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HPA status predicts age-‐related prefrontal impairments

Adrenocortical status predicts age-related deficits in prefrontal structure plasticity and working memory

Rachel Anderson

The University of Iowa Department of Psychology

Behavioral and Cognitive Neuroscience Academic Advisor: Jason J. Radley

Dec. 9th, 2013


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Abstract

Aging is associated with a gradual and steady decline in cognitive abilities,

although considerable variability exists throughout the population. Previous work

has linked individual differences in hypothalamo-pituitary-adrenal (HPA) activity

and glucocorticoid secretion with age-related alterations in hippocampal

dependent function, however the idea that these endocrine differences may

predict vulnerability to other types of cognitive processes has largely been

ignored. In the present study we interrogated the relationship between variations

in HPA function in aging with prefrontal cortical structure and function. Aged (20

month-old) rats that underwent repeated blood sampling across a 24-hour period

generally showed elevated AM and PM levels of corticosterone levels compared

with 3 month-old rats, although variability was evident across both groups.

Although aging was marked by a significant loss of dendritic spines and

morphologic alterations in mPFC neurons, animals bearing elevated HPA output

exclusively accounted for group differences. In a separate experiment,

adrenocortically characterized young and aged rats were tested on a spatial

working memory task that is dependent on prefrontal cortex function.

Impairments in performance resulted from an interaction between aging and

increased HPA activity. These data suggest that age-related structural plasticity

in mPFC and impairments in working memory may be largely accounted for by

elevations in adrenocortical activity, and define a set of synaptic features that

may help explain individual differences in cognitive functional capacity in aging.


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INTRODUCTION

Aging is associated with a gradual and steady decline in cognitive abilities.

Human and animal aging studies have shown impairment in prefrontal dependent

functions such as working memory and decision-making (Gallagher and Rapp,

1997; Grady, 2008). This deterioration in cognitive functioning varies within the

aged population, with some individuals experiencing significant impairments and

others maintaining cognitive abilities similar to that of a healthy adult who are

then classified as “successful agers” (Rowe and Kahn, 1987, 1997; Britton et al.,

2008; Fernández-Ballesteros et al., 2008; Hung et al., 2010). A considerable

body of research suggests that it is not a loss of neurons necessarily, but a

disruption in synaptic plasticity in the prefrontal cortical and hippocampal circuitry

that may underlie impairments in aging (Morrison and Baxter, 2012). To date, a

number of studies have identified a significant structural synaptic reorganization

in the prefrontal cortex in the aging brain that likely reflects impaired plasticity.

Dendritic spines compromise sites of postsynaptic contact for the vast majority of

excitatory synapses made onto cortical pyramidal neurons and are important

sites for structural plasticity (Spacek and Hartmann, 1983; Harris and Stevens,

1989). Dendritic spine loss has been documented in the aging prefrontal cortex,

most notably with thin spine subtypes. The fact that thin spines exhibit the

greatest potential for plasticity for synapse strengthening relative to other spine

geometries (Nimchinsky et al., 2002; Kasai et al., 2003, 2010a, 2010b), has led

to the suggestion that they are critical for learning (Kasai et al., 2003, 2010a;


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Matsuzaki, 2007). In this regard, thin spine loss in the aged macaque prefrontal

cortex has been correlated with impaired cognitive performance (Dumitriu et al.,

2010), suggesting a potential mechanism for the working memory decline in

aging.

Along with impairments in cognition, aging is characterized by elevated

hypothalamo-pituitary-adrenal (HPA) activity (Landfield et al., 1978; Sapolsky et

al., 1983; DeKosky et al., 1984). The HPA axis is a neuroendocrine pathway that

regulates the stress response, along with other homeostatic body processes,

with the end product being the secretion of glucocorticoids (cortisol in the human,

corticosterone in the rat). Aged rats and humans generally display increased

basal adrenocortical activity as compared with young cohorts (Issa et al., 1990;

Montaron et al., 2006). Prolonged elevations in circulating glucocorticoids are

thought to be responsible, or may exacerbate, a variety of neuropsychiatric and

systemic illnesses. Repeated corticosterone (CORT) administration leads to

structural impairments in the rodent medial prefrontal cortex (mPFC) (Wellman,

2001; Cerqueira et al., 2007; Liu and Aghajanian, 2008), whereas experimental

manipulations that induce prolonged HPA axis activation, such as chronic stress,

result in a similar dendritic reorganization (Cook and Wellman, 2004; Radley et

al., 2004) and dendritic spine loss in mPFC neurons (Radley, 2005; Radley et al.,

2008, 2013). Chronic stress-induced structural reorganization in mPFC has also

been shown to predict the degree of impairment in several prefrontally-mediated

behavioral tasks (Liston, 2006; Dias-Ferreira et al., 2009).


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The sensitivity of prefrontal cortical neurons to both aging and corticosteroids

raises the possibility that age-related structural and functional impairments in the

prefrontal cortex may be accounted for by cumulative exposure to glucocorticoids

across the lifespan. Here we investigate whether individual differences in basal

adrenocortical activity are predictive of impairments in dendritic spine plasticity in

mPFC and delayed alternation in aging. To assay for adrenocortical function,

young and aged rats (3 and 20 month-old, respectively) underwent repeated

blood sampling across a 24-hour period to determine the circadian rhythmicity of

CORT secretion. Aged rats generally show increased AM and PM CORT

secretion as compared with young counterparts, although considerable individual

variation was evident within both groups. Next dendritic spine density and

morphology were examined in neurons in the prelimbic area (PL) of the mPFC as

a function of both aging and adrenocortical status. Whereas aged animals

showed the expected decrease in dendritic spine density in PL neurons, aged

animals with elevated CORT levels showed a more pronounced attrition of these

morphologic indices. In a follow-up experiment, interactive effects of aging and

adrenocortical status were tested in delayed alternation in a T-maze. These data

suggest that age-related structural plasticity in mPFC and impairments in working

memory may be largely accounted for by elevations in adrenocortical activity,

and define a set of synaptic features that may help to explain individual

differences in cognitive functioning in aging.


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MATERIALS AND METHODS

Animals. The animals used in this study were male Sprague-Dawley albino rats

at ages of 3 and 20 months (designated as young and aged, respectively; Harlan

Laboratories, Indianapolis, IN). Animals were housed in pairs and maintained on

a 12:12 hour light/dark cycle (lights on at 7 am), with free access to food and

water. The health of the aged animals was verified upon arrival from the

supplier, and monitored regularly by the attending veterinarian to ensure that

they were free of any spontaneous tumors or other overt physiologic or

immunologic signs of distress. After 2 weeks of acclimatization in the animal

housing facility, rats were habituated to human contact by handling each for 5

min each day, over 7 days.

Blood collection and radioimmunoassay. Basal adrenocortical activity was

measured by obtaining blood samples from the tail vein of rats at 6 time points

over a 24-h period starting at 8 am. For blood collection, rats were restrained

briefly (~15-30 s), and a small longitudinal incision was made at the distal tip of

the tail with a sterile blade. Blood samples (~200 µl) were collected into chilled

plastic microfuge tubes containing EDTA and aprotinin, centrifuged, and

fractionated for storage of plasma at -80 °C until assayed. Plasma corticosterone

was measured without extraction, using an antiserum raised in rabbits against a

corticosterone-BSA conjugate, and 125I-corticosterone-BSA as tracer (MP


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Biomedicals, Solon, OH). The sensitivity of the assay was 0.8 µg/dl; intra- and

interassay coefficients of variation were 5 and 10%, respectively.

Experiment 1 – Fluorsescent dye-filling dendritic spine morphometric

analysis in prelimbic cortical pyramdial neurons

Histology and tissue processing. Rats were anesthetized with chloral hydrate

(350 mg/kg, ip) and perfused via the ascending aorta with 100 ml 1%

paraformaldehyde and 0.125% glutaraldehyde in 0.1 M phosphate buffered

saline (PBS; pH 7.4) followed by 500 ml of 4% paraformaldehyde and 0.125%

glutaraldehyde in 0.1 M PBS (pH 7.4), at a flow rate of 55 ml/min. The

descending aorta was clamped to limit the flow of fixative to the head and upper

extremities, and to also prevent fixation of the adrenal glands. Immediately

following perfusions, the adrenal glands were dissected and weighed, and brains

were removed and postfixed for 3-4 hours. After postfixation, the pregenual pole

of the cortex was sectioned coronally into 250 µm-thick slabs using an oscillating

tissue slicer (VT-1000S, Leica), and was stored in 0.1 M PBS containing 0.1%

sodium azide at 4 °C until the time of cell loading.

Intracellular dye injections. The procedure for iontophoretic intracellular dye

injections described is based upon previous reports (Radley et al., 2004; Radley

et al., 2006; Radley et al., 2008). Coronal tissue slabs were treated in the DNA-

binding fluorescent stain 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen) to

distinguish between nuclear lamination patterns that distinguish PL from other


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adjacent-lying prefrontal cortical subfields. DAPI- treated sections were mounted

on nitrocellulose filter paper and submerged in a tissue culture dish containing

0.1 M PBS, and viewed under fluorescence using a fixed-stage microscope

(Leica DM5500). Injections of 5% Lucifer yellow (Life Technologies) were made

by iontophoresis through micropipettes (1–2 µm inner diameter) under a DC

current of 1–6 nA for 5-10 minutes. Neurons in layers 2 and 3 were selected for

the PL dye injection procedure was based upon previous descriptions of the

cytoarchitectonic features of PL (Krettek and Price, 1977; Swanson and Cowan,

1977; Vogt and Peters, 1981). Briefly, PL was differentiated from its dorsal and

ventral counterparts by a more densely packed layer 2, and a broader layer 5.

The general technique for cell filling involved carefully observing the passive

diffusion of LY resulting from application of a neglibly small amount of current

from the advancing micropipette tip under 40X magnification; LY diffuses

amorphously until hitting a dendritic process or cell body, whereby the dye

becomes restricted intracellularly. After several neurons were filled

intracellularly, tissue sections were mounted onto glass slides and coverslipped

in Vectashield (Vector Laboratories).

Neuronal and dendritic reconstructions. An experimenter unaware of the

treatment condition for each animal performed neuronal reconstructions and data

analyses. Pyramidal neuron dendritic arbors were reconstructed in 3D using a

computer-assisted morphometry system consisting of a Leica DM4000R

equipped with an Applied Scientific Instrumentation MS-2000 XYZ computer-


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controlled motorized stage, a QImaging Blue digital camera, a Gateway

computer, and morphometry software (MBF Biosciences). Neurons were

visualized, and the dendritic tree was reconstructed using a Leica Apochromat

40X objective with a numerical aperture (N.A.) of 1.4 and Neurolucida software

(MBF Biosciences).

In order to be considered for analysis, LY-filled PL neurons had to exhibit

complete filling of the dendritic tree, as evidenced by well-defined endings. A

series of strict criteria were employed for inclusion of pyramidal neuron apical

and basal dendrites for morphologic analysis (Radley et al., 2004; Radley et al.,

2005). For apical dendrites, the fact that the primary shaft generally coursed

parallel, or gently downward from the top surface of the section (i.e., sections

were flipped in instances where apical dendrites coursed upward out of the top

surface of the section) optimized the probability for retaining complete dendritic

arbors. However, since dye-filling procedure was carried out in the sections were

only 250 µm thick, it was virtually impossible to retain an entirely intact apical

dendritic arbor with no truncations. Thus, apical dendrites included in the

analysis retained intact secondary and tertiary branches, with truncations

permitted only in collateral branches that appeared to be nearing the point of

termination, or, deemed unlikely to make any significant bifurcations (a sense of

this was derived from examination of intact collaterals from analysis of several

hundred dendritic arbors from LY filled neurons). PL neurons in layers 2 and 3

were also appreciated to exhibit some qualitative differences; deeper-lying layer

3 neurons possessed apical dendrites with elongated primary dendrites prior to


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the first truncation point (~75-125 µm; notwithstanding collateral branching),

whereas more superficially-situated layer 2 neurons contained shorter distances

to the first branch point (~25-75 µm; e.g., compare images in Figs. 2 and 3).

Nevertheless, consideration of these subsets as morphologically distinct

subpopulations in ancillary analyses failed to demonstrate any quantitative

differences that warranted partitioning them into distinct groupings. For basal

dendrites, it was common to retain an average of 1-3 entirely intact arbors for a

given LY-filled neuron, such that analyses on intact branches were performed for

this category.

Confocal laser scanning microscopy. Two-dimensional renderings for each

neuron were obtained using Neurolucida software, and a radial distance of 150

µm from the soma was selected as a boundary delineating proximal and distal

portions of the dendritic tree. Within these regions, 1-2 branches were randomly

selected for each neuron for an average of: 3 segments per neuron; average of 5

neurons region for each animal. The following criteria were adopted for the

selection of dendritic segments for confocal imaging, and based upon previous

reports (Radley et al., 2006; Radley et al., 2008): (1) possess a diameter of <3

µm, as larger diameter dendrites in PL pyramidal neurons exhibit greater

variability in spine density values; (2) reside within a depth of 70 µm from the top

surface of the section, due to the limited working distance of the optical system;

(3) to be either parallel to, or course gently relative to the coronal surface of the

section (i.e., this helps to minimize z-axis distortion and facilitate the

unambiguous identification of spines); (4) have no overlap with other branches


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that would obscure visualization of spines. z-Stacks for segments that met these

criteria were collected on a Leica SP5 confocal laser-scanning microscope

equipped with an argon laser and a 100X, 1.4 N.A. oil immersion objective, using

voxel dimensions of 0.1 X 0.1 X 0.1 µm3. Settings for pinhole size (1 airy disc),

gain, and offset were optimized initially and then held constant throughout the

study to ensure that all images were digitized under similar illumination

conditions at a resolution of 512 X 512 pixels. With these settings, dendritic

segments ranged between 52-59 µm in length. All confocal stacks included at

least 1 µm above and below the identified dendritic segment. For presentation,

composites from deconvolved optical stacks were exported first to ImageJ for

adjustments to optimize/balance contrast and brightness and then to Canvas

(version 10; Deneba Systems) for assembly and labeling.

Dendritic spine analyses. Images were deconvolved with AutoDeblur (Media

Cybernetics), and spine analyses were performed using the semi-automated

software NeuronStudio (Rodriguez et al., 2003; Rodriguez et al., 2006; Radley et

al., 2008)(http://research.mssm.edu/cnic/tools-ns.html), which analyzes in 3D

dendritic length, spine density, and morphometric features (i.e., head/neck

diameter, length) for each dendritic spine. Deconvolution improves both the axial

and lateral resolution of 3D images, and reduces the smearing of the image in

the z-axis. NeuronStudio also classifies dendritic spines into categories (thin,

mushroom, stubby) based upon user-defined parameters. Spines were classified

as thin or mushroom if the ratio of the head diameter-to-neck diameter was


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greater than 1.1. If their ratio exceeded this value, spines with a maximum head

diameter greater than 0.4 µm were classified as mushroom, or else were

classified as thin. Spines with head-to-neck diameter ratios less than 1.1 were

also classified as thin if the ratio of spine length-to-neck diameter was greater

than 2.5, otherwise they were classified as stubby. A fourth category, filopodial

spines, were considered to have a long and thin shape with no enlargement at

the distal tip, were very seldom observed, and were classified as thin. To ensure

that NeuronStudio appropriately classified dendritic spines into their respective

categories, an observer scanned each z-stack manually, and manual corrections

were made as necessary. Total and subtype spine densities were determined by

dividing the total number of spines by the length of each dendritic segment. In

this study, a total of 346 dendritic segments from fluorescent dye-labeled PL

neurons were analyzed for spine density and morphometric analysis (150 young,

196 aged), and 22,650 and 26,504 dendritic spines in young and aged animals,

respectively.

Experiment 2 - Assessment of prefrontal functionality using delayed

alternation

A separate group of young and aged rats were trained on a delayed alternation

task using a T-maze, a prefrontal-dependent spatial working memory task (Divac,

1971; Bubser and Schmidt, 1990; Aultman and Moghaddam, 2001). Animals

were subjected to repeat blood sampling for assessment of basal adrenocortical

activity (as above), and given a week to recover, while still being handled daily.


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For T-maze training, rats were habituation to the maze over a several day period

until miniature chocolate chips were eaten from the goal arms in less than one

minute. Next, animals were subjected to a forced alternation period of training

where they were only rewarded with chocolate after entering the opposite goal

arm that they were in previously. Between trials the maze was wiped clean with

70% ethanol to prevent olfactory cues from determining choice. After 3

consecutive days of 10 trials, animals were tested for spontaneous alternation

(i.e., chocolate was present in both goal arms, although rats were only rewarded

for entering the opposite arm from the previous trial). The delay between trials

was increased until animals could successfully alternate at a 15 s interval with

>70% accuracy for 10 trials.

Rats were tested on 8 trials a day for 6 consecutive days. The delays between

each trial were randomly varied between 30 seconds, 60 seconds, or 120

seconds (on a given day the same pattern was used for all animals). During

delay rats were placed in the holding cage. After each run, the maze was wiped

clean with 70% ethanol to prevent olfactory cues from factoring into decision-

making.

Statistics.

Group data from the corticosterone radioimmunoassay (12 young and 14 aged

animals) were compared with a mixed design analysis of variance with one

between- (young, aged) and one within- (time) group variable, followed by post


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hoc comparisons at each time point. Data are expressed as the mean +/± SEM.

Next, integrated values for corticosterone secretion were obtained for each

animal by averaging plasma titers from all six time points, and a median split was

performed within each age to give subgroupings of high and low corticosterone.

Group data for morphologic analyses were obtained by averaging values from

each animal (average of 3 segments/neuron, 5 neurons per region, 12 animals/

group) in neurons from PL in young and aged animals.

Group data for the morphometric analyses were obtained by averaging

values from each animal (average of 5 neurons per region, 5-7 animals per

group) in LY-labeled PL neurons as a function of both age and adrenocortical

status. The effects on overall dendritic length, number of branch endings,

dendritic spine and subtype densities were compared using a two-way analysis

of variance, with factors of age (young, aged) and adrenocortical status (low,

high corticosterone). Post hoc comparisons were made using a Bonferroni

correction, with significance set at p < 0.05, and data are expressed as mean +

SEM. Cumulative distribution differences were evaluated using the Kolmogorov-

Smirnov test with Matlab software.

For the behavioral data analyses, percent correct for each delay period (30, 60,

120 seconds) for the full testing period were collected for each animal and then

averaged among group (either age (n =10) or young (n = 10) and then age + high

CORT, age + low CORT, young + high CORT, or young + low CORT (n = 5 for


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each group). A two-way ANOVA was performed on the data and post-hoc

comparisons were made using a Bonferroni correction, with significance set at p

<0.05 and data are expressed as mean + SEM.

RESULTS

Characterization of adrenocortical activity in young and aged rats

Previous work has shown that aging is marked by alterations in adrenocortical

function, with elevations in HPA activity implicated in age-related cognitive

decline (Landfield et al., 1978; Sapolsky et al., 1983; DeKosky et al., 1984). More

recent evidence has directed attention to mPFC as a target of the effects of

stress and aging. It has been widely documented that pyramidal neurons in

layers 2/3 of mPFC undergo dendritic shortening and spine synapse loss

following prolonged stress and/or glucocorticoid exposure (Wellman, 2001;

Radley et al., 2004, 2013; Cerqueira et al., 2007; Sterner and Kalynchuk, 2010).

Aging per se, is marked by overall decreases in dendritic spine density and shifts

in spine geometry in mPFC pyramidal neurons (Dumitriu et al., 2010; Bloss et al.,

2011). These observations led us to examine whether age-related alterations in

dendritic spine plasticity in mPFC pyramidal neurons may be accounted for by

naturally occurring variations in HPA axis functioning.

First we characterized adrenocortical secretory activity in 3-month and 20-

month old rats across the light-dark cycle (Fig. 1, top). Blood samples were

collected from the tail vein of animals starting at 4 am, and were repeatedly


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sampled at 4-h intervals, through 12 am on the following day. Repeated

measures ANOVA showed main effects for within factors (time of sample

collection: (F(5,142) = 15.79, p < 0.05), and no main effects for between (age

status: (F(1,24) = 0.45, p = 0.51) or any significant interaction between these

variables (F(5,142) = 1.76, p = 0.13). Nevertheless, comparisons at each time

point revealed significant age-related elevations in plasma levels of

corticosterone at 12 pm (by 127%; t(24) = 2.14, p < 0.05) and 12 am (by 53%;

t(24) = 2.27, p < 0.05). While aged animals also exhibited a trend toward

reduced plasma titers of corticosterone at the 4 am time point, this trend did not

reach significance (p = 0.1). For evaluating individual differences in

adrenocortical activity, corticosterone values from all six sampling intervals were

averaged to a single value for each animal (Fig. 1, bottom). Young and aged

animals possessing plasma corticosterone levels above and below the median

value for each group were selected to represent high and low levels of

adrenocortical activity, respectively.

Aging and adrenocortical status contribute to dendritic spine loss in mPFC

Dendritic spine density. Individual neurons in PL were selected for intracellular

injection of Lucifer yellow in young and aged rats that had been previously

characterized for basal corticosterone activity. Different regions of the dendritic

tree (<150 µm apical, >150 µm apical, <150 µm basal) were selected for high-

resolution confocal LSM imaging of dendritic segments (Fig. 2, top). Digital

renderings (z-stacks) of dendritic segments made in 3D were deconvolved,


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followed by the analysis of spine density and morphology (NeuronStudio

software) by an experimenter that was unaware of the treatment conditions.

We observed an 11% decrease in overall dendritic spine density (i.e., the

average of all three regions of the dendritic tree analyzed) in PL in aged relative

to young animals (two-way ANOVA, age status: F(1,19) = 5.34, p < 0.05)(Fig. 2,

lower left). Further examination of main effects for overall spine density nearly

reached significance for corticosterone status (F(1,19) = 4.01, p = 0.06), although

not for an interaction (F(1,19) = 0.43, p = 0.52). Pairwise comparisons made

between young and aged groups in the context of low and high corticosterone

status revealed a profound loss of spines in aged animals classified in the high

adrenocortical subgrouping (i.e., aged + high CORT). These animals displayed

22% and 16% reductions in spine density relative to young low and high CORT

groups, respectively (p < 0.05), and a downward trend that nearly reached

significance (by 14%, p = 0.056) relative to the aged + low CORT group (Fig. 2,

bottom). By contrast, aged animals with low levels of corticosterone did not show

any reduction in spine density relative to either low or high corticosterone

subgroups of young animals (p = 0.82 and p = 0.28, respectively).

In a follow-up analysis, adrenocortical status was plotted as a function of

dendritic spine loss in PL neurons for young and aged groups. Despite the

existence of clear group differences in medial prefrontal spine loss between low

and high CORT groupings, we did not find any evidence for a significant

correlation between these indices in aged animals (r = -0.46, p = 0.30).

Interestingly, the analysis of young animals yielded a stronger tendency toward


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variations in adrenocortical activity predicting differences in PL spine plasticity (r

= -0.68), however this was not significant (p = 0.09).

Subsequent comparisons of group effects for alterations in dendritic spine

density within regions of the dendritic tree were also carried out using a two-way

analysis of variance. For proximal apical dendrites in PL neurons (i.e., <150 µm

apical), a main effect was evident with regard to age, however there were no

significant effects of adrenocortical status or interaction (age: F(1,19) = 8.32, p <

0.05; CORT status: F(1,19) = 2.02, p = 0.17; interaction: F(1,19) = 0.58, p =

0.46). Pairwise comparisons revealed the same downward trend of spine density

in proximal apical dendrites in the aged + high CORT group or rats, showing 23%

and 20% reductions in spine density relative to young low and high CORT

groups, respectively (p < 0.05 for each), whereas the aged + low CORT group

did not show any significant decrement in this index as compared with young

animals (Fig. 2, bottom).

For the most part, no main effects were observed in spine density in the two

other portions of the dendritic tree sampled (>150 µm apical - age: F(1,19) =

2.73, p = 0.12; CORT status: F(1,19) = 4.78, p < 0.05; interaction: F(1,19) = 0.90,

p = 0.35; <150 µm basal - age: F(1,19) = 1.69, p = 0.21; CORT status: F(1,19) =

1.82, p =0.19; interaction: F(1,19) = 0.01, p = 0.93). Nevertheless, pairwise

comparisons further reinforced the overall trend that aged rats bearing high

levels of corticosterone showed a greater vulnerability to spine loss than their

age-matched counterparts. In more distal aspects of the apical dendritic tree

(>150 µm), spine density was diminished in the aged + high CORT animals


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relative to both young + low CORT (by 17%) and aged + low CORT groups (by

15%; p < 0.05 for each; differences relative to young + high CORT animals did

not reach significance; p = 0.07)(Fig. 2, bottom). Further evidence for aging

effects differentiated by adrenocortical status are further endorsed by the lack of

any spine loss in the aged + low CORT group relative to young animals at any

region of the dendritic tree analyzed. Whereas these data are in concordance

with the age-related decreases in prefrontal dendritic spine density described in

previous studies (Dumitriu et al., 2010; Bloss et al., 2011), they suggest that

adrenocortical status is a critically important underlying factor accounting for

cortical spine loss in aging.

Dendritic arborization patterns. In a follow-up analysis, we examined treatment

effects on dendritic arborization patterns (overall length, branch number) in the

same PL neurons analyzed for alterations in spine plasticity (Fig. 3). A two-way

analysis of variance design was implemented as above, with age and

corticosterone status as the treatment variables. With regard to apical dendrites,

there were no significant main effects or interactions for total length (age status:

F(1,19) = 1.75, p = 0.20; CORT status: F(1,19) = 1.81, p = 0.20; interaction:

F(1,19) = 0.96; p = 0.34), or number of branch endings (age status: F(1,19) =

0.40, p = 0.85; CORT status: F(1,19) = 0.63, p = 0.44; interaction: F(1,19) = 0.48;

p = 0.50)(Fig. 3, bottom). Basal dendritic measures of length (age status: F(1,19)

= 2.87, p = 0.11; CORT status: F(1,19) = 0.44, p = 0.52; interaction: F(1,19) =

0.05; p = 0.83), and the number of branch endings (age status: F(1,19) = 0.35, p


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= 0.56; CORT status: F(1,19) = 0.01, p = 0.91; interaction: F(1,19) = 0.03; p =

0.87) also failed to reveal any significant differences. These data suggest that

dendritic arbors remain relatively resistant to such age-related alterations,

suggesting neither a homeostatic response of increased dendritic growth that

could compensate for spine synaptic compromise, nor further spine loss that

could be compounded by dendritic shortening.

Interactive effects of age and HPA status on PL dendritic spine morphology

Aging is associated with the loss of thin spines. Several previous studies have

shown that age-related dendritic spine loss in the prefrontal cortex is most

prominent within thin subtypes (Dumitriu et al., 2010; Bloss et al., 2011). Thin

spines are largely representative of immature excitatory synaptic population in

cortical pyramidal neurons, exhibiting high rates of turnover as compared to large

spines (Nimchinsky et al., 2002; Kasai et al., 2003), and have decreased rates of

turnover following prolonged glucocorticoid exposure (Liston et al., 2013). Thus,

we addressed the possibility that age-related decreases in thin spine subtypes in

PL may be exaggerated in animals presenting elevated adrenocortical activity.

Aging per se, failed to result in a significant decrease in the overall density of

thin spines (F(1,19) = 2.68, p = 0.12), and no significant effects were noted for

adrenocortical status (F(1,19) = 0.02, p = 0.93), or interaction (F(1,19) = 1.27, p =

0.27). Nevertheless, some attrition of thin spines was observed in aged animals

bearing high adrenocortical activity. Pairwise comparisons revealed a significant

decrease (by 17%) of thin spines between aged + high CORT animals relative to


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the young + high CORT group (p < 0.05)(Fig. 4). Plots of individual values from

young and aged animals also failed to demonstrate an inverse correlation

between adrenocortical activity relative to thin spine density in PL neurons

(young: r = -0.49, p = 0.26; aged: r = -0.35, p = 0.44; data not shown).

Regional analyses of thin spine density highlighted age-related losses as

being most prominent in proximal aspects of the apical dendritic tree (<150 µm

apical - age: F(1,19) = 8.06, p < 0.05; CORT status: F(1,19) = 0.56, p = 0.46;

interaction: F(1,19) = 0.97, p = 0.34), however, no effects were noted in the other

two regions analyzed (>150 µm apical - age: F(1,19) = 1.06, p = 0.32; CORT

status: F(1,19) = 0.16, p = 0.69; interaction: F(1,19) = 0.14, p = 0.72; <150 µm

basal - age: F(1,19) = 0.41, p = 0.53; CORT status: F(1,19) = 0.004, p = 0.95;

interaction: F(1,19) = 2.10, p = 0.16)(Fig. 4). In proximal apical dendrites, aged +

high CORT animals demonstrated a 25% and 26% loss of thin spines in proximal

apical dendrites as compared to low and high CORT groups, respectively (p <

0.05 for each), whereas the aged + low CORT group failed to show any loss of

thin spines relative to either subgroup of young animals. In summary, these data

lend support to an association between aging and the loss of thin spines in apical

dendrites PL pyramidal neurons; however, these effects show less dependency

on individual variations in adrenocortical status.

Loss of stubby spine subtypes is predicted by aging and adrenocortical status.

Although the functional implications of stubby spines are less well understood,

the available evidence suggests that these subtypes may reflect a distinct


22

subpopulation of immature excitatory synapses from thin spines (Spacek and

Hartmann, 1983; Nimchinsky et al., 2002; Kasai et al., 2003). These data raise

questions as to whether aging effects that have been previously documented in

thin spines (Dumitriu et al., 2010; Bloss et al., 2011), may extend to this other

purported immature subtype. In considering all three regions of the dendritic tree

in toto, aged animals did not show any frank reduction in stubby spine density (F

(1,19) = 0.89, p = 0.36). However, we observed significant effects of

adrenocortical status (F (1,19) = 10.20, p < 0.05) and interaction (F (1,19) = 7.64,

p < 0.05)(Fig. 4). Pairwise comparisons of aged + high CORT animals revealed

a significant loss of stubby spines compared to both young + low and high CORT

groups (by 21% and 20%, respectively, p < 0.05), and as compared with aged +

low CORT animals (by 27%, p < 0.05). Correlation of individual values for stubby

spines as a function of corticosterone status did not reveal any significant inverse

relationship within the young (r = 0.26, p = 0.57) or aged groups (r = -0.58, p =

0.17).

Regional analyses of stubby spine densities displayed the same trend as for

overall effects, with aged + high CORT animals being selectively vulnerable to

stubby spine loss throughout the apical dendritic arbor. Distal apical dendrites

showed a profound abrogation of stubby spines, not as a function of age (F

(1,19) = 0.60, p = 0.45), or corticosterone status (F (1,19) = 3.52, p = 0.08),

instead as the interaction of these two factors (F (1,19) = 6.74, p < 0.05). Follow-

up pairwise comparisons showed that aged animals with high corticosterone

levels were exclusive in bearing fewer stubby spines, as compared with young +


23

high CORT (by 37%) and aged + low CORT groups (by 44%, p < 0.05 for each;

differences relative to the young + low CORT group were not significant; p =

0.09). Analysis of stubby spine density in other aspects of the dendritic tree were

not significant (apical <150 µm - age: F (1,19) = 3.75, p = 0.07; CORT status: F

(1,19) = 4.11, p = 0.06; interaction: F (1,19) = 0.80, p = 0.38; basal <150 µm -

age: F (1,19) = 0.94, p = 0.34; CORT status: F (1,19) = 1.67, p = 0.21;

interaction: F (1,19) = 0.47, p = 0.50). However, in proximal apical dendrites, the

aged + high CORT group showed a selective attrition of stubby spines as

compared with all three groups (by 27% and 20% relative to young low and high

CORT, and by 21% compared with aged + low CORT, p < 0.05 for each)(Fig. 4).

Some spine parameters remain stable during aging and HPA fluctuations.

Mushroom spines are representative of a mature excitatory synaptic phenotype,

and possess enlarged postsynaptic densities, stronger AMPA receptor currents,

and a greater ability to sequester biochemical-signaling molecules, such as

calcium (Nimchinsky et al., 2002; Hongpaisan and Alkon, 2007; Hongpaisan et

al., 2013). Some evidence suggests that mushroom spines are the principle

subtype important for memory storage (Matsuzaki, 2007; Yasumatsu et al., 2008;

Kasai et al., 2010a), and the fact that these subtypes largely appear to remain

stable in the aged brain is consistent with behavioral data suggesting that aged

individuals show relatively stable long-term memory. Here we assessed

mushroom spine density throughout the apical and basal dendritic trees in young

and aged animals bearing high levels of adrenocortical activity, however, no


24

effects for age (F(1,19) = 1.94, p = 0.18), adrenocortical status (F(1,19) = 3.41, p

= 0.08), or interaction (F(1,19) = 1.62, p = 0.22) were evident in this spine

subtype (Fig. 4, bottom). More targeted analyses focused within apical and basal

dendritic subregions also failed to reveal any significant trends or effects (data

not shown). Correlational analyses of mushroom spine density as a function of

adrenocortical status were suggestive of a negative trend in young animals (r = -

0.51, p = 0.24), however, not in aged animals (r = 0.11, p = 0.81).

To provide an additional measure of aging and HPA effects on spine

morphologic alterations, we analyzed treatment effects on mean spine head

diameter. The principal analysis entailed collecting and averaging head

diameters from all three spine subtypes as a function of experimental treatment.

Our analyses failed to reveal any overall effects for this index (age: F (1,19) =

0.004, p = 0.95; CORT status: F (1,19) = 0.02, p = 0.90; interaction: F (1,19) =

1.27, p = 0.27)(Fig. 4, bottom), and follow-up analyses within different dendritic

regions also failed to demonstrate any regional trend (data not shown). Thus, in

spite of the substantial decrements in stubby and thin spines observed in the

aged + high CORT group, mean spine head diameters remain relatively stable.

Interactive effects of age and HPA status on mushroom spine head diameter. As

a follow-up to our examination of the repercussions of aging and adrenocortical

status on dendritic spine subtype in PL pyramidal neurons, we queried whether

structural alterations might be manifest within each designated spine category.

An initial indication of these effects is derived from the analysis of spine head


25

diameter (see above), although since thin spines comprise the majority of the

overall population of spines (60-65%), remodeling within thin subtypes per se,

might conceal alterations in other subtypes. Thus, we examined average spine

head diameter and cumulative frequency plots of spines within each spine

category (thin, stubby, mushroom).

Of the three subtypes subject to this analysis, mushroom spines exhibited a

strong bimodal shift in head diameter as a function of aging and HPA status.

While there were no group differences in average spine head diameter within

mushroom spine subtypes (age: F(1,19) = 0.24, p = 0.63; CORT status: F(1,19) =

1.23, p = 0.25; interaction: F(1,19) = 0.55, p = 0.47)(Fig. 5, top), when aged

animals were partitioned into high and low corticosterone subgroupings, a

prominent cumulative frequency shift was revealed. Cumulative frequencies for

mushroom spine head diameter were significantly shifted to the left in the aged +

high CORT group as compared with young animals (Kolmogorov-Smirnov test, p

< 0.0001)(Fig. 5, bottom). By contrast, aged rats with low adrenocortical activity

display a rightward shift in the cumulative frequency distribution for mushroom

head diameter relative to young animals (Kolmogorov-Smirnov test, p <

0.00001)(Fig. 5, bottom). Cumulative frequency analysis for other spine

subtypes did not reveal any significant trends in spine head diameter (data not

shown). As recent evidence suggests that mushroom spines in cortical neurons

may be vulnerable to high levels of circulating glucocorticoids (Michelsen et al.,

2007), the leftward shift of the aged + high CORT group suggests that cumulative

exposure to high levels of glucocorticoids may impart a restraining influence on


26

mushroom spine size, whereas lower levels may be permissive for greater

stability in these mature spine subtypes.

Interactive effects of age and HPA status in spatial working memory

Given that aging and HPA status interact to induce regressive alterations in

structural plasticity in the mPFC, we next examined the extent to which they

extend to prefrontally-dependent cognitive impairment. Separate groups of

young adult (n =10) and aged (n =10) male Sprague-Dawley rats were first

assayed for basal adrenocortical function and partitioned into low and high

subgroups for corticosterone activity (see above). After a habituation period,

young and aged rats were tested for spatial working memory using a delayed-

alternation task on a T-maze, although throughout behavioral testing the

experimenter remained unaware of the hormonal status of each animal.

Consistent with previous reports (Ando and Ohashi, 1991; Bimonte et al.,

2003; Ramos et al., 2003), aging resulted in impaired spatial working memory

performance. Main effects for age were evident at the 60 s delay interval (F(1,16)

= 44.273, p < 0.001), and were accompanied by main effects for adrenocortical

status (F(1,16) = 5.127, p < 0.05), and interaction between these indices (F(1,16)

= 5.778, p < 0.05). At this delay interval, both aged subgroups displayed

significant deficits in the percentage of correct choices in alternation relative to

both subgroups of young animals (p < 0.05 for each pairwise comparison)

whereas the aged + high CORT group showed even greater learning deficits

relative to its aged + low CORT counterpart (p <0.05) (Fig. 6). By contrast, age-


27

related impairments were exclusive only to within the aged subgroup bearing

high levels of corticosterone activity. At the 30 second delay interval, no main

effect was found for age (F(1,16) = 1.685, p = 0.213), adrenocortical status

(F(1,16) = 0.959, p = 0.342), or interaction (F(1,16) = 0635, p = 0.437). However,

post-hoc pairwise comparisons revealed a selective impairment in delayed

alternation in the aged + high CORT group relative to young animals (p< 0.05

with respect to high and low CORT subgroups). No main effects were evident

(age: (F(1,16) = 1.815, p = 0.197; CORT: (F(1,16) = 3.206, p = 0.092), although

nearly reached significance for the interaction between age and adrenocortical

status (F(1,16) = 3.846, p = 0.068). Nevertheless, the aged + high CORT group

showed a selective deterioration of delayed alternation at the 120 s interval

relative to aged + low CORT counterparts and both young subgroups (p <0.05 for

each).

Collectively, these data lend support to the idea that a significant component of

age-related spatial working memory impairment may be accounted for by

elevated adrenocortical activity. The fact that CORT levels exerted no affect on

delayed alternation performance in the young animals at any time point, suggests

that it is the cumulative exposure to CORT that leads to age-related impairments

in this type of cognitive functioning.

Given the testing paradigm involved in training animals to an equivalent level of

performance on the delayed alternation task (i.e., a threshold of at least 70%


28

correct responses to a delay interval of 15 s), the latency to reach performance

criterion provided an additional measure for behavioral assessment (e.g. see

Ramos et al., 2003). Although aged animals required a longer number of training

sessions on average (2.1 sessions) to reach performance criterion, this effect

was not significant (p = 0.3). Correlational analyses also failed to reveal any

monotopic relationship between integrated CORT values and number of training

sessions to criterion with either young (r = 0.41, p = 0.24) or aging animals (r = -

0.03, p = 0.93). Finally, analyses of individual differences of percentage of correct

responses at each delay interval as a function of integrated CORT values failed

to reveal any significant effects (p = 0.9 for all three).

These data lend support to the idea that a significant component of age-

related spatial working memory impairment may be accounted for by elevated

adrenocortical activity. The fact that CORT levels exerted no effect on delayed

alternation performance in the young animals at any time point, implicates the

cumulative exposure to CORT in underlying age-related impairments in this type

of cognitive functioning.


29

DISCUSSION In the present study, we have extended previous work showing that aging is

marked by a deterioration of prefrontally-mediated cognitive functions (Ando and

Ohashi, 1991; Bimonte et al., 2003; Ramos et al., 2003) and accompanying

regressive synaptic changes (Dumitriu et al., 2010; Bloss et al., 2011). First, we

verified that aging results in reduced dendritic spine density in PL cortical

pyramidal neurons. Digital reconstruction and 3D analysis of spine geometry

revealed that losses in immature subtypes (i.e., thin and stubby spines)

accounted for the overall decrements in spine density (Dumitriu et al., 2010;

Bloss et al., 2011). However, to our knowledge, this is the first study to show that

nearly all age-related dendritic spine alterations in the rat PL can be understood

in the context of elevated circulating glucocorticoids. While mushroom spines

have previously been shown to remain stable in the face of aging (Dumitriu et al.,

2010; Bloss et al., 2011), when we performed population analyses of spine head

diameter for this subtype this also uncovered shrinkage in this spine category as

a function of aging and high CORT secretory activity. Whereas subsequent

behavioral analyses verified at least some degree of impairment in spatial

working memory in aged rats, these effects were compounded in animals with

high HPA activity. Since immature spines exhibit the greatest potential for

synaptic plasticity and learning (Nimchinsky et al., 2002; Kasai et al., 2003,

2010a), the loss of this subpopulation has been hypothesized to account for age-

related impairments in cognitive function (Dumitriu et al., 2010; Bloss et al.,


30

2011). The present findings suggest that glucocorticoids may be a driving force

underlying dendritic spine remodeling in the prefrontal cortex that may be

precipitate cognitive impairment during aging.

HPA-dependence of dendritic spine depletion in aging mPFC

Our findings extend previous work showing depletion of dendritic spine density in

the prefrontal cortex of aging animals (Bloss et al., 2011). Similar to findings by

Bloss and colleagues (2011), aged rats in the current study showed significant

decreases in overall spine density in mPFC compared to young rats. However,

we have shown that dendritic spine loss is exclusive to aged animals with high

basal CORT levels, whereas aged animals with lower CORT secretion

maintained dendritic spine densities indistinguishable from young animals. The

present results are also consistent with the observation that thin spine subtypes

are selectively vulnerable to the effects of aging, although mature phenotypes,

such as mushrooms spines, remain relatively stable (Dumitriu et al., 2010; Bloss

et al., 2011). Additionally, we identified stubby spines as another vulnerable

subtype. Stubby spines are also considered as an immature, plastic spine

phenotype similar to thin spines (Harris and Kater, 1994; Boyer et al., 1998;

Petrak et al., 2005) so it is possible that our results can be understood from the

standpoint that the effects of aging broadly target immature spines. Most

importantly, our data suggest that attrition of both classes of these immature

prefrontal cortical spines in aging is glucocorticoid-mediated. While no effect was

seen on mushroom spine density based on age or CORT levels, cumulative


31

frequencies of mushroom spine head diameter indicate that aged animals with

high CORT secretion have mushroom spines with smaller head diameters than

young animals. This shift suggests that cumulative exposure to high levels of

CORT has a specific effect on mushroom spines, and that lower exposure allows

this phenotype to remain more relatively stable.

Behavioral correlates of prefrontal structural plasticity

A vast literature has described hippocampal-dependent cognitive deficits in

animals (and humans) with high levels of CORT (Lupien et al., 1997; McEwen,

1999; Lupien and Lepage, 2001). In humans, high levels of cortisol are

associated with significant impairments in declarative memory (Lupien et al.,

1997; McEwen, 1999) and administration of CORT has been shown to block LTP

in the hippocampus and to significantly impair spatial memory in animal studies

(Filioini et al., 1991; Diamond et al., 1992; McEwen, 1999). In spite of this,

interrogation of the relationship between the interactive effects of aging and

adrenocortical status on prefrontal structural and functional plasticity has not

been given much consideration. Due to the strength of the relationship between

elevated glucocorticoids and dendritic spine alterations in the aging mPFC, we

were compelled to test whether a prefrontal-dependent spatial working memory

task would show parallel disruptions (Divac, 1971; Bubser and Schmidt, 1990;

Aultman and Moghaddam, 2001). In delayed alternation, we found that high HPA

activity was associated with impaired performance exclusively in aged rats at

each delay interval tested (30, 60, and 120 s). However, at the 60 s delay


32

interval, aged animals bearing lower adrenocortical activity were also impaired

relative to younger animals, though less than aged-matched high CORT animals.

These results suggest that learning impairments in aged animals are not

exclusively dependent on HPA status. Thus at longer delay intervals, it appears

that aged animals with high basal levels of CORT are at a particular

disadvantage compared to aged animals with low CORT levels and young

animals in general.

Although our data strongly implicate an interaction between aging and

glucocorticoids in the deterioration of prefrontal structural and behavioral

plasticity, they do not directly show these structural alterations lead to behavioral

impairment. Previous work has shown that dendritic spines are constantly in a

labile state and can change quickly in response to physiological activation and a

variety of environmental stimuli (Nimchinsky et al., 2002; Hongpaisan and Alkon,

2007; Kasai et al., 2010b; Liston et al., 2013). With the delayed alternation task,

animals were trained over a period of several weeks, so it seems reasonable that

this prolonged learning period could lead to changes in prefrontal dendritic spine

morphology and function that may confound our current observations.

Age-related cognitive impairments following cumulative CORT exposure

There is a long-standing literature linking the cumulative exposure of

glucocorticoids across the lifespan and brain aging (Roth, 1979; Landfield et al.

1978; Yau and Seckl, 2012; Garrido, 2011). Studies suggest that adrenocortical


33

status predicts plasticity of dendritic spines in the prefrontal cortex of the aged

rat. Studies focusing on Sapolsky’s (1985) neurotoxicity of glucocorticoid

exposure hypothesis suggest that cumulative glucocorticoid exposure increases

damage to neurons in the hippocampus and impairing cognition (Kudielka et al.,

2009; Lupien et al., 2009). Many studies have shown that chronically elevated

levels of CORT significantly impair hippocampal function (Kirschbaum et al.,

1996; Lupien and McEwen, 1997; Lupien and Lepage, 2001; Lupien et al., 2009),

and our studies suggest the prefrontal cortex is also sensitive to this chronic

stress (Radley et al., 2004, 2008, 2013; Radley, 2005). Though we cannot make

mechanistic conclusions between the neurobiological measures we have taken

and the impairments in working memory we see in high CORT animals, a

potential mechanism can be elucidated. Aged rats with high basal levels of

CORT showed a significant loss of dendritic spine density, with this loss being

encompassed by the plastic thin and stubby spine phenotypes, potentially

impairing working memory. However, the smaller head diameter of the stable,

strong mushroom phenotype seen in these animals could also possibly play a

role in the working memory deficits. Collectively, these data highlight new targets

for developing therapeutic treatments that prevent or reverse age-related

cognitive decline in susceptible individuals.


34


35

References

Ando S, Ohashi Y (1991) Longitudinal study on age-‐related changes of working and

reference memory in the rat. Neurosci Lett 128:17–20.

Aultman JM, Moghaddam B (2001) Distinct contributions of glutamate and dopamine receptors to temporal aspects of rodent working memory using a clinically relevant task. Psychopharmacology (Berl) 153:353–364.

Bimonte HA, Nelson ME, Granholm A-‐CE (2003) Age-‐related deficits as working memory load increases: relationships with growth factors. Neurobiol Aging 24:37–48.

Bloss EB, Janssen WG, Ohm DT, Yuk FJ, Wadsworth S, Saardi KM, McEwen BS, Morrison JH (2011) Evidence for Reduced Experience-‐Dependent Dendritic Spine Plasticity in the Aging Prefrontal Cortex. J Neurosci 31:7831–7839.

Boyer C, Schikorski T, Stevens CF (1998) Comparison of Hippocampal Dendritic Spines in Culture and in Brain. J Neurosci 18:5294–5300.

Britton A, Shipley M, Singh-‐Manoux A, Marmot MG (2008) Successful Aging: The Contribution of Early-‐Life and Midlife Risk Factors. J Am Geriatr Soc 56:1098–1105.

Bubser M, Schmidt WJ (1990) 6-‐Hydroxydopamine lesion of the rat prefrontal cortex increases locomotor activity, impairs acquisition of delayed alternation tasks, but does not affect uninterrupted tasks in the radial maze. Behav Brain Res 37:157–168.

Cerqueira JJ, Taipa R, Uylings HBM, Almeida OFX, Sousa N (2007) Specific Configuration of Dendritic Degeneration in Pyramidal Neurons of the Medial Prefrontal Cortex Induced by Differing Corticosteroid Regimens. Cereb Cortex 17:1998–2006.

Cook SC, Wellman CL (2004) Chronic stress alters dendritic morphology in rat medial prefrontal cortex. J Neurobiol 60:236–248.

DeKosky ST, Scheff SW, Cotman CW (1984) Elevated corticosterone levels. A possible cause of reduced axon sprouting in aged animals. Neuroendocrinology 38:33–38.

Diamond DM, Bennett MC, Fleshner M, Rose GM (1992) Inverted-‐U relationship between the level of peripheral corticosterone and the magnitude of hippocampal primed burst potentiation. Hippocampus 2:421–430.


36

Dias-‐Ferreira E, Sousa JC, Melo I, Morgado P, Mesquita AR, Cerqueira JJ, Costa RM, Sousa N (2009) Chronic Stress Causes Frontostriatal Reorganization and Affects Decision-‐Making. Science 325:621–625.

Divac I (1971) Frontal lobe system and spatial reversal in the rat. Neuropsychologia 9:175–183.

Dumitriu D, Hao J, Hara Y, Kaufmann J, Janssen WGM, Lou W, Rapp PR, Morrison JH (2010) Selective changes in thin spine density and morphology in monkey prefrontal cortex correlate with aging-‐related cognitive impairment. J Neurosci Off J Soc Neurosci 30:7507–7515.

Fernández-‐Ballesteros R, García LF, Abarca D, Blanc L, Efklides A, Kornfeld R, Lerma AJ, Mendoza-‐Nuñez VM, Mendoza-‐Ruvalcaba NM, Orosa T, Paúl C, Patricia S (2008) Lay Concept of Aging Well: Cross-‐Cultural Comparisons. J Am Geriatr Soc 56:950–952.

Filioini D, Gijsbers K, Birmingham MK, Dubrovsky B (1991) Effects of adrenal steroids and their reduced metabolites on hippocampal long-‐term potentiation. J Steroid Biochem Mol Biol 40:87–92.

Gallagher M, Rapp PR (1997) The use of animal models to study the effects of aging on cognition. Annu Rev Psychol 48:339–370.

Geinisman Y, Detoledo-‐Morrell L, Morrell F, Heller RE (1995) Hippocampal markers of age-‐related memory dysfunction: behavioral, electrophysiological and morphological perspectives. Prog Neurobiol 45:223–252.

Grady CL (2008) Cognitive Neuroscience of Aging. Ann N Y Acad Sci 1124:127–144.

Harris KM, Kater SB (1994) Dendritic Spines: Cellular Specializations Imparting Both Stability and Flexibility to Synaptic Function. Annu Rev Neurosci 17:341–371.

Harris KM, Stevens JK (1989) Dendritic spines of CA 1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics. J Neurosci 9:2982–2997.

Hongpaisan J, Alkon DL (2007) A Structural Basis for Enhancement of Long-‐Term Associative Memory in Single Dendritic Spines Regulated by PKC. Proc Natl Acad Sci U S A 104:19571–19576.

Hongpaisan J, Xu C, Sen A, Nelson TJ, Alkon DL (2013) PKC activation during training restores mushroom spine synapses and memory in the aged rat. Neurobiol Dis 55:44–62.


37

Hung L-‐W, Kempen GIJM, De Vries NK (2010) Cross-‐cultural comparison between academic and lay views of healthy ageing: a literature review. Ageing Soc 30:1373–1391.

Issa AM, Rowe W, Gauthier S, Meaney MJ (1990) Hypothalamic-‐pituitary-‐adrenal activity in aged, cognitively impaired and cognitively unimpaired rats. J Neurosci 10:3247–3254.

Kasai H, Fukuda M, Watanabe S, Hayashi-‐Takagi A, Noguchi J (2010a) Structural dynamics of dendritic spines in memory and cognition. Trends Neurosci 33:121–129.

Kasai H, Hayama T, Ishikawa M, Watanabe S, Yagishita S, Noguchi J (2010b) Learning rules and persistence of dendritic spines. Eur J Neurosci 32:241–249.

Kasai H, Matsuzaki M, Noguchi J, Yasumatsu N, Nakahara H (2003) Structure–stability–function relationships of dendritic spines. Trends Neurosci 26:360–368.

Kirschbaum C, Wolf O., May M, Wippich W, Hellhammer D. (1996) Stress-‐ and treatment-‐induced elevations of cortisol levels associated with impaired declarative memory in healthy adults. Life Sci 58:1475–1483.

Kudielka BM, Hellhammer DH, Wüst S (2009) Why do we respond so differently? Reviewing determinants of human salivary cortisol responses to challenge. Psychoneuroendocrinology 34:2–18.

Landfield PW, Waymire JC, Lynch G (1978) Hippocampal Aging and Adrenocorticoids: Quantitative Correlations. Science 202:1098–1102.

Liston C (2006) Stress-‐Induced Alterations in Prefrontal Cortical Dendritic Morphology Predict Selective Impairments in Perceptual Attentional Set-‐Shifting. J Neurosci 26:7870–7874.

Liston C, Cichon JM, Jeanneteau F, Jia Z, Chao MV, Gan W-‐B (2013) Circadian glucocorticoid oscillations promote learning-‐dependent synapse formation and maintenance. Nat Neurosci 16:698–705.

Liu R-‐J, Aghajanian GK (2008) Stress Blunts Serotonin-‐ and Hypocretin-‐Evoked EPSCs in Prefrontal Cortex: Role of Corticosterone-‐Mediated Apical Dendritic Atrophy. Proc Natl Acad Sci U S A 105:359–364.

Lupien SJ, Gaudreau S, Tchiteya BM, Maheu F, Sharma S, Nair NPV, Hauger RL, McEwen BS, Meaney MJ (1997) Stress-‐Induced Declarative Memory Impairment in Healthy Elderly Subjects: Relationship to Cortisol Reactivity. J Clin Endocrinol Metab 82:2070–2075.


38

Lupien SJ, Lepage M (2001) Stress, memory, and the hippocampus: can’t live with it, can’t live without it. Behav Brain Res 127:137–158.

Lupien SJ, McEwen BS (1997) The acute effects of corticosteroids on cognition: integration of animal and human model studies. Brain Res Rev 24:1–27.

Lupien SJ, McEwen BS, Gunnar MR, Heim C (2009) Effects of stress throughout the lifespan on the brain, behaviour and cognition. Nat Rev Neurosci 10:434–445.

Matsuzaki M (2007) Factors critical for the plasticity of dendritic spines and memory storage. Neurosci Res 57:1–9.

McEwen BS (1999) Stress and Hippocampal Plasticity. Annu Rev Neurosci 22:105–122.

Michelsen KA, van den Hove DL, Schmitz C, Segers O, Prickaerts J, Steinbusch HW (2007) Prenatal stress and subsequent exposure to chronic mild stress influence dendritic spine density and morphology in the rat medial prefrontal cortex. BMC Neurosci 8:107.

Montaron MF, Drapeau E, Dupret D, Kitchener P, Aurousseau C, Le Moal M, Piazza PV, Abrous DN (2006) Lifelong corticosterone level determines age-‐related decline in neurogenesis and memory. Neurobiol Aging 27:645–654.

Morrison JH, Baxter MG (2012) The ageing cortical synapse: hallmarks and implications for cognitive decline. Nat Rev Neurosci 13:240–250.

Nimchinsky EA, Sabatini BL, Svoboda K (2002) Structure and Function of Dendritic Spines. Annu Rev Physiol 64:313–353.

Petrak LJ, Harris KM, Kirov SA (2005) Synaptogenesis on mature hippocampal dendrites occurs via filopodia and immature spines during blocked synaptic transmission. J Comp Neurol 484:183–190.

Radley J., Sisti H., Hao J, Rocher A., McCall T, Hof P., McEwen B., Morrison J. (2004) Chronic behavioral stress induces apical dendritic reorganization in pyramidal neurons of the medial prefrontal cortex. Neuroscience 125:1–6.

Radley JJ (2005) Repeated Stress Induces Dendritic Spine Loss in the Rat Medial Prefrontal Cortex. Cereb Cortex 16:313–320.

Radley JJ, Anderson RM, Hamilton BA, Alcock JA, Romig-‐Martin SA (2013) Chronic Stress-‐Induced Alterations of Dendritic Spine Subtypes Predict Functional Decrements in an Hypothalamo–Pituitary–Adrenal-‐Inhibitory Prefrontal Circuit. J Neurosci 33:14379–14391.


39

Radley JJ, Rocher AB, Rodriguez A, Ehlenberger DB, Dammann M, McEwen BS, Morrison JH, Wearne SL, Hof PR (2008) Repeated stress alters dendritic spine morphology in the rat medial prefrontal cortex. J Comp Neurol 507:1141–1150.

Ramos BP, Birnbaum SG, Lindenmayer I, Newton SS, Duman RS, Arnsten AF (2003) Dysregulation of protein kinase a signaling in the aged prefrontal cortex: new strategy for treating age-‐related cognitive decline. Neuron 40:835–845.

Rowe JW, Kahn RL (1987) Human Aging: Usual and Successful. Science 237:143–149.

Rowe JW, Kahn RL (1997) Successful Aging. The Gerontologist 37:433–440.

Sapolsky RM, Krey LC, McEwen BS (1983) Corticosterone receptors decline in a site-‐specific manner in the aged rat brain. Brain Res 289:235–240.

Spacek J, Hartmann M (1983) Three-‐dimensional analysis of dendritic spines. I. Quantitative observations related to dendritic spine and synaptic morphology in cerebral and cerebellar cortices. Anat Embryol (Berl) 167:289–310.

Sterner EY, Kalynchuk LE (2010) Behavioral and neurobiological consequences of prolonged glucocorticoid exposure in rats: Relevance to depression. Prog Neuropsychopharmacol Biol Psychiatry 34:777–790.

Wellman CL (2001) Dendritic reorganization in pyramidal neurons in medial prefrontal cortex after chronic corticosterone administration. J Neurobiol 49:245–253.

Yasumatsu N, Matsuzaki M, Miyazaki T, Noguchi J, Kasai H (2008) Principles of Long-‐Term Dynamics of Dendritic Spines. J Neurosci 28:13592–13608.


40

Figure 1. Top: Mean ± SEM plasma corticosterone (CORT) levels in young and

aged animals sampled at 4-h intervals across the light-dark cycle. Aged animals

showed elevated adrenocortical activity at 12 pm and 12 am time points as

compared with the young group. *, p < 0.05, differs significantly from control

animals . n = 12-14 per group. Bottom: Mean + SEM for plasma CORT levels

averaged across all six time points sampled (left), and plots of individual values

(right). Although young and aged animals did not significantly differ in terms of

overall adrenocortical activity, these exists a considerable degree of variability in

AgedYoungAged

Young

CORT(ng/ml/h)

0

20

40

60

CORT(ng/ml)

0

20

40

60 Young (3 mos)Aged (20 mos)

4 am 8 am 12 pm 4 pm 8 pm 12 am

∗∗


41

glucocorticoid secretory output within each group. These differences provided

the basis for dividing animals within each age category into subgroupings of high

and low adrenocortical activity (horizontal line in each indicates the median

value) for the assessment of age-related structural plasticity in mPFC as a

function of this endocrine index.


42

Figure 2. Top: Example of a layer 2 PL pyramidal neuron iontophoretically filled

with Lucifer yellow (LY, left), and (right) deconvolved confocal laser scanning

microscopy images of dendritic segments from different treatment groups.

Young Aged Young AgedApical<150µm

Young AgedApical>150µm

Young AgedBasal<150µm

dendriticspines/µm

1.0

2.0

3.0

4.0 Low CORTHigh CORT

Overall

LY neuron in layer 2 of PL

Young Low CORT

Young High CORT

Aged High CORT

Aged Low CORT

dendriticspines/µm

1.0

2.0

3.0

4.0

!

Young AgedOverall

!!

!! !

!


43

In this example, the apical dendritic tree points upward toward the pial surface

and curves to the right, whereas the axon and basal dendrites radiate from the

opposite pole of the cell body. The dashed circle demarcates the 150-µm

boundary used to partition the dendritic tree for spine analyses. Bottom: Mean +

SEM of dendritic spine density as a function of treatment group. Disregarding

differences in HPA status (lower left), aged animals (n = 13) show a significant

reduction in overall spine density (i.e., all three sites averaged into one value)

relative to young animals (n = 10). When aged and young animals are divided

according to adrenocortical status (lower left), the aged + high CORT group

exhibits a selective vulnerability for dendritic spine loss. Data represent mean +

SEM for each group, and are based on animal averages (n = 4-7 animals/ group;

n = 4 segments/ neuron; n = 5 neurons/ animal). Scale bar: 5 µm (upper right). *,

p < 0.05.


44

Figure 3. Top: Example neuron in layer 3 of PL that was iontophoretically filled

(left), and the rendering of its dendritic tree (right) utilizing computer-assisted

Young AgedApical

Young AgedBasal

dendriticlength(x10

2µm)

0

10

20

30

Young AgedApical

Young AgedBasal

numberofbranchendings

0

5

10

15

20

LY neuron in layer 3 of PL ddeennddrriittiicc mmaapp

Low CORTHigh CORT

Low CORTHigh CORT


45

morphometry. The apical dendritic tree (green) is pointing upward, and basal

dendrites (white) radiate from the opposite pole of the soma. Bottom:

Histograms for dendritic length and number of branch endings for apical and

basal dendrites. Aging or HPA status failed to result in any significant decreases

in the dendritic indices examined. Data represent mean + SEM for each index,

and are based on overall animal averages (i.e., n = 4-7 animals/ group; n = 1

arbor/ neuron; n = 5 neurons/ animal).


46

Figure 4. Top row: Example high-resolution deconvolved optical z-stack of a

dendritic segment utilized for spine analysis with NeuronStudio software. Open

stubbymushroom

thin

Young Aged Young AgedApical<150µm



dendriticspines/µm

1.0

1.5

2.0

Overall

2.0Thin subtype:

dendriticspines/µm

1.5

1.0

Young AgedOverall

length(µm)

0.3

0.4

0.2

Low CORTHigh CORT

length(µm)

0.3

0.4

0.2 Young AgedOverall

Spine head diameter:

Young Aged

dendriticspines/µm

0.5

0

1.0

Overall

Low CORTHigh CORT

dendriticspines/µm

0.5

0

1.0

Young AgedOverall

Mushroom subtype:

Young Aged

dendriticspines/µm

0.5

1.0

0 Young AgedApical<150µm



dendriticspines/µm

0

0.5

1.0Stubby subtype:

Young AgedOverall

Young AgedOverall

Low CORTHigh CORT

Low CORTHigh CORT

stubbymushroom

thin

Overall

***

***

**

**


47

colored circles designate spine subtypes based upon user-defined parameters in

the software (see methods). Bottom panel: Histograms showing the effects of

aging and adrenocortical status on mushroom, stubby, and thin spine density,

and mean spine head diameter. Whereas aging per se, did not result in any

decreases in overall densities for these subtypes (left column), aged animals

bearing high adrenocortical activity showed selective losses in thin and stubby

spines in overall measures and in aspects of the apical dendritic tree. *, p < 0.05.

Scale bar: 5 µm (applies to both images).


48

Figure 5. Top left: Mean + SEM for mushroom spine head diameters as a

function of treatment group. No group differences were evident in this analysis,

however these data are suggestive of a differential effect of corticosterone status

on head diameter in aged animals (aged + low CORT vs. aged + high CORT, p =

0.15). Top right: Cumulative frequency distributions of mushroom spine head

diameters in young animals do not indicate any change in the size in these

subtypes as a function of adrenal steroid activity. Lower left: The aged + high

CORT group shows a leftward shift in mushroom spine head diameter relative to

0.5

0.55

0.45 Young AgedOverall

mushroomheaddiam.(µm)

0.6 0.8 1.00.4

0.5

1.0

0

Aged+low CORTYoung-combined

K-Sp<0.000001

spine head diameter (µm)

cumulativedistribution

Young AgedOverall

LowCORT

LowCORT

HighCORT

HighCORT

1.0

Young+low CORTYoung+high CORT

K-Sp=0.8

0.4 0.6 0.8spine head diameter (µm)


0.5

1.0

0

0.6 0.8 1.0spine head diameter (µm)

0.4


0.5

1.0

0

K-Sp<0.0001

Aged+high CORTYoung-combined


49

young animals, suggesting that high levels of corticosterone may underlie an

increase in the number of smaller spines within this category. By contrast, aged

+ low CORT animals for this index show a cumulative frequency that is right-

shifted relative to young animals. Thus, aged animals with low CORT exhibit a

greater frequency of mushroom spines with large head diameters relative to the

young group. K-S, Kolmogorov-Smirnov test.


50

Figure 6. Histograms demonstrating the percentage of correct responses for

delayed-alternation performance using a T-maze task. At 30 s (left) and 120 s

(right) delay intervals, selective impairments are evident only within the aged +

high CORT group with respect to the other three groups. By contrast, interactive

effects of aging and adrenocortical status were observed at the 60 s delay

interval (middle). Whereas aging resulted in an attrition in the percentage of

correct response relative to both young subgroups after a 60 s delay period

regardless of adrenocortical status, this impairment was exacerbated in aged +

high CORT animals relative to age-matched animals with low adrenocortical

activity. Data represent mean + SEM for each index, and are based on overall

animal averages (i.e., n = 10 animals/group, 5 per subgroup).*, p < 0.05, differs

significantly from both young groups; †, p < 0.05, differs significantly from the

aged + low CORT group (applies to all).

percentcorrect

50

75

100

Young Aged30 s delay

Young

Aged

30 s delay

*

* †


60 s delay

percentcorrect

50

75

100

* †


Young

Aged

120 s delay

Low CORTHigh CORT

*

*

Young

Aged

percentcorrect

50

75

100Low CORTHigh CORT

Low CORTHigh CORT

*

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