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Detecting memory-associated modifications in the hippocampus

Victor Akujobi
Packer Collegiate Institute
Abstract:
Memory, the process of the brain storing daily experiences, has been rigorously studied
over the last half century, one major question still remains: where are declarative memory traces
stored exactly? There is overwhelming evidence that the hippocampal formation is responsible
for the processing, encoding and storing of long-term declarative memories. However, research
has been unable to pinpoint where exactly these memory traces are stored, what they look like
in the hippocampus, or whether different memories are processed similarly or differently within
the hippocampus. An investigation was conducted to determine how memory affects a
population of neurons. It was hypothesized that memory formation will recruit a measurable
fraction of synapses in the trained mouse hippocampus and this synaptic recruitment will result
in a widespread modulation of synaptic responses in the hippocampus. It was found that
memory causes an increase in synaptic responses across thirty percent of the
CA1-hippocampus.

Introduction:
Memories are found in the cortical region that is associated with the function that is being
stored. The hippocampus, or hippocampal formation, is the structure within the medial temporal
lobe most associated with declarative, semantic, and spatial-navigational memory function
because of structures within the hippocampus: neurons. Neurons are special cells in the brain
that are responsible for transporting signals and messages to other neurons through action
potentials, electric transmissions. However, synapses, small structures on the end of dendrites
are pivotal to the transmission of these action potentials. Synapses are made up of two
components: the presynaptic terminal, in the dendritic spine of one cell, and the postsynaptic
terminal, in the axon terminal of another cell. When action potentials are triggered, they pass
through synapses.
Depending on the rate of stimulation, synapses have the ability to strengthen or weaken
based on the input stimulation. A strong stimulation propagates a larger action potential, which
causes the release of more neurotransmitters and a greater response in the postsynaptic cell.
This can cause structural changes in the synaptic circuitry that can persist as long as that
circuitry is consistently in use. This process is known as long-term synaptic potentiation (LTP)
and may underlie long-term memory function.
So, how great are the structural and electrophysiological changes caused by LTP? There are
two main notions that scientists suggest could be possible: either the formation of memory can
have a widespread effect on the synaptic efficacy in the hippocampus, or the changes caused
by memory formation can be small and restricted.
Figure 1: Two notions classify the scale of synaptic function during memory formation in the
hippocampus: restricted (A) vs. widespread (B).
It was hypothesized that trained mice and untrained mice will have a measurable,
significant difference in synaptic response. There are four results that may be observed after
conducting the experiment, if we assume that trained mice will show higher synaptic efficacy in
accordance with previous data. First, there could be widespread increases in synaptic response.
So, every synapse that was recorded in the trained peristimulus histograph is activating at a
higher level than the untrained peristimulus histograph. Second, there could be only one
synapse, out of all the synapses that were recorded in the peristimulus histograph that is
operating at a much higher rate than the synapse in the untrained. This would imply that
memory formation creates small cortical changes in the brain. Third, though unlikely, there could
be no changes between the rate of response between the trained and the untrained. And finally,
we could see something in between all the other three possibilities.

A) B)

C) D)
Figure 2: Four predictive models for the comparison of the post synaptic response results
across the three locations for the three distinct conditions.

Methodology:
In this experiment, mice will be trained to learn to avoid a shock zone. Then they will be
sacrificed for the purpose of harvesting their brain slices. After the slices have been obtained,
they will go off to two separate procedures: immunohistochemistry staining and
electrophysiology analysis. And by interpreting the data from these procedures, I will be able to
draw conclusions.
There are two main training conditions: trained and untrained. Trained mice are released
in the rotating arena, and subjected to a shock-avoidance paradigm, where they are forced to
actively avoid a shock-zone by using various cues around the room. In this way, animals must
associate the shock area with particular cues, resulting in the formation of a memory. Untrained
mice are exposed to the arena with the shock off, so although they can acknowledge the
various cues around the room, they can make no associations; thus no memory is formed.

Figure 3: The apparatus consisted of a rotating arena with local arena and distal (room) cues.
Trained mice needed to learn to actively avoid a shock-zone, while untrained mice were
exposed to the arena with the shock off.

Positive and negative reinforcements to those variables were investigated as well, in


order to see how synapses would be affected when the system was over stimulated and under
stimulated. Overstimulated mice were subjected to the conflict test paradigm, which switched
the shock-zone after two trials, so the animal would be forced to perform two cortical processes:
suppressing a memory, and forming a new memory. Theoretically, this should result in an
elevated level of synaptic response. Under stimulated mice simply stayed in their home cage,
which, theoretically would have resulted in a diminished synaptic responses.

Figure 4: A table depicting the four training conditions. Home cage mice never see the arena.
Untrained mice are in the arena but the shock remains off. Memory trained mice are in the
arena with the shock on for one hour per trial. Conflict test mice are in the arena with the shock
zone for one hour per trial, but on the last trial the shock zone is switched.

After mice are trained, their hippocampal brain slices are harvested. To do so, mice are
anesthetized using isoflurane, and then euthanized by beheading. Once the hippocampal brain
slices are retrieved using a Vibratome slicer, they are restored and kept alive using artificial
cerebral spinal fluid, a hearty mice of chemicals that prolongs cell functions.
Brain slices then undergo two separate procedures: immunohistochemistry and
electrophysiology. Immunohistochemistry is the process of staining antibodies that react with
particular proteins. When these proteins are stained, they emit colors that compel the visual
quantification of synapses in the CA1-hippocampus. Electrophysiology is the process of sending
action potentials through axons to record responses from synapses in the CA1-hippocampus.
These responses were recorded using a program called Clampex, and then analyzed using
Clampfit. The results from these procedures were used in conjunction to determine the
percentage of synapses recruited during the process of memory formation.

Results:

Figure 5: Peristimulus Histograph analysis of synaptic efficacy measured as the maximum


value from post synaptic response curves recorded at each dendritic location (N,C,S) from each
behaviorally conditioned group of mice.

Untrained mice had the smallest postsynaptic potentials, or synaptic responses out of all
the conditioned mice. In the north, synapses from the CA1-hippomcapus of untrained mice
recorded responses that averaged to approximately 0.01 volts. In the center, synapses recorded
responses that averaged to approximately 0.015 volts. In the center, synapses recorded
responses that averaged to about 0.02 volts. For home cage mice, in the north, synapses from
the CA1-hippocampus had responses that averaged to about 0.02 volts. In the center, however,
synaptic responses were approximately 0.03 volts. And in the southern section of the
CA1-hippocampus, the synaptic responses averaged to about 0.02 volts. For conflict test mice,
or the positive reinforcement, synapses in the northern section of the CA1-SR had responses
that averaged to about 0.03 volts. In the center, the responses were about 0.035 volts. And in
the south, they were about 0.025 volts. For memory trained mice, the synaptic responses in the
northernmost section of the CA1-SR were about 0.035 volts. In the center, they were 0.05 volts.
And in the south, they were approximately 0.03 volts. The data shows that the synaptic
responses of the trained mice were significantly greater than the synaptic responses of the
untrained mice.

Figure 6: A representation of the percent change in synaptic efficacy in the CA1-SR in trained
and untrained mice in a PSH model. It is also accompanied by the estimated number of actual
synapses that are recruited during memory formation.

In the CA1-SR hippocampus, there are about 105,000 synapses in total, meaning that
there are approximately 35,000 synapses in each location, north, center, and south. Of the
synapses in the north, 11,235 are recruited during the process of memory formation, which is
29.1%. In the center, 31.9% of synapses were recruited, or 11,165 out of the 35,000 synapses
in the region. And finally in the southern area of the CA1-SR hippocampus, 11,235 synapses
were recruited, which is roughly 32.1%.

Discussion:
It was hypothesized that trained mice and untrained mice will have a measurable,
significant difference in synaptic response. Our data suggest that memory causes a
semi-widespread activation of synaptic circuits. This view is in opposition to the conventional
notion that memory changes only a small or restricted fraction of synaptic circuits. The training
conditions had varying synaptic responses across all three of the locations in following the
order: UT < HC < CT < MT. After quantifying the number of synapses in the CA1-SR, and
calculating the percent of change of synaptic responses between trained mice and untrained
mice, it was found that trained mice recruited around thirty percent more synapses in each
section of the CA1-SR. This means that trained mice recruit ten thousand more synapses in
each section than untrained mice do, which equates to over thirty thousand more synapses that
are recruited in trained mice. This data overwhelmingly agrees with the widespread notion of
synaptic recruitment during memory formation.
So, the formation of memory leads to a widespread increase in synaptic function in the
CA1-hippocampus. This is means that memory formation requires a much more complex and
integrated system than many scientists have suggested. The data shows that memory, in fact,
incorporates a cognitive schema, or a framework of knowledge in order to recall or remember
various engrams. This means that, in the hippocampus, past memories are highly invested in
the process of forming new memories because they provide the context that is needed to
understand and comprehend new stimuli, which would explain the widespread increase in
synaptic response that the data shows. Ultimately, memory formation seems to be a cumulative
progression of experiences, building upon each other constantly. Perhaps, this should prompt a
change in the way that scientists research and understand synaptic plasticity and memory
formation.

Acknowledgements:
This research was made possible by the oversight and instruction of Juan Marcos
Alarcon, the help of Rudolf Abdelmessih, Kunal Khanna, Joey Bukai, David Mandil, and the
direction and support of Ms. Erin Schmitz at the Packer Collegiate Institute.
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