers chose to test this theory by taking RNA from a trained animal and placing it into an untrained animal to see if it generates that same long-term memory from the trained animal. They used sea slugs Aplysia for the study. The training was for the Aplysia to undergo long-term sensitization. In this process, repeating a stimulus will cause an enhanced reaction over time, becoming more sensitive to the stimulus. The Aplysia were placed into separate tanks and all were implanted on bilaterally on their tail with Teflon-coated platinum wires. They sensitized the Aplysia’s Siphon-Withdrawal Reflex by lightly stimulating the tubular portion on the Aplysia known as the siphon with a Grass stimulator. This reflex is to retract the siphon when being disturbed. Three pretests were given to the sensitized Aplysia 25 minutes before the training once every 10 minutes. For the training, the duration of the reflex was timed and recorded two times with a twenty-four-hour gap between. Five rounds of shocks were given at twenty-minute intervals and a twenty-four hours later a posttest was performed. For the RNA injections portion of the test, untrained animals were given three pretests like the trained animals before they were injected followed by a posttest twenty-four hours later. To prepare for RNA injection, four to five of the trained and four to five of the untrained animals had their pleural-pedal and abdominal ganglia removed. The RNA was then removed from the ganglia and homogenized. Then it was centrifuged to separate the upper aqueous phase which holds the nucleic acids of RNA. All the RNA from trained animals was combined into a tube and the same for RNA from untrained. Each animal was injected with 70 ug of either trained or untrained RNA. Electrophysiological measurements were taken from Pleural sensory neurons and small siphon motor neurons which were separated from the animals into a cell culture. Each cell culture could have had sensory neurons or motor neurons, or a synaptic pairing of the two. The separate neurons were in the culture for five days while the synaptic paired neurons were in the culture for three days before recording. They were recorded by being impaled with micropipettes filled with 1.5 M potassium acetate, .5M potassium chloride, and .01 M HEPES. This would allow for the recording of voltage by a molecular device and digitized for analysis.

The Action potential threshold was determined by injecting 2-s current at incremental intensity levels. Sensory neurons whose resting membrane potential was more depolarized than -35mV were excluded. Motor neurons whose membrane potentials were more depolarized than -30mV were also excluded. Their excitability was recorded by sending positive current pulses of .1, .2, or .3 nA. Once electrophysical measurements were finished, the cultures were treated with RNA or a control solution. For the synaptic paired cultures, the amplitude of an EPSP produced by a single action potential was recorded on the first day of the experiment. The sensory and motor neurons in the culture were impaled with microelectrodes. The motor neuron needed to be held at a resting membrane potential between -80mV and -85mV by a negative current of .3-.8 nA to ensure it wouldn’t fire spontaneously. A positive current of .2-.8 nA was used to elicit an initial EPSP. Once this testing was complete, the cultures were treated with either RNA or the control solution. With all electrophysiological measurements complete, the cultures were randomly assigned to treatment with RNA from trained animals or RNA from untrained animals or a solution without RNA.

The results of the study were statistically analyzed by the software system SigmaStat. This software can compare effects among groups before and after repeated measures. To compare to independent groups, the Mann-Whitney test was used. This test declares if samples will differ from each other when randomly selected, testing the null hypothesis. The Wilcoxon and paired t-test were used with dependent groups. An ANOVA or Kruskal-Wallis test was used to measure three independent groups. The normal distribution was tested with a Shapiro-Wilk test. Leven’s test was used to find the homogeneity of variance in the synaptic experiments.It was found that the siphon withdrawal reflex was significantly more in the trained RNA group than the untrained RNA group. The duration of the reflex was also significantly longer in the trained RNA recipients during the posttest. This reveals that only the RNA of sensitized animals induces the reflex more heightened onto recipients. DNA methylation was determined to be needed for RNA to be able to enhance the siphon withdrawal reflex. This was determined after running a test of inhibiting DNA methylation and observing if that affected the sensitization in RNA from trained animals, it did. Because of the long-term sensitization, sensory neurons were found to have a long-term increase in excitability as well in the somata, shown by the increase in the number of action potentials. Animals treated with RNA from sensitized animals produced more action potentials in sensory neurons. It was determined that there was no overall increase in the excitability of motor neurons. This reveals that the RNA’s excitatory ability is only applicable to sensory neurons. EPSPs in the RNA from sensitized animals had a greater variance, revealing that there is a variable effect on the synaptic strength of the synaptic combined cultures of sensory and memory neurons.

This study aimed to see if epigenetics encodes memory and found that it did. This discovery was done by multiple processes. The article did a good job of complying all the data into four figures and explaining what the results meant. Their footnotes really help any reader put the data into plain English sentences. I was skeptical at first that you could actually transfer the engram from one animal to another, but it makes sense that you can. When you theorize about it, Memory is crafted mostly by your sensory neurons and meaning you put behind the memory. In animals, retracting from danger is important for them to survive. So, the sensitization of the siphon withdrawal reflex in the Aplysia, a survival tactic, is memorable and therefore worthy of being encoded and stored. The only skepticism I have left is if all long-term sensitization is transferable, does it have to be a worthy thing to be memorable? Is this possible in humans? So many questions.