Sparser Representation of Experiencein Aged Rat Lateral Entorhinal Cortex

Item Type text; Electronic Thesis

Authors Comrie, Alison Emelie

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this materialis made possible by the University Libraries, University of Arizona.Further transmission, reproduction or presentation (such aspublic display or performance) of protected items is prohibitedexcept with permission of the author.

Download date 21/05/2018 19:15:27

Link to Item http://hdl.handle.net/10150/624949

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Abstract

The hippocampus undergoes biological changes with age that mediate memory

dysfunction. The hippocampus is highly connected to lateral entorhinal cortex (LEC), which is

thought to represent non-spatial features of experience, including odors. We aimed to discover if

LEC neuronal populations are selectively activated by distinct odors, and hypothesized that

aging alters these activity patterns. After training adult and aged rats to run laps around a track,

one behavioral group experienced two run sessions, 20 minutes apart, with the same set of

odors (AA) added around the track each time, while another group had distinct odor sets (AB).

mRNA of the immediate-early gene Arc is localized to discrete neuronal compartments based

on the time since activation. We used fluorescence in situ hybridization and confocal

microscopy to visualize the subcellular distribution of Arc mRNA to identify the neurons

activated during each session. The behavioral experiences induced elevated LEC activity, but

population activity failed to distinguish between distinct odor sets. This suggests that LEC

populations stably represent higher order features of experience. Additionally, a lower

proportion of LEC neurons participated during behavior in aged than in young rats. A decrease

in neuron activation could reflect a reduction or refinement of LEC network function.

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Acknowledgements

This work was supported by R01 AG003376, F32 AG033460, the McKnight Brain

Research Foundation, and HHMI 5200006942. Additionally, I would like to thank Dr. Carol

Barnes for extensive mentoring, Dr. Jim Lister for conducting the behavioral portion of the

experiment and for guidance, Dr. Monica Chawla for histological training, Doug Cromey for

miscroscopy training, Jeri Meltzer and Hana Davis for assistance with image analysis, Dr.

Rachel Samson for assistance with statistical modeling, Michael Montgomery for technical

assistance, and Michelle Carroll, Michelle Albert, and Luann Snyder for administrative

assistance.

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Introduction

Normal aging in the absence of pathology entails cognitive changes including memory

deficits in humans and other mammals. The great majority – 86 percent – of people over the

age of 71 are not demented, but have healthy cognitive aging (Plassman et al., 2007).

Therefore, it is critical to establish an understanding of the biological changes that underlie

normal age-related memory decline.

The hippocampus is a brain region that undergoes characteristic changes with age that

mediate episodic memory dysfunction in rodents and humans. For example, aged rats have

impaired spatial memory. The neural basis of this behavioral problem involves hippocampal

place fields that inappropriately remap when a rat explores a familiar environment (Barnes,

Suster, Shen, & McNaughton, 1997) and fail to remap in a novel environment (Sava & Markus,

2008). These and other age-related changes in hippocampal neural population activity may

arise in part from altered activity in connected brain regions such as the entorhinal cortex (EC).

Despite the fact that the EC provides major inputs into many subregions of the

hippocampus, the aging of the EC remains poorly characterized. The EC and hippocampus are

anatomically heterogeneous, but the hippocampus primarily receives projections from

superficial layers of EC and sends back-projections to deep layers of EC (Figure 1). Therefore,

examining the activity of superficial EC neurons informs us about the influence of EC projections

to the hippocampus, while studying the activity of deep EC neurons provides insight into the

influence of hippocampal projections to EC. The EC has two functional subdivisions: the better-

studied medial EC (MEC) and understudied lateral EC (LEC). MEC plays a role in spatial

information processing and spatial memory, however, the basic functions of LEC are not

understood. In contrast to neurons in MEC, LEC neurons do not predominantly fire in a

spatially-selective manner (Deshmukh & Knierim, 2011; Hargreaves, Rao, Lee, & Knierim,

2005). LEC neurons receive significant inputs from olfactory areas, piriform cortex, and

perirhinal cortex (Figure 1), and the rodent and monkey connectivity patterns are comparable

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(Burwell & Amaral, 1998; Canto, Wouterlood, & Witter, 2008; Insausti, Herrero, & Witter, 1997).

These regions are implicated in processing information about odors and objects, corroborating

the idea that the LEC responds preferentially to local, non-spatial, external attributes of

experiences (Knierim, Neunuebel, & Deshmukh, 2014). For example, neurons in LEC have

been reported to fire in response to individual features of the environment including objects and

odors (Young, Otto, Fox, & Eichenbaum, 1997; Zhu, Brown, & Aggleton, 1995). It remains

unclear whether a distributed population code represents items in an environment, but this

would be consistent with the finding that some LEC neurons responded to a specific subset of a

group of objects in an open exploration task, rather than to one or all objects (Deshmukh &

Knierim, 2011). Furthermore, the stability of LEC responses across multiple experiences is not

understood, and may be impacted by environmental complexity, brain state, attention, context,

stimulus novelty, and salience (de Curtis & Paré, 2004; Kajiwara, Takashima, Mimura, Witter, &

Iijima, 2002; Rodo, Sargolini, & Save, 2017). If any of the functional properties of superficial

layers of LEC are disrupted in aging, then the hippocampus would be subject to altered inputs,

which may impact output and therefore deep layers of LEC.

Figure 1 Simplified summary of relevant circuitry. Superficial lateral entorhinal cortex (LEC) receives

inputs from piriform cortex (PIR) and perirhinal cortex (PRC). Superficial LEC projects to different

subregions of the hippocampus (DG, CA1, CA3), while deep LEC receives hippocampal back-projections.

Deep LEC sends projections down to cortical areas including PIR and PRC.

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During normal aging, LEC and hippocampal neuron numbers are preserved, although in

the hippocampus, synaptic connections are compromised with age and are associated with

cognitive decline (Geinisman, de Toledo-Morrell, Morrell, Persina, & Rossi, 1992; Merrill, Chiba,

& Tuszynski, 2001; Nicholson, Yoshida, Berry, Gallagher, & Geinisman, 2004; Rasmussen,

Schliemann, Sørensen, Zimmer, & West, 1996; Smith, Adams, Gallagher, Morrison, & Rapp,

2000). In contrast, the LEC is vulnerable to neurodegeneration in early stages of Alzheimer’s

disease for unknown reasons (Braak & Braak, 1992). Biochemical changes have been observed

in the aged EC that could mediate population-level alterations, including reduced reelin and

synaptophysin expression (Stranahan, Haberman, & Gallagher, 2011). A better understanding

of behaviorally-relevant age-related changes in EC neural population activity will be important

for understanding how the aged EC may impact hippocampal function, and how EC function

may be impacted by back-projections from the aged hippocampus.

Interestingly, odor and object discrimination of similar stimuli are impaired with age,

although odor detection thresholds are not (Burke & Wallace, 2011; Yoder et al., 2017). This

age-related impairment could be due in part to LEC dysfunction, as fine odor discrimination is

lost upon LEC silencing by muscimol (Chapuis et al., 2013). Additionally, odor memory and odor

perception deficits are characteristic preclinical signs of a number of neurodegenerative

diseases, including Alzheimer’s Disease (Devanand et al., 2000; Mesholam, Moberg, Mahr, &

Doty, 1998; Segura et al., 2013). Studying how LEC ages in conjunction with behaviors that

involve olfaction may therefore be useful in the development of diagnostic tests and novel

therapies for aged individuals.

The LEC is clearly well-positioned to represent features of an environment that are

combined with spatial information in the hippocampus to form multimodal episodic memories

that are impaired with age. However, the role of the LEC in representing and discriminating odor

experiences and how the corresponding neural population activity may change with age remain

unknown.

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To probe these questions, we employed a histological method to examine the activation

of populations of neurons in adult and aged rats during olfactory experiences. We monitored

activity-dependent expression of the immediate early gene Arc to assess the neural ensembles

activated at two behavioral time points per rat. In contrast to high-density electrophysiological

recordings, this approach provided a large sample size of tens of thousands of neurons.

Additionally, we could ensure that we were assessing principal neuron activity, as Arc is

expressed only in principal neurons, not interneurons or glia (Vazdarjanova et al., 2006).

Cellular compartment analysis of temporal activity by fluorescence in situ hybridization

(catFISH) enabled us to fluorescently label Arc mRNA that is expressed by strongly activated

neurons (Guzowski, McNaughton, Barnes, & Worley, 1999). As time passed between the first

and second behavioral epochs, the Arc mRNA was translocated with tightly regulated kinetics

from the nuclei to the cytoplasm of the neurons. The labelled tissue was visualized and neurons

were characterized based on the localized, subcellular distribution of Arc mRNA (see Methods).

Neurons activated in the first behavioral epoch had cytoplasmic Arc signal, neurons activated in

the second behavioral epoch had nuclear Arc mRNA foci, and neurons activated in both were

“double-labelled” in the cytoplasm and nucleus.

The behavioral design included two epochs with distinct or repeated odors, intended to

induce LEC neural population activation (and reactivation) in adult and aged rats. Rats were

trained to run in alternating clockwise and counterclockwise laps on a circular track in a constant

spatial environment. After training, one behavioral group (AA) experienced the same set of 6

odors mixed in sand in ramekins in the same order around the track during two 5-minute

running sessions (adult mean = 7.4 laps/session, aged mean = 9.8 laps/session) separated by

20 minutes. A second group (AB) also experienced two running sessions, but the odor stimuli

were completely distinct between the first and second time points.

We quantified the proportion of total neurons activated in each epoch under the

hypothesis that neural population activity in LEC would stably represent repeated odor

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experiences and selectively represent distinct odor experiences – and that these patterns would

be impacted by age. Detection of a significant proportion of double-labeled neurons in the AA

condition would provide evidence for stable representation of repeated experience, while

double-labeled neurons in the AB condition would provide evidence that the same population of

cells can represent distinct odor experiences. A higher proportion of single-labeled (cytoplasmic

or nuclear positive) neurons in the AB condition would support the idea that different sets of

LEC neurons were activated by different odorants. These patterns may be affected by age and

may differ between the superficial and deep layers of LEC. For example, deficits in superficial

LEC could drive changes in the hippocampus. Alternatively, effects of age may be exaggerated

in deep layers of LEC due to defective inputs from the aged hippocampus. These age-related

changes may together contribute to memory deficits observed in aged animals.

Methods

Subjects and behaviors

Twenty-three adult (9 months) and 24 aged (24 months) experimentally naïve male

Fischer 344 rats (from the National Institute of Aging’s colony, Wilmington, MA) participated in

the experiment. Rats were individually housed in transparent cages (44 cm deep x 23 cm wide x

20 cm tall) in a colony room with a reverse 12-hour light-dark cycle. All behavioral testing took

place during the dark cycle. Prior to behavioral testing, each animal was handled by

experimenters for 10 minutes for 2 days. Rat weights were monitored throughout the course of

the experiment. Rats were provided water with ad libitum access for the duration of the

experiment, and rodent chow pellets (Teklad, Harlan Laboratories) with ad libitum access until

behavioral testing and diet restriction began.

Rats completed the spatial and visually-cued versions of the Morris water maze task as

previously described (Morris et al., 1982; Barnes et al., 1996) to assess spatial memory and to

test for blindness. Data were analyzed with ANY-maze software (data not shown here).

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After completion of the water maze protocols, food deprivation began, and health was

closely monitored for one week. Food was restricted to maintain 85% original body weight, and

diet consisted of a mash made of rat chow, STAT (PRN Pharmacal, Pensacola, FL) and apple

sauce (Motts’ brand, Plano, TX). Rats were each trained to run clockwise and counterclockwise

laps around a circular track (48 inches diameter). Before training, rats were familiarized with the

track environment for 15 minutes of free exploration with crushed food pellets sprinkled around

the track. Training took place for 10 minutes daily for 1 week prior to the experiment day. The

track had a divider to indicate the beginning and end of a lap, and rats were rewarded at both

ends with a mixture of mash and vanilla-flavored nutrition shake (Ensure brand, Abbott

Laboratories) that was dispensed by hand in plastic trays. On the last 2 training days, sand-filled

ramekins were placed on the track to acclimate the rats to the objects. The ramekins were small

enough that rats had more than half of the track width to pass the ramekins. However, the rats

were also close enough to the ramekins that they could whisk and explore them. This exposure

ensured that the ramekins were not novel objects on experimental days.

Animals from each age group were randomly assigned into positive control, negative

control, and two behavioral groups (AA and AB). On experiment day, the AA and AB rats each

completed two 5-minute epochs of running on the circular track for food reward, separated by

20 minutes in their home cages (Figure 2). Rats were transported between their home cages,

the behavioral testing room, and the room in which sacrifice took place in towel-lined pots to

minimize exposure to extraneous stimuli. Unlike during training, 6 distinct odors were mixed into

the ramekins around the track on experiment day. Rats in the AA group experienced the same

odors in the same order around the track for both track running epochs. These A odors were

nutmeg, coffee, sage, ginger, smoky paprika, and cumin. Rats in the AB group experienced a

distinct odor set during each epoch; first, the aforementioned A odors, and then the B odors.

The B odors were black pepper, cinnamon, baby powder, ground clove, pumpkin spice, and

garlic powder. One teaspoon of the powdered odor was mixed into 10 teaspoons of sand in

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each ramekin. The behavioral testing room and any potential visuospatial cues were constant

across behavioral epochs.

Figure 2 Behavioral paradigm. Adult and aged rats each completed clockwise and counterclockwise

laps on a circular track for food reward at both sides of the barrier for 5 minutes. 6 odors were spaced out

around the track in ramekins. After 20 minutes in the home cage, rats underwent a second behavioral

epoch with either the repeated A odors (A-F) or distinct B odors (G-L).

Upon completion of the second epoch of track running, rats were anesthetized in a bell

jar filled with isoflurane and then decapitated with a guillotine. Rats from the negative control

group were sacrificed from their home cages to assess baseline Arc expression (Cage Control,

CC), and rats from the positive control group underwent Maximal Electroconvulsive Shock

(MECS) 5 minutes before sacrifice to induce Arc expression in all possible neurons. All handling

and experimental procedures were in accordance with the University of Arizona Institutional

Animal Care and Use Committee protocols.

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Tissue processing

Brains were rapidly extracted and hemisected immediately upon sacrifice and flash-

frozen in -70°C isopentane in a mixture of dry ice and ethanol to preserve Arc mRNA. Left

hemispheres were stored in a -80°C freezer. Blocks of 8 hemispheres were made using OCT

Compound (Ted Pella, Inc., Redding, CA) and each 2 x 4 block included hemispheres to

represent each experimental condition; an adult and an aged animal of each control (CC,

MECS) and behavioral (AA, AB) group. Six total blocks were made. Blocks were cut into 20

micron coronal sections in a cryostat and sections were mounted on Super Frost Plus slides

(VWR), then stored at -80°C.

A cresyl violet Nissl stain was performed on a series of every tenth section (200 microns

apart) to identify sections containing lateral entorhinal cortex (LEC, Figure 3). Permount

(Thermo Fisher Scientific Inc.) was used to coverslip stained sections. Cytoarchitectonic

markers were used to identify LEC; ventral to the lateral sulcus, sparse layer I, large island cells

in layer II, sparse lamina dissecans layer IV, dense layers V/VI with smaller neurons. These

layers contrast the cytoarchitecture of the neighboring perirhinal cortex and pyriform cortex, and

were therefore used to select 3 consecutive sections containing LEC per hemisphere for

histology. Nonconsecutive sections were only used in the case of damaged tissue.

Figure 3 LEC image locations. 4 images were taken per section (see boxes); 2 superficial and 2 deep. 3

consecutive sections of left LEC were imaged per animal, resulting in 12 images collected in total per rat.

Paxinos & Watson, 2008

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Cellular compartment analysis of temporal activity by fluorescence in situ hybridization

(catFISH) was carried out for immediate-early gene Arc mRNA on the selected sections

(Guzowski et al., 1999). In summary, a plasmid containing Arc cDNA was used to generate a

riboprobe labeled with digoxigenin. The digoxigenin labeled Arc antisense riboprobe was

hybridized with tissue overnight. The probe was detected with anti-digoxigenin-HRP conjugate

and visualized with cyanine-3-labeled tyramide. Nuclei were counterstained with DAPI in

Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA) and sealed with nail

polish. The nuclear stain enabled differentiation of cytoplasmic and nuclear Arc mRNA.

Imaging and analysis

A Leica SP5-II resonant scanner confocal microscope (Leica Microsystems, Buffalo

Grove, IL) was used to take four image volumes per section; two superficial and two deep

images (Figure 3). Images were captured with a 40x/1.25NA PL Apo oil immersion objective at

1024 x 1024 pixel resolution through the depth of the entire section, in 1 micron steps.

The detector gain and amplifier offset settings for imaging the DAPI signal were adjusted

to easily differentiate non-neuronal cells from neurons. Non-neuronal nuclei have a smaller,

rounder shape, with bright and uniformly distributed signal. This contrasts with the larger, less

regular shape, and sparser signal distribution of neuronal nuclei (Vazdarjanova et al., 2006).

The detector gain and amplifier offset settings for Arc signal were adjusted to capture an

image that reproduced the signal observed through the eyepiece. The settings were established

for each slide based on controls, and were only adjusted across sections within a slide to

accommodate cases of uneven staining. The experimenter who collected images was blind to

the age and behavioral treatment that corresponded to each section.

Images were analyzed offline with ImageJ software. Only neurons with all or part of the

nucleus in the median 20% of the image z-stack and fully contained in the x and y planes were

characterized to avoid sampling error from partial cells. Neurons were differentiated from non-

neuronal cells using the DAPI signal before overlaying the Arc signal to avoid bias. The

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compartmentalization of the Arc signal was used to classify each neuron (Figure 4) as nuclear

positive (Nuc+), cytoplasmic positive (Cyto+), double-labelled (Dbl+), or negative (Neg). Nuc+

classifications were made when a neuronal nucleus contained one or two distinct puncta

spanning at least three optical planes. Cyto+ classifications were made when the Arc signal was

outside of and surrounding at least half of a neuronal nucleus. Dbl+ classifications were made

when both nuclear foci and cytoplasmic staining occurred on the same neuronal nucleus, and

Neg classifications were made when Arc signal was neither present in nor around a neuronal

nucleus. Image counters were blind to the age and experimental condition for each section.

Images from all age groups and behavioral conditions were distributed among people

classifying cellular localization of Arc mRNA in this study.

Figure 4 Neuron classification. (a) Double-labelled neuron with Arc mRNA (red) in the DAPI-labeled

nucleus (blue) and in the cytoplasm. This cell was active in both the first and second behavioral epoch.

(b) Cytoplasmic positive neuron with Arc mRNA in the cytoplasm of the neuron, surrounding the nucleus.

This cell was active during the first behavioral epoch. (c) Nuclear positive neuron with Arc mRNA foci in

the nucleus. This cell was active during the second behavioral epoch.

Counts in each category were converted to percentages of total number of neurons for

each image volume. A Generalized Linear Mixed-effects Model (GLMM) was employed in R.

The glmerMod function from the lme4 package was used (Bates, Mächler, Bolker, & Walker,

2015), applying the following general equation: η = Xβ + Zγ.

The GLMM differs from a standard generalized linear model in that there is a random

effects term (Zγ) in the linear predictor expression (in addition to the fixed effects term (Xβ)).

Such a model is appropriate for these data because it has multiple levels; the proportion of cells

in each Arc localization category for each image are nested at the level of the individual rat

a. b. c.

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brain, age group, and experimental condition. The proportion of activated neurons (η) has a

binomial error distribution. The matrix of the predictor variables (X) includes Arc localization

(nuclear positive, cytoplasmic positive, double-labelled, negative), age group (adult, aged), layer

(superficial, deep), and behavior (AA, AB, CC, MECS), and β is a vector of the regression

coefficients for the predictor variables. Z is the design matrix for the random effects and γ is a

vector of regression coefficients for the random effects (to account for testing at multiple time

points per rat). In the loss function, the squared error was weighted by the number of neurons

counted in each image. Significance was assessed using the log-likelihood ratio test. Kruskal-

Wallis tests were used in cases where GLMM residuals were not normally distributed.

Bonferroni corrections were made after each analysis. Normality in the distribution of the model

residuals was assessed using scatter plots and quantile-quantile plots.

Results

Total proportion of neurons expressing Arc

To examine the proportion of neurons in lateral entorhinal cortex (LEC) activated by

track running in the presence of odors, adult and aged rats underwent two 5-minute behavioral

epochs separated by 20 minutes in their home cages. Rats in the AA group experienced the

same set of 6 distinct odors around a circular track during both epochs, while rats in the AB

group experienced distinct sets of 6 odors around the track in the first and second epochs. The

mean number of laps completed per session by each behavioral group were: 8.7 for adult AA

rats, 6.2 for adult AB rats, 10.1 for aged AA rats, and 9.6 for aged AB rats. On average, aged

rats completed a slightly higher number of laps per session than did adult rats. The presence of

Arc mRNA was then used as a proxy for neural activity.

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Figure 5 Percentage of neurons expressing Arc. (a) Overall proportion of superficial LEC neurons with

Arc mRNA. (b) Overall proportion of deep LEC neurons with Arc mRNA. (a-b) Behavioral condition, layer,

and age, all significantly affected the percentage of LEC neurons that expressed Arc. Deep layers had

slightly but significantly higher percentages of Arc-expressing neurons than did superficial layers. For

both layers, rats in the AA and AB groups had higher percentages of Arc-expressing LEC neurons than

did rats in the CC group, and lower percentages of Arc-expressing neurons that did rats in the MECS

group. There was no difference in the proportion of Arc-expressing LEC neurons between the AA and AB

groups. Aged rats in the AA and AB groups exhibited a reduction in the percentage of LEC neurons

expressing Arc compared to adult rats, while aged rats in the CC group did not.

If LEC neural populations represented information about the experience of track running

in the presence of odors, then a higher percentage of neurons would express Arc during the AA

and AB conditions than during CC conditions. If distinct odor stimuli were represented by

different neural ensembles, and repeated stimuli reactivated stable neural ensembles, then the

AB condition would induce Arc expression in a higher total percentage of LEC neurons across

epochs than would the AA condition. We found that behavioral condition had a significant effect

on the total percentage of LEC neurons that expressed Arc (GLMM, X2(2) = 72.85, p < 0.0001).

As expected, rats in the MECS group had a higher proportion of Arc-expressing neurons

Superficial LEC Deep LEC a. b.

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compared to rats in the AA, AB, and CC groups (Figure 5). Additionally, rats in the CC group

had a significantly lower proportion of Arc-expressing neurons than rats in the AA, AB, and

MECS groups (Figure 5). There was no significant difference in the overall percentage of cells

that expressed Arc between the rats in the AA and AB groups (GLMM, superficial X2(1) = 0.16,

p > 0.5; deep X2(1) = 0.36, p > 0.5).

Age significantly affected the percentage of LEC neurons with Arc mRNA for rats in the

AA and AB groups (GLMM, superficial X2(1) = 7.38, p < 0.01; deep X2(1) = 5.25, p < 0.05), but

not for rats in the CC group after correcting for multiple testing (Kruskal-Wallis, superficial X2(1)

= 4.27, p > 0.05, deep X2(1) = 1.96, p > 0.1). Adult rats had consistently higher proportions of

LEC neurons with Arc mRNA than did aged rats during the track running behavior in the

presence of odors (Figure 5).

Layer also had a significant effect on the percentage of neurons that expressed Arc

(GLMM, X2(1) = 62.33, p < 0.0001). A significantly higher proportion of neurons had Arc mRNA

in deep LEC than in superficial LEC (Figure 5). However, there was no interaction between

layer and age (GLMM, X2(1) = 0.34, p > 0.5).

Distribution of Arc mRNA across subcellular compartments

To investigate how populations of neurons were activated by repeated (AA) and distinct

(AB) odor experiences while track running, we assessed the subcellular compartmentalization of

Arc mRNA. As a result of the tightly regulated kinetics of activity-induced Arc expression,

neurons activated during the first epoch exhibited cytoplasmic Arc mRNA (Cyto+), and neurons

activated during the second epoch exhibited nuclear Arc mRNA (Nuc+). Neurons activated at

both time points were double-labelled with nuclear and cytoplasmic Arc mRNA (Dbl+).

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Figure 6 Percentage of LEC neurons activated by two behavioral epochs (AA, AB) or non-

behavioral control conditions (CC). Nuc+ indicates activation during the second epoch, Cyto+ indicates

activation during the first epoch, and Dbl+ indicates activation during the first and second epochs. For rats

in the AA and AB groups, we observed a higher proportion of Nuc+ than Dbl+ neurons and a higher

proportion of Dbl+ than Cyto+ neurons, across both superficial and deep layers. Deep layers exhibited a

higher proportion of activated cells than did superficial layers. Adult rats exhibited higher percentages of

activated neurons than did aged rats in the AA and AB, but not CC, conditions.

A significant proportion of double-labelled LEC neurons in rats from the AA and AB

groups would be consistent with the stable population representation of the first and second

epochs. Behavioral condition did not have a significant effect on the percentage of neurons that

expressed Arc when assessing the AA and AB groups alone (Figure 6, GLMM, X2(1) = 0.074, p

> 0.5). But, the AA and AB rats’ Arc expression patterns were significantly different from those

of the CC rats (GLMM, X2(2) = 72.3, p < 0.0001). This reflects the same effect of behavioral

condition, but not odor condition, on localized Arc expression as on total percentage of Arc-

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expressing neurons in Figure 5. Rats in the CC condition had limited Arc expression in all

subcellular compartments, and the percentage of neurons with nuclear Arc mRNA was slightly

higher than the percentages of cytoplasmic positive and double-labelled neurons (Kruskal-

Wallis, X2(2) = 181.64, p < 0.0001). There was neither an effect of age nor layer on the

percentage of neurons expressing Arc for rats in the CC group (Figure 6, Kruskal-Wallis, age

X2(1) = 3.32, p > 0.06; layer X2(1) = 0.53, p > 0.4).

Age significantly affected the percentage of neurons with Arc mRNA in the nucleus,

cytoplasm, or both (Figure 6, GLMM, X2(1) = 8.16, p < 0.005). As in Figure 5, Figure 6 shows

that aged rats in the AA and AB groups had a lower proportion of nuclear positive, cytoplasmic

positive, and double-labelled neurons than did adult rats under the same conditions. An

exception to this pattern occurred in rats from the AA group; in deep LEC layers, adult rats did

not have a higher proportion of nuclear positive neurons than did aged rats. As aforementioned,

no age effect was observed in the rats in the CC group.

Layer also had a significant effect on the percentage of neurons with nuclear and

cytoplasmic Arc mRNA (GLMM, X2(1) = 65.42, p < 0.0001); deep layers had higher proportions

of neurons expressing Arc than did superficial layers (Figure 6). No interaction between age and

layer was observed (GLMM, X2(1) = 1.1, p > 0.2).

Arc mRNA localization within each neuron indicated the activity history of principal cells

during the first and second behavioral epochs. The subcellular compartmentalization of Arc

mRNA had a significant effect on the percentage of LEC neurons that expressed Arc (Figure 6,

GLMM, X2(2) = 2188.2, p < 0.0001). A higher percentage of LEC neurons had Arc mRNA in the

nucleus than in the cytoplasm (Figure 6). Furthermore, the proportion of nuclear positive cells

was greater than the proportion of double-labelled cells (GLMM, X2(1) = 513.17, p < 0.0001),

and the proportion of double-labelled cells was greater than the proportion of cytoplasmic

positive cells (GLMM, X2(1) = 627.86, p < 0.0001). This effect held in all age groups and layers

for rats in the AA and AB groups.

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Discussion

The main novel findings of this experiment are that (1) track running in the presence of

odors elevates the activity of neurons in lateral entorhinal cortex (LEC) in both young and aged

rats, above that of non-behavioral controls. Interestingly, a higher proportion of neurons are

activated in the deep layers of LEC, which receive hippocampal input, than in the superficial

layers. (2) Changing the composition of the odors between the first and second behavioral

epochs does not alter the proportion of active neurons. But, surprisingly, more cells reach Arc

activation thresholds during the second epoch than the first. (3) The proportion of neurons

activated by this behavioral experience was decreased in aged compared to adult rats. There

are at least two possible explanations for these data: the higher order representation of

multimodal experience by LEC is either sharpened or reduced by aging. This problem awaits

future investigation.

LEC population activity is elevated during behavior

Traversing a track in the presence of odors induced an increase in the proportion of

neurons that expressed Arc in superficial and deep LEC in adult and aged rats. This is

consistent with the idea that LEC processes incoming non-spatial information from olfactory

areas and perirhinal cortex (PRC). In fact, 30% of neurons in LEC were activated during the AA

and AB behavioral conditions, above the 6% activated by the non-behavioral control condition.

In medial entorhinal cortex (MEC), identical AA and AB behavioral conditions induced a similar

increase in neuron activity of 32% between behavioral groups and the non-behavioral control

group (Liang, Lister, & Barnes, 2013 thesis). This finding is consistent with our expectations and

implicates the LEC in participating in the representation of features of the behavioral

experience.

LEC population activity is not affected by altered odor stimuli

An objective of this investigation was to determine how complex odor experiences are

represented on a population scale, if at all, by neurons in LEC, which receive substantial inputs

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from olfactory cortex. Contrary to our expectations, the proportion of neurons with Arc mRNA,

and thus the proportion of neurons activated during the two behavioral epochs, failed to

discriminate between repeated (AA) and distinct (AB) behavioral conditions. Because altering

the odor sets did not affect the overall population activity, the active circuit composition in LEC is

unlikely to directly represent information about specific odorants. The similarity of neural

activation patterns between these two behavioral conditions is not likely to have resulted from

an inability to discriminate the A and B odor sets on a perceptual level, as the odor stimuli were

quite diverse in their chemical structures. In fact, aged rats only show deficits in behavioral

discrimination of odorants when the stimuli are extremely similar in structure, and when carbon

chain length differs by 3 or less carbons (Yoder et al., 2017). Additionally, the failure of

population activation to differentiate repeated and distinct odor conditions does not necessarily

imply that LEC does not contribute to odor information processing; rather, it suggests that LEC

circuits may stably represent complex odor experiences regardless of their unique chemical

constituents. This aligns with the view that, unlike primary sensory areas, neural representations

by higher order association cortices including LEC do not directly correspond to individual

sensory stimuli.

While the catFISH method does reflect active circuit composition, it does not reflect how

many action potentials were generated by a given neuron. It is conceivable, however, that

modulated firing rates could contribute to a more complex representation of odor combinations

through a rate coding mechanism. PRC circuits feed directly into LEC circuits, so it would not be

surprising if these regions shared analogous coding mechanisms. Akin to PRC object fields,

LEC principal neurons increase their firing rates at the locations of objects (Deshmukh,

Johnson, & Knierim, 2012). The persistent activity of neurons across the A and B epochs in the

present experiment may reflect stable representation of the ramekins, which did not differ

between the two experiences, but are still likely to be represented as proximal objects by LEC

Comrie 20

(Tsao, Moser, & Moser, 2013). Nevertheless, the population representation of 3D object stimuli

in LEC remains unknown.

More neurons participated in the second behavioral epoch

A benefit of the Arc catFISH approach over traditional electrophysiological methods is

the ability to track the activity of the same individual neurons – and tens of thousands of them –

across time points. An unexpected finding in these data was the markedly higher proportion of

nuclear positive cells compared to cytoplasmic positive cells in the AA condition. Even though

the environmental stimuli and amount of exposure were constant across the two epochs, 12% of

neurons were activated during the first epoch, while 27% were activated during the second. The

participation of more than double the number of LEC neurons during the second epoch

suggests that LEC neuron activation may be sensitive to some sort of priming by a similar past

experience. For example, the first exposure to the environment may induce action potentials

and Arc expression in a subset of LEC neurons and may induce subthreshold depolarization of

another subset of neurons. The subthreshold depolarizations could mediate increased

excitability of those principal cells, which could then be more likely to spike and transcribe Arc

mRNA during the second epoch.

This pattern of activation was also observed in an unpublished study of rat LEC with a

different behavioral paradigm (Lister, Clasen, Hartzell, Burke, & Barnes, 2011 poster). Lister et

al. showed that a higher proportion of neurons expressed Arc during the second epoch of an

object exploration task than during the first epoch, independent of the objects present. This

pattern of activation is consistent with our findings in LEC during track running. However, unlike

in LEC, in MEC, equal proportions of neurons were activated in the first and second epochs of

an identical track running task (Liang et al., 2013 thesis). This corroborates the functional

distinction between medial and lateral subdivisions of EC. Moreover, the participation of more

LEC neurons upon a second exposure to an environment is not specific to track running, and

may generalize to other behaviors.

Comrie 21

Aging reduced the proportion of active LEC neurons

Another primary aim of this work was to ascertain how aging affects the participation of

large-scale neural circuits during behavior. We found that aged rats in both behavioral

conditions had a reduced proportion of neurons that expressed Arc. There was no age

difference in Arc-expression in non-behavioral controls. Thus, the age-related hypoactivity in

deep and superficial LEC was specific to the behavior conditions. These data suggest that aged

rats have a sparser representation of the track running experience. The reduction may reflect a

more refined and efficient population code, or a weaker representation with diminished

information content. In either case, aging of the LEC may alter the behaviorally-relevant inputs

from superficial LEC to the hippocampus. Furthermore, aging may modify the outputs from deep

LEC to neocortical regions as well as subcortical areas such as the septum, amygdala, striatum,

and thalamus.

The behaviorally-induced age effect cannot be explained by aged rats completing fewer

laps and having fewer exposures to the environment than did adult rats. In fact, on average,

aged rats completed two more laps per run session than did adult rats. The age effect was also

not due to a simple reduction in number of LEC cells in aged rats, as LEC neuron number is

preserved in aged Fischer 344 rats (Merrill et al., 2001). Age-related changes in MEC activity

are also unlikely to cause the effect observed in LEC, because under the same track running

behavioral conditions, aging failed to alter the proportion of activated MEC neurons (Liang et al.,

2013 thesis).

One possible cause for the age-related decrease in LEC principal cell recruitment during

track running is a reduction in excitation and long-range inhibition from PRC. Aged rats’ PRC

putative interneurons and excitatory neurons both have reduced firing rates compared to those

of adults during track running with objects on the track (Maurer, Burke, Diba, & Barnes, under

revision). Furthermore, as in LEC, fewer principal neurons are recruited during behavior in aged

rat PRC (Burke et al., 2014). PRC neurons are known to target GABAergic LEC neurons, and

Comrie 22

GABAergic PRC neurons in particular have been shown to terminate on GABA-negative

neurons in LEC (Apergis-Schoute, Pinto, & Paré, 2007; Pinto, Fuentes, & Paré, 2006). No direct

evidence for PRC-LEC disinhibition has been shown as of yet. The idea that muted excitation of

PRC neurons would carry forward to LEC principal cells would align with our findings of

hypoactivity in aged LEC. Dampened long-range inhibition from PRC interneurons to LEC

interneurons would also be consistent with our findings, although there is no report at present

that this disinhibitory mechanism exists. The effects of feedforward inhibition from PRC to

principal cells in LEC is less easily explained by our results. An understanding of LEC-PRC

circuitry on this level is critical to clarify how aging in PRC may mediate changes in LEC and

thereby hippocampal circuit activity in the aging brain.

Another possible explanation for the reduced proportion of Arc-expressing neurons after

behavioral experiences could be diminished transcriptional activity by aged LEC principal cells.

In fact, in aged hippocampal granule cells, there is a decrease in levels of Arc transcription in

addition to a lower number of neurons that express Arc during a behavioral task (Penner et al.,

2011). No such behavioral correlates of the present age effect were identified in this experiment,

but it is possible that age-related activity changes in LEC may contribute to cognitive-behavioral

deficits such as in fine odor perception discrimination (Yoder et al., 2017).

LEC changes may contribute to hippocampal dysfunction in aging

This study revealed a difference between the behaviorally-induced activity in deep and

superficial LEC, with a consistently higher proportion of neurons activated in deep than in

superficial layers of LEC. This layer effect was not modulated by age, so it is likely to reflect the

different anatomical circuit relationships and functional roles of superficial versus deep LEC.

The relatively higher proportion of activated neurons in deep layers is consistent with the idea

that efficient and sparse hippocampal codes become more distributed in subsequent levels of

output (Barnes, McNaughton, Mizumori, Leonard, & Lin, 1990). Yet, it is essential to point out

that LEC circuitry is not a simple superficial-input/deep-output region for the hippocampus. The

Comrie 23

layers of LEC are interconnected by both principal cells’ axons and interneurons, and some

inputs target apical dendrites of deep, not just superficial, EC principal neurons (Canto et al.,

2008). Nevertheless, we did not observe differences in the effect of age on superficial and deep

subdivisions of LEC, so we do not have evidence that age-related changes in hippocampal

circuits were reflected by, or exaggerated in, deep LEC.

The LEC is in a position to contribute to the age-related dysfunctions known to occur in

the hippocampus proper. The hippocampus undergoes subregion-specific changes with age:

CA1 place cells inappropriately remap in familiar environments, while CA3 place cells fail to

remap in new environments (Barnes et al., 1997; Wilson, Ikonen, Gallagher, Eichenbaum, &

Tanila, 2005). CA3 also becomes hyperexcitable with age (Robitsek et al., 2015). It is unclear

how age-related behaviorally-induced hypoactivity in LEC fits into the age effects observed in

these varied subregions, because superficial LEC projects directly to DG, CA3, and distal CA1.

Future work that examines and manipulates LEC neural activity in a cell-type- and connectivity-

pattern-specific manner will be critical to dissect how aging of the diverse components of LEC

circuits alter subregional hippocampal functions.

In summary, these findings suggest that while LEC plays a role in representing features

of episodic experiences, changing odor stimuli fails to alter population activity. Activity of these

circuits is more likely to correspond to a complex representation of the experience rather than

specific stimulus features. The hypoactivity in aged LEC likely alters inputs to the hippocampus

and outputs descending to neocortical and subcortical areas. In contrast to PRC and MEC

neurons, LEC neurons are more likely to participate during a second behavioral exposure than

upon the initial behavioral experience. Furthermore, the effect of aging on behaviorally-relevant

LEC activation is neither observed in MEC nor echoed across other target hippocampal

subregions. These observations suggest that LEC is vulnerable to aging in a unique way. The

question of whether a sparser network representation results in maintained, improved, or

reduced cognitive and behavioral function across the lifespan awaits future investigation.

Comrie 24

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