Tracking memory-induced changes in the CA1 region of the hippocampal network to

compare the extent of its role across spatial memory tasks.

March 1, 2017, Hawthorne Ripley1,2and
Dr. Juan Marcos Alarcon.1
SUNY Downstate

Affiliations:
Department of Pathology, SUNY Downstate Medical Center, 450 Clarkson Avenue,
Brooklyn, NY 11203, USA.1
The Packer Collegiate Institute, 170 Joralemon Street, Brooklyn, NY, 11201.2

Abstract:
The hippocampus is a region of the medial temporal lobe that is known to be
essential for long-term memory operations, particularly encoding, consolidation, and
retrieval of declarative memories. The region also plays an important role in spatial
navigation. Neurons in the mammalian hippocampus have been shown to increase cellular
activity at particular locations within an environment. This suggests that there may be
location-specific regions that are active in the hippocampus for specific spatial memory
tasks. Because of this high level of specificity, the hippocampus can be further broken down
regionally based on the complex and distinct functions of individual neurons and engrams
in these tasks. The question is how exactly spatial memories and learning operations
represent themselves across these regions, and whether the same spatial tasks can be
consistently linked to the same subregions. This study will focus on the CA1, a subregion of
the hippocampus chosen due to its position at the end of the hippocampal formation, the
logic being that memory engrams will be more defined after passing through several other
subregions.
There are conflicting studies on the role of the CA1, and pyramidal neurons in the
region have been suggested to be critical to object differentiation in long-term memory.
The CA1 has also been proposed to be responsible for comparison between retrieved
information and sensory input. The function of the CA1 region during spatial memory
operations will be examined by comparing the number of neurons receiving action
potentials under a variety of behavioral spatial-navigational trainings.
Cre-recombinase/EYFP double transgenic mice will be conditioned in an active place
avoidance memory task, and the genetic manipulation of the mouse allows for a system
that tags neurons associated with different memory stages during this training. Based on
this information, the activation of the region can be compared across several behavioral
conditions of trained and untrained mice, which will suggest in what contexts the CA1
serves the hippocampus and the brain as a whole along the process of acquiring,
consolidating and retrieving spatial-navigational memory engrams. It is hypothesized that
the CA1 will be most active during the acquisition of conflicting spatial memory, and that
the results will show more active cell bodies in mice that have undergone Conflict Test
behavioral training (in which mice undergo behavioral training that differs from
habituation conditions), based on evidence that proposes the CA1s responsibility in
comparing a matchmismatch of memory retrieval sensory input.

Introduction:
The brain is a network, the links of which undergo constant changes through
molecular modulation (Kandel, 2012). These changes are a result of the acquisition of new
memory engrams (Long-term Potentiation) and deterioration of neglected ones
(Long-term Depression) (Eichenbaum, 2012). This ongoing process, called synaptic
plasticity, underpins how memories are stored and retrieved (Kandel, 2012).
The hippocampus in particular is known to play a role in the ability to form and
retain new declarative memories (Lavenex et al., 2006). This subset of recollected facts and
events refers to those memories that can be consciously recalled (or "declared"). It consists
of information that can be explicitly stored and retrieved, such as facts, experiences, and
concepts (Klein et. al. 2002). Studies in laboratory mice demonstrate that damage to the
hippocampus results in impairment of spatial memory (Kolb and Wilshaw, 1996). Research
on the human patient Henry Molaison, whose medial temporal lobe (including the
hippocampus) was removed bilaterally to treat his epilepsy, also indicates that while his
ability to acquire and maintain short-term memories was intact, he could not consolidate
them into long-term memory, signifying that the hippocampus is required for memory
consolidation (Corkin et al., 2002). As with all memory operations, neurons facilitate the
ability of the hippocampus to store and encode spatial-navigational engrams (Lavinex et al.,
2006). Still, the physical representations of memory throughout the brain is largely
unknown beyond understanding the basic importance of certain regions such as the
hippocampus.
In order to determine the precise role of the hippocampus in memory operations, it
is necessary to determine the distinct significance of its subregions. This study will focus on
tracking neuronal activation during spatial learning in the CA1, the first region of the
hippocampal circuit. Since memory is thought to be represented by vastly interconnected
synaptic networks coordinated by molecular systems, it leaves a physical, traceable effect
on the neurons it touches (Dudai, 2004). Employing our knowledge of these traces in the
observation of hippocampal CA1 neurons using behavioral training of mice and
immunohistochemistry can provide an explanation for the hippocampuss important
position in declarative memory consolidation.
The CA1 is of particular interest because it contains a major output pathway to the
entorhinal cortex, the main interface between the hippocampus and the region of the brain
responsible for motor commands and spatial reasoning, the neocortex, and because it has
been proposed to be responsible for the comparison of retrieved information and sensory
input (Hesselmo, 2005). This study will investigate how spatial learning experiences
recruit neurons in the CA1. It is hypothesized that the CA1 plays an important role in the
hippocampuss ability to consolidate new spatial memories that conflict with stored
information, and that consolidation of spatial memories in an active place avoidance task
with a shock zone that has been relocated will significantly activate neurons in this region
as compared to control and regularly trained conditions, and activate the CA1 in the
comparison between the received shock zone and the sensed location of the current shock.

Project Description:
In recent decades, neuroscientists have worked tirelessly on locating the memory
trace, or in other words identifying how memories are encoded and stored on a regional,
molecular, and engram-level (Kandel, 2012). The goal of this project is to come one step
closer to understanding what a memory looks like, by tracking and comparing
memory-affected neurons in the hippocampus based on knowledge of the behavioral
training the mouse has undergone. Comprehensive knowledge of the subregions of the
mammalian hippocampal system is essential to gain a better understanding of how people
learn and remember; understanding how memory is retained could allow for more
targeted treatment of learning challenges and neurodegenerative diseases.
There are two major pillars that will allow these physical changes in the CA1 to be
tracked effectively. The first as that memory needs to be supplied through conditioning,
forcing learning to occur, and the second is that the changes associated with that specific
memory within the region of the hippocampus must be readily identifiable through
confocal microscopy of immunohistochemistry-treated hippocampal slices. This project
uses an Arc-Cre-driven tagging process to identify what neurons are active during four
different learning conditions in mice, so that the CA1 region of the hippocampus can be
observed across a four spatial memory scenarios and its purpose can be better understood.
There are conflicting theories on the role of the CA1 in matching entorhinal input (signal
that the CA1 receives) with retrieval of associations between environments and context
(Lee et al., 2005), (Hasselmo, 2005). This study hopes to clarify the distinctive role of the
region in spatial learning and memory retrieval.
Several potential trends for the results of this project are possible, all of which are
based on which conditioning expresses YFP in hippocampal slices most consistently and
intensely. Either (1) expression is strongest in untrained mice, (2) expression is strongest
in trained mice, (3) expression is strongest in conflict test mice, or (4) expression is
constant across behavioral trainings. If expression is strongest in mice that underwent
training without the shock zone (untrained mice), it could potentially mean that the CA1 is
a region that is integral to spatial awareness rather than learning, and that another region
would be activated to compensate for a lower activation of the CA1 during the shock-based
learning. If expression were strongest in conflict test mice, it would suggest that the region
is required more for comparison of mis-matched sensory input, rather than performing
more general spatial learning as in the trained mice. Strong expression in trained mice
would demonstrate a strong link between initial learning and retention rather than recall
or application of retained information. If expression were constant across all behavioral
trainings or in the control/home cage mice, it would suggest that the CA1 is an area that is
required for the hippocampus to be functional in the basic retention and acquisition of all
spatial-navigational memories, rather than one type of learning in particular, which would
mark it as a highly important region overall as compared to the CA3 and dentate gyrus.

Methodology:
In order to compare the activation of neurons under conditions of learning and
recall in spatial memory activity, Cre-recombinase EYFP double transgenic mice will be
injected with Tamoxifen in order to regulate the recombination of the Lox-p sequence of
the YFP gene. Activation of the Arc promoter will lead to the Cre driven expression of
enhanced yellow fluorescent protein (EYFP) in neurons that have been strongly activated
by the neural activity that accompanies, in this case, spatial learning and recall. These
double transgenic mice will be conditioned in an active place avoidance task, in which they
learn to avoid a shock zone in a spinning arena through memory of colored cues (see figure
1). The behavioral conditioning of the double transgenic mice will span from mice without
spatial experience of the spinning arena (home cage), to mice have been habituated in the
arena (untrained), to mice who have learned how to avoid the shock zone (trained).
Confocal imaging and immunohistochemistry will be used to observe YFP positive cells
(signaling their activation in this task) in the form of YFP-produced fluorescent tags, and
these active cells will be quantified using ImageJ particle analysis.
The first step of this process is behavioral training. Mice are placed on a rotating
arena with a shock zone covering one-sixth of the total area. The mice will learn to avoid
the shock zone through spatial cues attached to the walls of the behavioral room (see
Figure 1). The movement of the mice will be measured through camera tracking. Home
cage mice are left in an isolation chamber, while all others undergo habituation in the
behavioral room, before being placed on the rotating arena for 30 minutes with the shock
zone turned off. On the third day, trained mice will be subjected to three 30 minute training
runs in which they learn to avoid the shock zone on one side of the arena. The untrained
mice that will be habituated to the arena on the previous day will be subjected to training
with the shock zone off.
Figure 1: Visual representation and photograph of the training apparatus. (A) A model of
the training apparatus. (B) A top view of the training apparatus. The three shapes in the
corners signify spatial cues.

Figure 2: Table showing a total of four behavioral tests; two of which are untrained
(controls) and two that are trained. Habituation ensures that unrelated stimuli from
general surroundings are not taken as false learning during day two. (1) Home cage
condition controls for lack of spatial-navigational memory, as mouse is never placed in the
arena. (2) Untrained condition subjects the mouse to only the first day protocol
(habituation with shock-zone off). (3) Trained mice experience the shock zone and later
undergo a retention test with the shock zone turned off. (4) Conflict test moves the shock
zone to the other side of the arena (signified by flipped lightning bolt). Tamoxifen is
injected before the second training runs on day two, and before the mouse is put back into
the arena on the third day.
Figure 3: The learning pattern of mice in an APA task based on motion sensing. (C) the
average behavioral data acquisition on training day one. (D) the average behavioral data
acquisition on day three.

For the collection of slices, double transgenic mice will be anesthetized and
euthanized by decapitation. The brain will then be dissected and the cerebellum and frontal
lobe removed. After being secured on a metal stage and placed on a slicing machine, the
medial temporal lobe will be cut into 200-micrometer slices. These will each be placed into
artificial cerebrospinal fluid (ACSF) wells to keep the cells alive. Next, they will be treated
with paraformaldehyde (PFA) to decrease their fragility. The slices will then be transferred
into new wells containing phosphate buffer solution (PBS) and those wells will be covered
with foil. Slices will next be washed on a shaker 15 minutes. Afterwards, they will be placed
into new wells containing PBST for permeabilization. The new wells will be placed onto the
shaker for 15 minutes, three times, each time with new wells and PBS solution. 500uL of
DAPI solution (staining for nucleic acids) will be added into each well to tag all nucleoli.
The wells will then be placed on a shaker for 15 minutes. They will then be transferred into
new wells with PBST and placed on a shaker for another 15 minutes. Primary and
secondary antibodies will be bound to the YFP antigens by being placed in wells with the
slices on a shaker, one after the other, each for 15 minutes. The slices will then be mounted
onto glass slides, and left to dry for 30 minutes. Antifade solution will then be placed on the
slices and a coverslip is placed on top of the slices. Confocal imaging uses laser power to
stimulate the flourophores in EYFP expressed in the neurons of the mounted slices,
allowing for the visibility of neurons tagged by EYFP in the CA1. Because EYFP is expressed
as a result of the activation of Cre Recombinase, whose promoter is the early gene Arc-Cre,
the fluorescent light around the cell bodies will represent the number of neurons tagged
with EYFP in the hippocampus.
 In order to quantify these results, ImageJ particle analysis technology will be used
with a threshold with limits 0-57. A 300 by 150 pixel area will be selected in the CA1 region
of the hippocampus, and cells that fit inside the threshold will be counted by the ImageJ
software (see Figure 4). This method was chosen over manual counting or a cell-counter
pipeline program due to its objectivity and ability to differentiate between cell bodies and
the dendrites seen on the right of figure 4, and to other visual noise before beginning
color-based quantification. The grayscaled image will be divided into a grid with each box
at two square pixels. This will allow for a comparison of neuron activation across the CA1
by comparing the coordinates of the activated neurons across different conditions. This will
allow for further distinction beyond what memory operations activate the CA1 the most,
answering the question of whether the location of activation changes in the CA1 based on
memory tasks.

Figure 4: 10x hippocampal confocal image before greyscaling and particle analysis (left)
and after greyscaling and particle analysis with analyzed area (CA1) highlighted by yellow
box (right). In this example, the ImageJ software counted 23 active cells in the selected CA1
region.

Figure 5: A plot of the CA1 region from Figure 4 to demonstrate how active neurons will be
locationally profiled based on coordinates. For example, the red circled neuron is at (14, 3).
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