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The Effects of Inhibiting Neurons in Layer II of the Medial

Entorhinal Cortex on Hippocampal Place Cells in CA1 and CA3

Roshan Chikarmane

Kentros Lab

A BIOCHEMISTRY MAJOR RESEARCH REPORT

Presented to the Department of Chemistry & Biochemistry

at the University of Oregon

in fulfillment of the requirements for the degree of

Bachelors of Science, May 2017

Clifford Kentros, Principal Investigator

1

ABSTRACT

The hippocampus and medial entorhinal cortex (MEC) are brain regions important for spatial

memory formation and retrieval. Problems with hippocampus and MEC underlay

neurodegenerative disorders like Alzheimer’s Disease and other forms of dementia. Therefore, it

is important that the mechanisms by which these brain regions function and interact are

elucidated. Neurons called place cells in the hippocampal CA1 and CA3 regions fire whenever

an animal occupies a certain location, relative to landmarks in that local environment. The MEC

Layer II (MEC-LII), a brain region that receives inputs from many regions of the cerebral cortex

and projects directly into the hippocampus, also contains spatially responsive neurons called grid

cells. While the behaviors of place cells and grid cells have been well characterized, it is unclear

whether place cells utilize information from grid cells. This study will focus on how the activity

of place cells in the CA1 and CA3 hippocampal regions respond to inhibition of MEC-LII

neurons. To investigate this relationship, we will use a transgenic line of mice that expresses

hM4 receptors exclusively in the MEC-LII. Administration of the hM4 ligand clozapine-n-oxide

(CNO) decreases neuronal activity. Using this method, we can decrease MEC-LII activity and

simultaneously measure the activity of place cells downstream in vivo while mice freely explore

an environment. This experiment will measure the transfer of information between two important

brain regions that give rise to learning and memory.

2

INTRODUCTION

The hippocampus and medial entorhinal cortex (MEC) are brain regions important for the

formation and retrieval of memory. Extensive clinical studies have shown that accumulation of

neurofibrillary tangles, a hallmark of neuron loss in Alzheimer’s Disease, occurs in the

entorhinal cortex, CA1 hippocampal region, and CA3 hippocampal region during Stage I, Stage

IV, and Stage V progressions, respectively. (Braak & Braak, 1991). Significant neuron loss in

layer II of the MEC (MEC-LII) distinguishes even the mildest forms of Alzheimer’s Disease

from individuals without any forms of dementia (Gómez-Isla et. al. 1996). Therefore, a thorough

understanding of how the hippocampus and MEC brain regions function and interact as a

memory system is of paramount importance.

Neurons in the hippocampal CA1 and CA3 regions called place cells fire whenever an

animal occupies a specific location within a local environment, known as a place field (O’Keefe

& Dostrovsky, 1971). Populations of place cells have corresponding place fields that, in

aggregate, represent the full area in a local environment, forming a systematic map of two-

dimensional space (O’Keefe, 1976). A major portion of excitatory inputs into the hippocampus is

comprised of neurons from the MEC-LII called grid cells, which are also spatially responsive.

Each grid cell has multiple firing fields that are separated by a fixed distance and are arranged in

a repeating triangular pattern that covers the local environment (Fyhn et. al., 2004). This grid-

like arrangement has led some to suggest that grid cells are elements of a cognitive metric system

for spatial navigation (Hafting et. al., 2005). Taken together, grid cells in the MEC-LII represent

general two-dimensional space while hippocampal place cells represent specific locations that an

animal occupies within that space.

Curiously, spatial representations in CA1 and CA3 place cells persist after considerable

lesioning of intrahippocampal networks, which has led many to believe that place cell activity

may be generated by direct grid cell inputs from MEC-LII (McNaughton et. al., 1989; Brun et.

al. 2002). Theoretical models illustrate how information from arrays of grid cells may combine

to form place field representations in the hippocampus (O’Keefe & Burgess, 2005; Fuhs &

Touretzky, 2006; McNaughton et. al., 2006). However, there is currently a lack of empirical

evidence that demonstrates the influence MEC-LII inputs have on CA1 or CA3 place cells.

Recent findings in the Kentros Lab show that depolarization of MEC-LII neurons alter

spatial representations in CA1 place cells (Kanter et. al. 2017). Place fields that occurred in one

location prior to MEC-LII depolarization, shifted to a new location in CA1 place cells.

Additionally, CA1 place cells showed increases in place field size and firing rate, with a

conserved firing location, in response to depolarization of MEC-LII neurons. Interestingly,

transformations in place field location have also been observed when salient extrinsic cues in a

familiar environment have been moved and when the size or shape of the geometric borders have

been altered (Muller & Kubie, 1987). This study aims to examine whether inhibition of MEC-LII

neurons also causes changes in firing rate and firing location in CA1 and CA3 place cells.

3

RESULTS

Transgenic Mouse Lines with MEC-LII Specific Control of Neuronal Activation

In order to investigate how the activity of place cells in the CA1 and CA3 hippocampal regions

respond to inhibition of MEC-LII neurons, it was necessary that a system be established to

selectively inhibit neurons in Layer II of the MEC. This required experimental control over two

factors: neuronal activity and expression specificity to the MEC-LII.

To achieve experimental control over neuronal activity, we utilized a hM4Di-tetO line of

transgenic mice that constitutively expressed hM4Di Designer Receptors Exclusively Activated

by Designer Drugs (DREAD) in the brain (Armbruster et. al. 2007). These modified muscarinic

Gi-protein coupled receptors (GPCRs) are potently activated exclusively by the synthetic small

molecule clozapine-N-oxide (CNO), which is pharmacologically inert in wild-type mice, to

induce membrane hyperpolarization. This method is capable of efficaciously causing neuronal

silencing upon intraperitoneal (IP) administration of CNO in live mice.

To achieve expression profiles that are specific to the MEC-LII domain, we utilized a

tTA-EC driver line (Yasuda & Mayford, 2006). These mice strongly express a targeted gene

primarily in layer II of the MEC as well as the pre- and para-subiculum regions, which project

strongly into the superficial layers of the MEC but very weakly into the hippocampus (van Groen

& Wyss, 1990; Honda & Ishizuka, 2004; Köhler, 1985). In contrast, expression ranges from very

weak to not at all in neighboring regions including the deep layers of the MEC, the lateral

entorhinal cortex, the visual cortices, and the caudal regions of the retrosplenial agranular cortex.

We crossed the hM4Di-tetO DREADD line with the tTA-EC driver line to yield double-

positive offspring that express hM4Di DREADDs exclusively in the MEC-LII. Henceforth, we

will refer to these offspring as hM4 mice. To visualize this localization, we performed in situ

hybridization on coronal brain slices using an RNA probe that targeted hM4Di mRNA

transcripts (Figure 1). In accordance with previous findings, hM4Di expression is strong in

MEC-LII neurons, but minimal in neighboring brain regions. Qualitatively, our expression

profile matches that of Kanter et. al. (2017), who quantitatively determined that a similar

expression scheme targeted approximately 27% of layer II stellate cells.

Figure 1. Expression of hM4Di DREADD

receptors in transgenic mice limited to MEC-LII

in right hemisphere of mouse brain in coronal

slice. Visualized by in situ hybridization using

hM4Di-specific RNA probes that had previously

been Nissl stained with cresyl violet.

D, dorsal; L, lateral relative to midline; MEC-LII,

layer II of medial entorhinal cortex; DREADD,

Designer Receptors Exclusively Activated by

Designer Drugs. Magnification is 3×. Tissue

viewed by Olympus BX61 microscope, BX-UCB

control box, Prior ProScanIII motorized stage, and

Lumen200Pro light source. Images captured by

DP72 camera and processed in Photoshop CS4

(Adobe Systems, CA).

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Screening and Experimental Protocols for Place Cell and Grid Cell Recordings

To measure the activity of place cells we implanted multi-channel adjustable-depth tetrode arrays

(four spun iridium/platinum filaments 18 microns in diameter with a polyamide coating and

exposed ends) into the CA1 or CA3 regions of hM4 mice and allowed seven days of recovery

prior to screening. A similar procedure was followed for the recording of grid cells, but the

tetrodes were instead implanted into the MEC of hM4 mice.

Each tetrode was secured in the channel of an EIB-16 electrode interface board, or drive,

with gold pins, which made contact with a tethered HS-18MM operational amplifier during

recordings of neuronal activity (Neuralynx, Bozeman, MT). Recordings in CA3 mice utilized a

four-channel drive with a Teflon housing and three screws (0-80 3× 3/8”), which fixed the drive

to the skull and facilitated the simultaneous adjustment of tetrode depth in all channels.

Recordings in the CA1 and MEC-LII mice utilized a seven-channel VersaDrive-4 microdrive

(Neuralynx) that facilitated the adjustment of tetrode depth in each individual channel.

During screening sessions, implanted mice were allowed to freely roam a familiar

environment. CA1 and CA3 mice roamed a circular environment with a diameter of 60 cm.

Since the place field for place cells are known to orient relative to salient extrinsic cues that are

arranged along a geometric boundary (Müller & Kubie, 1987), the cylindrical wall of the

environment was black except for a white rectangle (8.5” × 11”) at the north end, which served

as the dominant cue. MEC-LII mice roamed a 100 × 100 cm square environment with black

west, east, and south walls but a white north wall.

A digital camera located above the environment recorded the position of the mouse by

tracking the movements of two light-emitting diodes, oriented along the anteroposterior axis of

the mouse. Concurrently, multi-unit neuronal activity was recorded and stored with the Cheetah-

16 system (Neuralynx). Potential changes within a certain range (minimum: 50 µV; maximum:

250-600 µV) were automatically amplified and spikes were band-pass filtered (600-6000 Hz).

Subsequent to recording, the activities of single units were manually separated with the MClust

spike-sorting software for MATLAB using the selection criteria outlined in Kentros et. al.

(2004). The same software was used to correlate place cell or grid cell activity with the mouse’s

position.

Experimental sessions began with a 30-minute recording of baseline activity followed by

an intraperitoneal injection of 10 mg/kg CNO (1.0 mg/mL in 10% DMSO/saline). Shortly after

injection, the mouse was placed in the same environment and neuronal activity was recorded for

120 minutes. After 12-24 hours, the mice were once again introduced into the same environment

and data was collected for 30 minutes.

5

Inducible Hyperpolarization of Grid Cells in MEC-LII in hM4 Mice

A cohort of hM4 mice with microelectrode implants in MEC-LII were first recorded for baseline

activity (30 minutes) the 100 × 100 cm square environment described in the previous subsection

(‘Screening and Experimental Protocols for Place Cell and Grid Cell Recordings’). The mice

were then administered with 10 mg/kg CNO and were recorded in the same environment 120

minutes immediately following administration. Finally, 12-24 hours after CNO, the mice were

recorded once again for 30 minutes. Grid cells included in this analysis had a mean rate of less

than 10 Hz and a peak firing rate of above 0.1 Hz.

In the 120-minute recording session subsequent to CNO, grid cells in the MEC-LII

showed a significant decrease in mean firing rate (hM4 MEC-LII n = 5, p < 0.001; ANOVA),

although activity was restored to its baseline levels 12-24 hours after CNO (Figure 2). These

findings have two important implications for later experiments. Firstly, if 120-minute post-CNO

recordings are conducted on hM4 mice in the same time frame but, instead, place cells are

recorded it can reliably be assumed that hyperpolarization is occurring in MEC-LII grid cells.

Therefore, changes in place cell activity can be attributed to CNO-induced hyperpolarization of

MEC-LII grid cells. Secondly, grid cell hyperpolarization is reversible, as seen by the restoration

of mean firing rate to its basal level 12-24 hours after CNO. Therefore, multiple place cell

experiments can be conducted on the same mouse without disruption to baseline grid cell

activity, as long as those experiments are separated by at least 12 hours.

Figure 2. Administration of 10 mg/kg CNO reversibly induces decrease in mean firing rate in

grid cells within MEC-LII. Mean ± SEM. *p < 0.001

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1

2

3

4

BL 0-30 min 30-60 min 60-90 min 90-120 min +12 hr

Mea

n F

irin

g R

ate

(Hz)

*

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Figure 3 shows the rate maps of representative grid cells in hM4 mice. These rate maps

show a top-down view of the 100 × 100 cm square environment with corresponding grid cell

activity represented by different colors. Grid cells in the initial (Figure 3a) and latter (Figure 3b)

portions of the 120-minute post-CNO recording sessions show hyperpolarization, but show

restoration of basal activity after 12 hours.

Figure 3. Rate maps of representative MEC-LII grid cell shows that firing rate decreases in the

(A) initial and (B) latter portions of the recording sessions following administration of CNO

(10 mg/kg), relative to baseline. Time period following CNO injection and mean firing rate is

represented above and below each rate map, respectively. Baseline rate maps occur prior to

CNO injection. Red regions correspond to regions that the mouse occupied when the cell fired

maximally, blue regions correspond to regions of neuronal silence, and white regions

correspond to areas not explored by the mouse within the specified timeframe.

7

Hyperpolarization of Neurons in MEC-LII Does Not Alter Firing Rate or Firing Location

in CA1 or CA3 Place Cells

A cohort of hM4 mice with microelectrode implants in CA1 or CA3 were first recorded for

baseline activity (30 minutes) in the circular environment with a diameter of 60 cm described in

the previous subsection (‘Screening and Experimental Protocols for Place Cell and Grid Cell

Recordings’). The mice were then administered with 10 mg/kg CNO and, immediately after

administration, were recorded in the same environment for 120 minutes. Finally, 12-24 hours

after CNO, the mice were recorded once again for 30 minutes.

Place cells included in this analysis had a mean rate between 0.1 and 7 Hz, a peak firing

rate of above 1 Hz, and a spatial correlation value greater than 0.5 between the baseline and the

12-24-hour session. Place fields were defined as having at least 20 contiguous pixels (80 cm2)

with a firing rate greater than 20% mean rate. As a precautionary measure, only the 90-120

minute portion of the 120-minute post-CNO time period was taken into account. Normalized rate

changes were calculated as shown in Equation 1 below.

𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑅𝑎𝑡𝑒 𝐶ℎ𝑎𝑛𝑔𝑒 = 𝑀𝑒𝑎𝑛 𝑅𝑎𝑡𝑒𝐵𝑎𝑠𝑒𝑙𝑖𝑛𝑒 − 𝑀𝑒𝑎𝑛 𝑅𝑎𝑡𝑒90−120 min after CNO injection

𝑀𝑒𝑎𝑛 𝑅𝑎𝑡𝑒𝐵𝑎𝑠𝑒𝑙𝑖𝑛𝑒 + 𝑀𝑒𝑎𝑛 𝑅𝑎𝑡𝑒90−120 min after CNO injection

Neither CA1 nor CA3 place cells in hM4 mice showed changes in firing rate (Figure 4)

or firing location (Figure 5) in response to hyperpolarization of MEC-LII cells, relative to wild-

type mice (Wild-type CA1 n = 9, hM4 CA1 n = 33, hM4 CA3 n = 18; two-sample t-test). Their

rate changes and spatial correlations did not vary significantly from the rate changes and spatial

correlations seen in wild-type mice, which presumably did not experience depolarization in

MEC-LII neurons following CNO.

Figure 4. Hyperpolarization of MEC-LII neurons does not change mean firing rate in control

group, CA1 and CA3 place cells. All groups hM4, except for Wild-type. Mean ± SEM.

-0.2

-0.15

-0.1

-0.05

0

0.05

0.1

0.15

0.2

Wild-type CA1 (90-120 min) CA3 (90-120 min)

No

rmal

ized

Rat

e C

han

ge

(1)

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Figure 5. Hyperpolarization of MEC-LII neurons does not cause changes in firing location of

place cells in control group, CA1 and CA3 place cells. All groups hM4, except for Wild-type.

Mean ± SEM.

Figure 6 shows the rate maps of representative place cells in hM4 mice. These rate maps

show a top-down view of the circular environment (diameter 60 cm) with corresponding place

cell activity represented by different colors. Place cells in the CA1 (Figure 6a) and CA3 (Figure

6b) hippocampal regions show mean rate and firing location similar to basal activity in the 120-

minute session immediately after CNO.

Figure 6. Rate maps of representative (A) CA1 place cell and (B) CA3 place cell show that

firing rate and location remains constant following hyperpolarization of MEC-LII cells. Time

period following CNO injection and mean firing rate is represented above and below each rate

map, respectively. Baseline rate maps occur prior to CNO injection. Red regions correspond to

regions that the mouse occupied when the cell fired maximally, blue regions correspond to

neuronal silence, and white regions correspond to areas not explored by the mouse within the

specified timeframe.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Wild-type CA1 (90-120 min) CA3 (90-120 min) CA1 (+12 hr) CA3 (+12 hr)

Spat

ial C

orr

elat

ion

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DISCUSSION

This study demonstrates that, upon hyperpolarization of neurons in layer II of the medial

entorhinal cortex, the firing rate and firing location of place cells in the CA1 or CA3

hippocampal regions remain relatively stable. Our outcome is, paradoxically, surprising because

the MEC is a major source of excitatory inputs into the hippocampus (Zhang et. al., 2013), but

expected because other studies report spatial tuning in CA1 place cells after extensive lesioning

of the MEC (Hales et. al., 2014). Spatial tuning in the hippocampal CA1 and CA3 regions must

be sustained through alternate parahippocampal inputs, which should be explored further. The

stability of CA1 and CA3 place cells could also, conceivably, stem from the fact that

hyperpolarization occurred in a fraction of MEC-LII neurons and that those neurons input to

CA1 and CA3 place cells, which evaded detection.

While MEC-LII inputs are not necessary for spatial tuning in hippocampal place cells,

depolarization of MEC-LII inputs are sufficient to induce changes in CA1 place representations,

namely changes in firing location and bidirectional changes in firing rate (Kanter et. al., 2017).

However, we found that these changes do not occur when MEC-LII neurons are hyperpolarized.

In combination, these findings serve a dual purpose.

Firstly, they demonstrate that MEC-LII neurons and hippocampal place cells act as a

functionally connected system rather than as independently functioning entities. Whether or not

that system exclusively involves grid cells as the source of spatial modulation in place cells has

yet to be determined, since other spatially selective cells reside in the MEC, namely border cells

and head-direction cells (Sargolini et. al., 2006; Solstad et. al., 2006). Secondly, they clarify the

mechanistic role of MEC-LII firing rate on spatial firing in the hippocampus.

In a next step, we should record and include an analysis of border cell and head-direction

cell hyperpolarization in response to CNO, as it would add nuance to the functional types of cell

that are or are not necessary for spatial representations in CA1 or CA3. While it is not yet

possible to isolate expression of inhibitory receptors, like hM4Di, or excitatory receptors, like

hM3Dq, exclusively in grid cells, head-direction cells, or border cells without expression in

surrounding tissues, it would be interesting to examine the effect of rate manipulation

exclusively in those functional cell types on spatial representations in the hippocampus.

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