PiRNAs Involved in the Memory Formation of Fear-conditioning

Tests Migrating from the Brain to the Germline

Lin Li

Bromsgrove School, Birmingham, U.K.

Keywords: Pirna, Odor Fear-Conditioning, Olfactory, Glomerulus, Learning Memory.

Abstract: Small piRNAs regulate and manipulate gene expressions and are important for forming memories. These

piRNAs are thought to be germline-specific and can help offspring inherit memories from their parents. In

this paper, we conducted odor fear-conditioning tests to identify a piRNA that increased in abundance and is

involved in the memory formation of the fear-conditioning test to determine how the offspring can inherit

memory. A mutant piRNA is created using a virus vector and introduced into the mice brains to see if it can

migrate from the brain to the germline. If the the mutant piRNA is found in the sperm cells, then we know

that the piRNAs can migrate from the brain to the sperm cells and thus inherit the memory of the odor used

in the odor fear conditioning test.

1 INTRODUCTION

In a recent study, Moore et al. reported that C.elegans

worms, upon exposure to PA14 (Moore, 2019), can

transmit avoidance memory to their offspring for

several generations. This transgenerational memory

can provide their offspring with advantageous

mechanisms to increase their chances of survival.

Besides, the study has helped us think of a possible

way how memories can be transferred from parent

animals to their offspring i.e., via piRNAs.

piRNAs are a class of 26-28 nucleotide small non-

coding RNAs and are associated with piwi proteins to

regulate gene expression and form memories

(Rajasethupathy, 2012). In Kandel’s study, it was

identified that piRNAs increased in amount in the

neurons of the Aplysia adult brain and thus amplified

its sensitivity to serotonin by inhibiting the

transcription of CREB2. Furthermore, the offspring

of the Aplysia were tested to see if the neurons can

also display long-term potentiation (LTP) from

serotonin stimulus, and was found that the offspring

will also display LTP. However, it is still unclear how

this transgenerational memory is achieved.

In this paper, we are going to create a mutant

piRNA to the piRNA that is involved in the memory

formation of odor-fear conditioning in the olfactory

bulb and see if it can be detected in the germline. If it

is detected and the progeny has the memory, then we

can suggest that transgenerational memory is

achieved via piRNAs.

We hypothesize that if the specific piRNA

associated with the fear-conditioning neuronal circuit

changes in abundance in the olfactory bulb, the sperm

cells would be able to get these piRNAs that migrated

from the brain region and thus inherit the memory of

the odor used in the odor fear conditioning.

2 MATERIALS AND METHODS

2.1 Odor-Fear Conditioning Tests

All mice used in the experiments are M71-GFP mice

strains. These mice are transgenic and have

fluorescent neurons when M71 receptors sense

acetophenone in the olfactory bulb. In this way, we

can observe changes and take out these specific

neurons more easily. The adult mice were kept in

standard cages with a 12-hour light/dark cycle with

free access to food and water.

The experiment utilized 2 different odorants to see

if the mice can discriminate between them: the first

odorant consisted of 10% acetophenone and the

control odorant consisted of 10% propanol in

propylene glycol.

Before the fear-conditioning tests, the mice were

given 3 weeks to habituate to the startle chambers to

ensure a strong odor-shock association. They

Li, L.

PiRNAs Involved in the Memory Formation of Fear-conditioning Tests Migrating from the Brain to the Germline.

DOI: 10.5220/0011208000003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 361-365

ISBN: 978-989-758-595-1

361

received 2 training sessions per week and each

training session consisted of 5 trials of 10 s odor

conditioned stimulus followed by a 0.24 s, 0.4 mA

electric footshock. The trials were separated by a 120

s time gap (Jones, 2008).

When the solenoid switch is closed, clean air can

flow freely with no difference in airflow. Backflow

of the air is prevented by several one-way valves

(yellows arrows). To remove the odor an exhaust

hose (green arrow) is utilized. The electric shock is

generated by a machine-driven animal shocker and

delivered through electric bars installed in the cage

floor. During the behavioral fear-conditioning

testing, the startle is recorded by a 105dB white noise

outburst, and the activity and startle amplitude are

measured by a piezoelectronic device underneath the

floor of the cage. (Jones, 2008)

The first experiment consisted of 6 mice with an

acetophenone stimulus + electric footshock. The

second experiment consisted of 6 control mice and

was given 10 s of acetophenone stimulus only. This

control experiment was carried out to see if any

changes in the neurons and glomeruli sizes were

found without given footshocks. The third

experiment also consisted of 6 control mice and was

given 10 s of propanol odor + electric footshock to

see if whether other odors can also be detected by the

M71 receptors.

Figure 1: The flow of the air through the odorant jar and

into the startle chamber is controlled using a SR-Lab

Response Software which uses a solenoid switch (red

arrows) to control the flow of compressed air.

Figure 2: The glomerular surface area and cross-sectional

area were larger in the acetophenone + shock trained group

than in the homecage group (p < 0/05) (Jones, 2008).

2.2

Sequencing and Identification of

piRNA

First, the glomeruli with acetophenone-sensitive

neurons are isolated. These should be easy to identify

as the neurons with the M71 receptors will fluoresce

in the presence of acetophenone. After obtaining the

RNA clusters a reverse transcriptase enzyme is used

to form cDNAs of the RNAs. These cDNAs are then

transferred into an Illumina high-throughput

sequencing machine to be sequenced. Following

having sequenced the cDNAs, we can identify the 26-

28 nucleotide long piRNAs.

This experiment needs to be done before and after

the fear-conditioning test. The second experimental

group of the fear-conditioning test (the group of mice

with exposure to acetophenone only) can be used to

find the initial amount of piRNA in the brain and the

first experimental group of the fear conditioning test

can be used to find the piRNA that is involved in the

formation of acetophenone memory. By using RNA

sequencing, the machine will be able to show which

piRNA has increased in an amount most, thus we can

find which piRNA was associated with the test. The

piRNA identified with the most significant increase

in abundance is then called piRNA-X.

As shown in figure 3 the glomelum should have

an increased M71 axon density and glomerular size

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

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after the odor fear conditioning test in the dorsal and

medial areas. Therefore, these acetophenone-

sensitive neurons can be identified and isolated to

find the piRNA that has increased in abundance.

Figure 3: Increased M71 axon density and glomerular size

with fear conditioning in the medial and dorsal pairs of M71

glomeruli (Jones, 2008).

2.3

Determination of the piRNA-X

To ensure that the found piRNA-X is indeed

associated with the neuronal circuit and memory

formation, the piRNA-X is conditionally removed in

the olfactory bulb. The piwi protein that works

together with the piRNA is knocked out by using

CRE recombinase to remove the gene encoding for

the piwi protein. Therefore, piRNA-X will no longer

have an effect. Lastly, a fear conditioning test is

conducted to see if the inhibition of the piRNA-X has

any effect on the memory formation of acetophenone.

2.4

Mutant piRNA

To see if piRNAs can migrate from the brain to the

germline a mutant piRNA-X is created using a virus

vector to add a piRNA DNA to a group of mice

embryos. The mutant piRNA-X differs from the

initially found piRNA-X by a nucleotide achieved by

using the CRE loxP system. Then the mutant piRNA

strand can be activated using the CRE recombinase

enzyme in the olfactory bulb region of the adult mice.

Following this, immunoprecipitation is used to

see if the mutant piRNA-X can still perform the same

task as the original piRNA-X. First, a crosslinker is

used to link the piwi protein to the mutant piRNA-X

and test if they can interact. Then specific antibodies

are used to separate the piwi-piRNA complexes from

the cells. We elute to separate the mutant piRNA-X

from the piwi protein and check if the mutant piRNA-

X is present with the RT-PCR test.

2.5

Testing for the Migration of

piRNA-X

The RT-PCR test is used to check if the mutant

piRNA-X is present in the sperm cells. First, a

constant sequence of nucleotides is joined to the 3'

end of the mutant piRNA-X. A primer

complementary to the constant region binds to it and

the reverse transcriptase can come along and form the

DNA strand. A second primer specific to the mutant

piRNA-X binds to the DNA strand and reverse

transcriptase forms the cDNA of the mutant piRNA-

X. The sample is then placed into the PCR machine.

2.6

Further Experiments + Controls

Lastly, the offspring of the experimental groups are

tested to see if they were able to inherit the memory

of the acetophenone fear-conditioning test. The first

group of mice consists of the offspring of parents who

have undergone the fear-conditioning test and have

the mutated piRNA-X. The second experimental

group consists of offspring of parents who have

undergone the fear-conditioning test but do not have

the mutant piRNA-X. The third experimental group

consists of offspring of parents who have not

undergone the FC test but have the mutant piRNA-X.

These experiments are to prove that the mutant

piRNA-X does not interfere with the regular function

of the piRNA-X and the original piRNA-X is

functional with the addition of the mutant piRNA-X

too.

3 POSSIBLE RESULTS

3.1

Odor Fear-conditioning

From the first FC tests, some predictions of the results

can be drawn. We expect that the first group of mice

will show strong freezing behavior when exposed to

acetophenone after the fear conditioning test. If the

mice do not display a freezing behavior the same

procedure will be repeated. The second group of mice

should not show sensitivity to the odor and the third

group of mice should not have fluorescent neurons

(Jones et al., 2008).

Figure 4 shows how the results of the freezing

behavior look like in the mice in response to odor

PiRNAs Involved in the Memory Formation of Fear-conditioning Tests Migrating from the Brain to the Germline

363

stimulus after training.

Figure 4: Freezing in response to CS presentation on Day 2

after training. (Sweatt, 2010).

3.2

Sequencing and Identification of

piRNA

After DNA sequencing, the machine should give us

the nucleotide base sequence of the piRNA that has

increased in abundance the most, the piRNA-X.

3.3

Determination of piRNA

We expect that after the inhibition of piRNA the mice

will not be able to form a memory of the odor FC test,

they will not display freezing behavior. Therefore, we

have found the correct piRNA that is involved in the

FC neuronal circuit.

3.4

Mutant piRNA

The PCR machine can give 2 different results:

positive or negative. If the PCR machine shows a

positive result then we know that the cDNA is present

in the sample and the mutant piRNA-X has migrated

from the brain to the germline. If the PCR machine

gives a negative result, then the cDNA is not present

in the sample and the piRNA has not migrated from

the olfactory bulb to the germline.

3.5

Offspring

We would expect that the offspring of the first and

second experimental groups would display similar

increased freezing behavior to acetophenone, whilst

the offspring of the third experimental group would

not display freezing behavior on exposure to

acetophenone.

4 DISCUSSION

One possible limitation of using DNA sequencing to

obtain the piRNA sequences is that several piRNAs

can be identified that have increased in amount. In

this case, we would have to choose which piRNA to

mutate and eliminate. If after the inhibition of the 1st

piRNA the mice can still exhibit enhanced sensitivity

to acetophenone then a 2nd piRNA must be inhibited

until the mice stops exhibiting freezing behavior and

the piRNA then can be mutated.

Furthermore, one other limitation of creating the

mutant piRNA is that changing the base sequence of

the piRNA can result in the mutant piRNA having a

different function to the original strand or can lead to

damage to the animal since piRNAs are only 26-28

nucleotides long and a slight change to the sequence

can lead to consequences.

5 CONCLUSION

In conclusion, if the mutant piRNA is present in the

sperm cells, then the piRNA associated with fear odor

conditioning has migrated from the brain to the sperm

cells and memory of the odor used in th fear

conditioning test can be inherited by offspring.

If the mutant piRNA is not present in the sperm

cells, then the piRNA associated with odor fear

conditioning has not migrated from the brain, and

offspring will not inherit the memory of

acetophenone.

This work could therefore confirm that piRNAs

are related to transgenerational memory and migrate

from the brain to the germline. In the future, the

pathway and mechanism of the migration of piRNAs

are nevertheless yet to be confirmed. We are currently

thinking that perhaps piRNAs are transported in

exosomes around the body or piwi-piRNA complexes

can travel around the body to the germline. Moreover,

future studies can also focus on structures such as the

hippocampus and amygdala which are known to be

involved in FC memory formations, and possibly the

transgenerational memory.

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