NORMAL AGING AND COGNITION: SYSTEM-SPECIFIC CHANGES TO G-PROTEIN
COUPLED RECEPTOR-MEDIATED SIGNAL TRANSDUCTION WITHIN THE
HIPPOCAMPUS
BY
JOSEPH A. MCQUAIL
A Dissertation Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES
in Partial Fulfillment of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
Neuroscience
May 2013
Winston-Salem, North Carolina
Approved By:
Michelle M. Nicolle, Ph.D., Advisor
Examining Committee:
David R. Riddle, Ph.D., Chairman
Allyn C. Howlett, Ph.D.
Mary Lou Voytko, Ph.D.
Scott E. Hemby, Ph.D. ACKNOWLEDGEMENTS
It was only with the greatest support and consideration from my advisor, Dr. Michelle
Nicolle, that I could ever have achieved anything at all as a student of science. Our shared
interest in cognitive aging was merely the beginning to what ultimately became my deepest
professional relationship. Her guidance and mentorship as a scientist are only exceeded by her
advice and concern as a friend. I owe her the greatest debt of thanks for welcoming me into her
lab, nurturing my research interests and helping me to become the scientist that I am today.
I would also like to thank each of my committee members. Dr. David Riddle was a
constant source of encouragement, generous with his advice and a polite devil’s advocate; I was
always grateful for his input. Dr. Mary Lou Voytko offered fantastic insights into neurocognitive
aging, but also helped me to learn about teaching others; I appreciate that she welcomed me
aboard as a teaching assistant so early in my career and continued to help me refine my teaching
abilities. Dr. Allyn Howlett helped me transform a small idea inspired by a single article into a
fully-fledged proposal and invited me into her lab to make it a reality; her support and
enthusiasm for my research never waned. Dr. Scott Hemby made his expertise and resources
readily available to address the countless questions and issues that cropped over the duration of
my time in the lab; he was a fantastic “lab neighbor” and his generosity enabled so much of my work. Individually and collectively, I am indebted to these great scientists for their direction and consideration.
I would like to thank all of the scientists who helped me along the way on my lengthy professional journey. Drs. Linda Werling, Joshua Burk, Pamela Hunt, Michela Gallagher and
ii Jessica Mong were all instrumental in shaping the course of my development as an aspiring
neuroscientist. They all helped me to arrive at this moment and achieve this great distinction.
Most importantly, I must thank my friends and family for their support throughout this experience. Stephanie Willard and Tamara Spence were my closest friends here at Wake Forest and together we shared and endured the challenges of graduate school. My closest friend from high school, Ana Oancea, was constant source of understanding and support as we compared notes our respective graduate education experiences. Finally, it is the love and encouragement of
two great women, my mother, Diane McQuail, and my girlfriend, Elizabeth Currin, that made this whole experience and everything that comes after seem truly meaningful. I owe them the greatest “thank you” of all.
iii TABLE OF CONTENTS
PAGE
LIST OF ABBREVIATIONS ……………………………………………………………………..v
LIST OF TABLES………………………………………………………………………………..ix
LIST OF FIGURES…………………………………………………………………………..…...x
ABSTRACT……………………………………………………………………………………...xi
CHAPTER
I. INTRODUCTION.……………………………………………………………………...1
II. A MODEL OF COGNITIVE AGING IN THE F344 × BROWN NORWAY F1 HYBRID RAT: RESULTS FROM A TRIAL-BASED ANALYSIS..………………….73 Supplementary Text from Neuropharmacology 70:64–73, 2013
III. NEUROINFLAMMATION NOT ASSOCIATED WITH CHOLINERGIC DEGENERATION IN AGED-IMPAIRED BRAIN…………………………………….93 Published in Neurobiology of Aging 32(12):2322.e1–2322.e4, 2011
IV. GABAB RECEPTOR GTP-BINDING IS DECREASED IN THE PREFRONTAL CORTEX BUT NOT THE HIPPOCAMPUS OF AGED RATS…….110 Published in Neurobiology of Aging 33(6):1124.e1–1124.e12, 2012
V. HIPPOCAMPAL Gαq/11 BUT NOT Gαo-COUPLED RECEPTORS ARE ALTERED IN AGING…………………………………………………………………146 Published in Neuropharmcology 70:64–73, 2013
VI. DISCUSSION………………………………………………………………………190
APPENDIX……………………………………………………………...……….……..228
CURRICULUM VITAE………………………………………………...... ……..…256
iv LIST OF ABBREVIATIONS
2+ 2+ [Ca ]i intracellular Ca concentration
ABC avidin biotin complex
AC adenylyl cyclase
ACh acetylcholine
ACh acetylcholine
AD Alzheimer's disease
AI aged-impaired
AMPA 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid
AMPAR AMPA receptor
ANOVA analysis of variance
APV (2R)-amino-5-phosphonovaleric acid
ARC Aging Rodent Colony
ATP adenosine-5'-triphosphate
AU aged-unimpaired
AUC area under the curve
BAPTA-AM 1,2-bis(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid, acetoxymethyl- conjugated
CA1 Cornu Ammonis 1
CA2 Cornu Ammonis 2
CA3 Cornu Ammonis 3 cAMP 3'-5'-cyclic adenosine monophosphate
CCh carbachol
ChAT choline acetyltransferase
CICR Ca2+-induced Ca2+ release
v CPA cyclopiazonic acid
CPM counts per minute
DAG diacylglycerol
DAPI 40,6-diamidino-2-phenylindole dihydrochloride
DG dentate gyrus
DHPG (S)-3,5-dihydroxyphenylglycine
DNMS delayed non-matching to sample
DR delayed recognition
EC50 half maximal effective concentration
EDTA ethylenediaminetetraacetic acid
EGTA ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid
EMAX maximum possible effect for agonist
ER endoplasmic reticulum
F0 initial fluorescence
F344 Fisher 344
FBNF1 F344 × Brown Norway F1 hybrid
fMRI functional MRI
GABA γ-Aminobutyric acid
GABAAR GABA A receptor
GABABRs GABA B receptor
GAD-67 67 kDa isoform of glutamic acid decarboxylase
GDP Guanosine diphosphate
GPCR G-protein coupled receptor
GTP Guanosine-5'-triphosphate
GTP-Eu europium-labelled GTP
vi GTPγS guanosine-5’-O-(3-thio)triphosphate
HC-3 hemicholinium-3
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
ICS intracellular Ca2+ store
IgG immunoglobulin G
IP inositol phosphate
IP3 inositol 1,4,5-trisphosphate
IP3R IP3 receptor
LE Long-Evans
LTD long term depression
LTP long term potentiation mAChR muscarinic acetylcholine receptor
MCI mild cognitive impairment mGluR metabotropic glutamate receptor
ML molecular layer
MRI magnetic resonance imaging
MS medial septum nAChR nicotinic acetylcholine receptor
NH Hill slope coefficient
NHP non-human primate
NIA National Institute on Aging
NMDA N-Methyl-D-aspartate
NMDAR NMDA receptor
NPY neuropeptide Y
OML outer molecular layer
vii PFC prefrontal cortex
PI phosphoinositide
PIP2 phosphatidylinositol 4,5-bisphosphate
PKA protein kinase A
PLC phospholipase C
RGS regulator of G-protein signaling
RMANOVA repeated measures ANOVA
RyRs ryanodine receptor
SC Schaeffer collatoral sIPSP slow inhibitory post-synaptic potential
SLI spatial learning index
SPA scintillation proximity assay
SR stratum radiation
SST somatostatin
TBS tris-buffered saline
VAChT vesicular acetylcholine transporter
VDB vertical diagonal band
VGCC voltage gated calcium channel
ΔF change in fluorescence
viii LIST OF TABLES
PAGE
CHAPTER I
Table 1.1. Classification and Properties of GPCRs in the Hippocampus…………………...72
CHAPTER IV
Table 4.1. Parameters of baclofen-stimulated GTP-binding in the hippocampus of young and aged F344 rats…………………...………….……………………144
Table 4.2. Parameters of baclofen-stimulated GTP-binding in the PFC of young and aged F344 rats………………………………..……………………………145
CHAPTER V
Table 5.S1. Results of correlation analyses between proximity scores and neurobiological parameters in young and aged rats………………………….…189
ix LIST OF FIGURES
PAGE
CHAPTER I
Figure 1.1. The interaction between chronological or biological aging and cognitive function (“cognitive aging”)……………………………………………………..69
Figure 1.2. Schematic illustration of the hippocampus and adjacent structures…………..…70
Figure 1.3. Key GPCRs in the hippocampus…………………………………………………71
CHAPTER II
Figure 2.1. Training trial performance of young and aged rats………………………………89
Figure 2.2. Quadrant-derived measures characterizing probe trial performance in young and aged rats……………………………………………………………...90
Figure 2.3. Savings scores following 24 hour delay imposed in each training block in young, aged-unimpaired and aged-impaired rats……………………………...91
Figure 2.4. Swim speeds of young and aged rats during training trial and probe trial conditions………………………………………………………………………...92
CHAPTER III
Figure 3.1. Morris water maze performance in young and aged rats……………..………...105
Figure 3.2. Quantitative measures of cholinergic neurons and activated microglia in young and aged rats within the MS/VDB……………………...……………….106
Figure 3.S1. Bright-field photomicrographs depicting ChAT- and CD68-immunostaining in the MS/VDB of young and aged rats…………………………………..……107
Figure 3.S2. Density of ChAT+ cells thorough rostro-caudal sampling distribution……...…109
CHAPTER IV
Figure 4.1. Spatial learning in young and aged rats………………………...………………138
Figure 4.2. Baclofen-stimulated GTP-binding in the hippocampus of young and aged rats…………………………………………………...……………………139
x
Figure 4.3. Baclofen-stimulated GTP-binding in the prefrontal cortex of young and aged rats…………………………………………...……………………………140
Figure 4.4. GABABR1 and GABABR2 protein levels in hippocampus of young and aged rats…………………………………………………...……………………141
Figure 4.5. GABABR1 and GABABR2 protein levels in the prefrontal cortex of young and aged rats……………………………………………………………………142
Figure 4.6. Ratios of GABABR1a:GABABR1b across PFC and hippocampus of young and aged rats……………………………………………………………………143
CHAPTER V
Figure 5.1. Performance of young and aged rats in the Morris water maze………………..178
Figure 5.2. GTPγS-binding to Gαq/11 in the hippocampus of young and aged rats…………179
Figure 5.3. Basal and oxotremorine-M stimulated GTPγS-binding to Gαq/11 are inversely correlated in the hippocampus of young and aged rats…………………………180
Figure 5.4. GTPγS-binding to Gαo in the hippocampus of young and aged rats…………...181
Figure 5.5. Oxotremorine-M-stimulated changes to intracellular Ca2+ concentration in CA1 of young and aged rats……………………………………….……………182
Figure 5.6. DHPG-stimulated changes to intracellular Ca2+ concentration in CA1 of young and aged rats………………………………………………….…………184
Figure 5.S1. Relationship between spatial learning and basal GTPγS-binding to Gαq/11 in DG after correcting for differences between age groups……………….……188
CHAPTER VI
2+ Figure 6.1. Summary of changes to GPCRs and modulation of [Ca ]i at aged hippocampal synapses.……………………………………………….…………226
Figure 6.2. Proposed model linking current findings to changes in synaptic plasticity.……227
xi ABSTRACT
JOSEPH A. MCQUAIL
NORMAL AGING AND COGNITION: SYSTEM-SPECIFIC CHANGES TO G-
PROTEIN COUPLED RECEPTOR-MEDIATED SIGNAL TRANSDUCTION WITHIN
THE HIPPOCAMPUS
Dissertation under the direction of Michelle M. Nicolle, PhD
Associate Professor of Internal Medicine, Section on Gerontology, and Physiology &
Pharmacology
Aging is associated with a general deterioration in the ability to form new memories, a neural process that depends upon the hippocampus and its interconnected brain regions.
However, the magnitude of age-related cognitive decline is highly variable, a portion of older humans exhibit no or little memory impairment whereas others exhibit obvious memory deficits that may or may not progress to more severe neurological illness such as Alzheimer’s disease.
Thus, there is a pressing need to understand the effects of normal aging separate from and before the manifestation of neuropathological disorders. Aged rats exhibit a qualitatively similar cognitive profile when tested for spatial learning ability in the Morris water maze; ~50% of aged rats exhibit robust cognitive impairment compared to young controls while the remaining 50% of aged rats perform on par with young. Using this naturally-occurring model, it is possible to examine neurobiological processes that not only changed with age, but also significantly associated with learning and memory. Parameters associated with neurotransmission are logical candidates for neurobiological analysis; including the numbers of cells that produce specific
xii neurotransmitters, the functionality of receptors and associated proteins that transduce
extracellular signals and the modulation of intracellular Ca2+, a key molecular messenger that
modifies synaptic strength. In Chapter II it is determined that F344 × Brown Norway F1
(FBNF1) hybrid rats are impaired at spatial learning by 24 months of age, relative to 6 month-old
controls. This finding is novel because it was previously assumed that this strain of rat maintains
cognitive function until somewhat older ages (i.e. 28-30 months). The basis for this impairment relates to inferior spatial learning in a subset of aged rats (aged-impaired) relative to young as well as aged rats that are behaviorally similar to young (aged-unimpaired). Importantly, aged- impaired rats exhibit deficits in acquisition, but retention of learned information is not affected.
Chapter III presents the stereologically-determined estimates of the total number of cholinergic
(ChAT+) neurons and activated (CD68+) microglia in the MS/VDB of young rats and aged-
impaired rats. These data show that there is no loss of cholinergic neurons in the aged-impaired
MS/VDB, despite increased numbers of activated microglia, suggesting a substantial elevation of
the local, basal inflammatory state. In Chapter IV, baclofen-stimulated GTP-Eu binding and
western blotting were used to measure the functionality and expression of GABABR proteins in
the hippocampus and PFC of young and aged rats. Results of this study reveal that aged-impaired
rats express lower levels of GABABR1 protein in the hippocampus compared to young and aged-
unimpaired rats while GABABR2 protein level was not changed. Significantly, loss of
GABABR1 did not impair baclofen-stimulated GTP-Eu binding in the hippocampus. However,
there is clear evidence for reduced GTP-Eu binding and expression of GABABR1 and
GABABR2 in the PFC of all aged rats, suggesting an age-, but not spatial-learning, related loss of
GABABR substrates and activity in this region. Chapter V describes the use of a modified
version of the GTPγS-binding assay to determine that aging is associated with greater basal
xiii binding of GTPγS-binding to Gαq/11 across all hippocampal subregions, while oxotremorine-M- stimulated GTPγS-binding to Gαq/11 tended to be lower in the aged hippocampus and was
2+ inversely related to basal activity. Also, in Chapter V, confocal imaging of [Ca ]i demonstrates
2+ oxotremorine-M-stimulated elevation of [Ca ]i is lower in the aged CA1 area compared to
2+ young while DHPG-stimulated changes to [Ca ]i are potentially dysregulated in the aged CA1.
Despite these receptor system-specific effects, aged CA1 cells in both experiments utilized ICS to a greater degree than young, suggesting that Ca2+ source, not simply magnitude, is an important factor in neuron aging. In conclusion, these studies demonstrate that changes to
GPCRs and their associated physiologic functions are significant targets for further evaluation in the context of aging and hippocampal-dependent cognition.
xiv CHAPTER I
INTRODUCTION TO “NORMAL AGING AND COGNITION: SYSTEM-SPECIFIC
CHANGES TO G-PROTEIN COUPLED RECEPTOR-MEDIATED SIGNAL
TRANSDUCTION WITHIN THE HIPPOCAMPUS”
Portions of this Chapter were published: McQuail JA, Nicolle MM (2012) Animal Models of
Aging and Cognition. Current Translational Geriatrics and Experimental Gerontology Reports.
1:21-28.
1 1.1. Statement of Problem
Aging is a risk factor for progressive cognitive decline as well as the manifestation of
neuropsychiatric disorders. However, not all older individuals will inevitably develop a neurological disorder of any sort, indicating that there is a normal aging process that is separate
from any disease. Thus, understanding the effects of normal aging on cognition may lead to the
discovery of associated neural changes that increase one’s likelihood for developing
neuropathological disorders. Importantly, the earliest indication of age-related changes to
cognitive function provides the ideal moment to begin offering preventative treatments provided the mechanisms that mediate cognitive decline are known and amenable to therapeutic modulation. Conversely, examination of neural processes associated with preserved cognitive function at advanced ages, or “successful aging”, provides a conceptual framework for the development of the next generation of molecular targets and treatment strategies. With these key considerations in mind, the current studies were designed to evaluate the relationship between early changes in cognitive function in a translationally relevant rodent model of aging and the status of particular subtypes of neural cell populations, therapeutically relevant G-protein
coupled receptors, and receptor-mediated changes to intracellular Ca2+.
1.2. Human Aging and Cognition
As our population grows and improved standard of living and medical innovation extend the human lifespan, more individuals will live to older ages, ages when cognitive well-being is a deciding factor in one’s level of independence and quality of life. The number of Americans over the age of 65 will more than double from slightly more than 40 million today to almost 90 million by 2050 (United States Census Bureau, 2009). What’s more, older Americans will
2 comprise a larger fraction of our population growing from 13% to 20% over the same time
interval. This growth is unique in fact because children and teenagers and adults between 20 and
64 years of age will actually come to represent a smaller fraction of the total population in 2050
when compared to current numbers. The absolute and relative increase in the number of older
individuals poses substantial medical, financial and social concerns. While, aging is generally associated with decreased cognitive ability, there is actually a great diversity in individual performance. Some older individuals perform on par with subjects half their age while others are not as efficient as young but perform as well as expected given their age. Critically, a significant portion of the aged population begin to exhibit greater than expected impairment relative to their own age group and the appearance of such impairments places one at greater risk for developing dementia, most commonly, Alzheimer’s disease (AD). Therefore, it is critical to understand how normal aging modulates neural substrates that support learning and memory and to differentiate this process from more severe forms of neuropathology that occur with increased frequency in the elderly.
1.2.1. Normal Aging and Memory
Memory loss is the most frequent cognitive complaint among older adults. Although not obvious, there is a subtle decline of memory function beginning at young adulthood and proceeding throughout the duration of the human lifespan (Salthouse, 2009). Memory also exhibits less stability at older ages, indicating that memory outcomes are highly variable with
advancing age (Morse, 1993; Salthouse, 2012). When making discrete comparisons of memory
among healthy humans grouped according to age, a significant deficit is noted as early as the
sixth decade (i.e. the 50s) relative to younger controls (those in their 30s), but even in the 70-80
year-old group, there are a subset of individuals that remain on par with young (Petersen et al.,
3 1992; also discussed in Albert, 1997), further underscoring that chronological age does not sufficiently characterize cognitive status. As the rate of memory decline varies from one individual to another, it is now understood that individuals with the greatest changes in memory are the most likely to progress to dementia (Rubin et al., 1998; Gamaldo et al., 2012), although the transition from normal, healthy age-dependent changes in memory to abnormal, pathological changes remains an imprecise boundary.
1.2.2. Mild Cognitive Impairment
The transition between the subtle effects of normal aging on memory and the severe behavioral impairments evident in dementia patients remains imprecise. To bridge these states, amnestic mild cognitive impairment (aMCI) was first developed as a clinical diagnosis to characterize individuals that exhibited a significant memory deficit, unaccompanied by deficits in other cognitive domains, that is greater than expected after adjusting for age, education and other health factors (Petersen et al., 1999). The aMCI construct is a useful tool as these individuals are at a greater likelihood of eventually being diagnosed with clinically probable
Alzheimer’s disease (AD) than the general age-matched population (10-15% per year for aMCI versus 1-2% for general population; Tierney et al., 1996). In fact, approximately 80% of aMCI patients will progress to AD within six years of initial diagnosis (Petersen et al., 2001), providing a significant population of patients for specialized, targeted care and treatment as well as clinical study and research. At this time, more than 160 clinical trials are recruiting MCI patients to either investigate basic parameters of the MCI brain or to test the therapeutic efficacy of pharmaceutical interventions (U.S. National Institutes of Health, 2013).
4 1.2.3. Dementia and Alzheimer’s Disease
Dementia is the most severe form of cognitive impairment afflicting the elderly. Such patients will, by clinical definition, exhibit not only deficits of memory but also impairments in
at least one other cognitive domain (i.e. executive function, language, visuo-spatial processing)
and lack the capacity to carry out basic activities of daily living such as feeding, bathing, dressing or otherwise caring for oneself (American Psychiatric Association, 2000). The causes of dementia are many-fold, but in the absence of any specific neurologic insult or mitigating health condition, older patients with dementia are clinically diagnosed with AD (McKhann et al., 2011).
Confirmation of bona fide AD is not possible until post-mortem examination corroborates that clinically evident dementia is accompanied by the presence of amyloid plaques and neurofibrillary tangles within the brain (Braak and Braak, 1991). From a treatment perspective, interventions initiated prior to (prodromal) or shortly after the onset of dementia yield minimal benefit to patients as approved treatments provide modest and temporary relief of symptoms, but cannot alter or reverse the underlying disease process.
1.2.4. Neural correlates of cognition in Normal Aging, MCI and AD
Characterization of susceptible brain regions or circuits aids in the understanding of the neurobiological mechanisms that drive changes to memory between normal aging, MCI and AD.
Much aging research focuses on evaluating the integrity of the hippocampus (reviewed in Burke and Barnes, 2010; Driscoll and Sutherland, 2005; Gallagher et al., 2010; Lister and Barnes,
2009; Rosenzweig and Barnes, 2003; Small et al., 2011) as patients with selective hippocampal damage exhibit memory deficits that are qualitatively similar to those observed in Alzheimer’s disease (Bright et al., 2006; Corkin et al., 1997; Jeneson et al., 2010; Kohl et al., 2011; MacKay et al., 1998; Race et al., 2011; Rempel-Clower et al., 1996; Scoville and Milner, 2000; Squire et
5 al., 2010; Stefanacci et al., 2000; Zola-Morgan et al., 1986). In healthy individuals these same regions are activated during tasks that require the encoding and recall of novel information
(Karlsgodt et al., 2005; Kumaran and Maguire, 2006; Motley and Kirwan, 2012; Rekkas and
Constable, 2005; Small et al., 2001).
The hippocampus is a bilateral structure that may be broadly subdivided into 3 primary regions, based upon cell morphology and synaptic connectivity; these subdivisions are the dentate gyrus (DG), and cornu ammonis (CA)3 and CA1 (reviewed in Amaral and Witter, 1989).
The hippocampus is comprised of a largely unidirectional network of pathways. Information enters the hippocampus via the perforant path, formed by the axons of neurons located in the entorhinal cortex, which terminates upon the dendrites of granule neurons in the DG. The granule cells send their axons to the CA3 via the mossy fiber pathway, to synapse upon dendrites belonging to pyramidal neurons located in the CA3. These CA3 pyramidal neurons give rise to the Schaeffer Collateral pathway that form synapses on CA1 pyramidal neurons. Finally, the neurons within the CA1 send their axons out of the hippocampus to the subiculum that, in turn, eventually connects back to the entorhinal cortex (Figure 2).
1.2.4.1 Hippocampal volume declines across the human lifespan
The hippocampus demonstrates a unique volumetric decline over the human lifespan; there is minimal shrinkage prior to the age of 50, but after this age, the hippocampus shrinks faster than any other brain region measured (Raz et al., 2004a) and it’s rate of shrinkage increases with age (Raz et al., 2004b). This decreased hippocampal volume is reliably correlated with impaired performance on neuropsychological measures of memory at older ages (Raz et al.,
1998; Rosen et al., 2003). Imaging approaches that distinguish among hippocampal subdvisions suggest that the DG and the subiculum are selectively reduced in size with age (Small et al.,
6 2002). Interestingly, post-mortem analysis of hippocampal tissue from healthy, aged adults also reveals a selective loss of neurons within the hilus, a heterogeneous grouping of neurons encircled by the DG granule cells, as well as the subiculum (West, 1993). But this pattern is distinct from AD patients who uniquely exhibit pronounced neuron loss in the CA1 subregion
(West et al., 2000) in addition to neuropathological changes in the adjacent entorhinal cortical regions (Thangavel et al., 2008). Collectively, these substantial differences between the normal aged and AD brain reveal that dementia is not the ultimate destination of brain aging, AD pathology follows its own unique disease progression.
1.2.4.2. Loss of cortical input to hippocampus and compensatory changes in Aging, MCI and AD
In contrast to neuronal cell loss observed in AD, changes to synaptic function appear to drive cognitive deficits in normal aging and MCI. Scheff et al (2007, 2006) has demonstrated that the number of synapses in the outer molecular layer (OML) of the DG and the stratum radiatum (SR) of the CA1, respective synaptic input zones for each subregion, are significantly correlated with delayed recall across a cohort of older adults including cognitively normal individuals as well as MCI and AD patients; greater recall was associated with greater synapse number. Yassa et al. (2010a) used in vivo ultra-high resolution diffusion tensor imaging to determine that decreased diffusivity, a measure of fiber integrity, along the perforant path, the tract that carries input to the hippocampus from cortex, was associated with worse delayed recall in older adults. Although synapses are lost in MCI patients, there is a paradoxical increase in hippocampal activation, measured using functional magnetic resonance imaging (fMRI), unique to MCI patients performing a memory task relative to normal controls or AD patients (Dickerson et al., 2005; Miller et al., 2008). This latter observation suggests that decreased cortical input to the hippocampus results in compensatory functional changes within the hippocampus of MCI
7 patients. However, this mechanism is not beneficial to memory function in older adults or MCI patients as excess activity of the DG/CA3 network observed by fMRI is associated with worse performance on a task that explicitly requires the differentiation of novel versus familiar visual stimuli (Yassa et al., 2011, 2010b).
1.2.5. Summary of human brain aging
Older humans are likely to experience some degree of memory loss with age, but the severity of this loss varies greatly from one individual to the next ranging from little or none to profound dementia. While differences between these extremes are quite obvious, the vast continuum of behavioral and neurological differences in between offers few clear boundaries.
Importantly, it is not conclusively known the extent to which memory loss is attributable to
“normal” aging or when and how disease processes diverge from this trajectory on their own unique etiological course. As the antecedents of AD may evolve over the course of many years, it can be difficult to differentiate normal cognitive aging from prodromal AD (Amieva et al.,
2005; Elias et al., 2000; Kawas et al., 2003). Conversely, not all MCI patients will necessarily develop dementia, further complicating the use of clinical criteria to class elderly memory patients. Thus, clinicians and scientists examining memory impairments in older humans are challenged to differentiate between normal, nonpathological aging and the possibility that subtle changes to memory are actually the first step in a progression towards dementia.
1.3. Animal Models of Cognitive Aging
Human brain aging is a complex process that involves interactions between biological aging as well as the cumulative effects of lifestyle, such as smoking, diet and exercise, and health issues, such as diabetes, obesity or cardiovascular disorders, that differentially affect general, as
8 well as cognitive health (reviewed in (Ahlskog et al., 2011; Biessels and Kappelle, 2005;
Colcombe et al., 2004; Kidd, 2008; Peters et al., 2008; Siervo et al., 2011). Consequently, researchers make use of animal models that provide a highly controlled organism for behavioral and neurobiological analysis to maximize the detection of age-related changes to cognition and brain substrates while minimizing cofounds associated with disease, peripheral health or differences in life history and prior experience. However, researchers must demonstrate that their selected model sufficiently recapitulates relevant aspects of the aging process to ensure that findings are of translational value to inform back to the human condition. Currently, the most commonly used model species for investigations of aging are non-human primates (NHP; monkeys such as the rhesus macaque) and rodents (particularly rats).
1.3.1. Non-Human Primate Model of Aging
Research findings obtained from monkeys are readily translatable given the close phylogenetic relationship between humans and monkeys compared to any other species in the animal kingdom, barring great apes of the family hominidae which are rarely used in scientific experimentation due to ethical concerns and commensurate regulatory restrictions (Kaiser,
2013). NHPs possess a well-developed, gyrencephalic cerebral cortex, can perform psychological tasks that closely mirror those used to measure human cognition and NHPs may be studied using medical and neuroimaging procedures that are almost identical to those used in human care in addition to more invasive procedures that may require euthanasia and necropsy for post-mortem analyses. Like humans, monkeys are relatively long-lived; researchers generally agree that monkeys age at approximately a 3:1 ratio compared to humans, thus a monkey is considered to be “aged” after 20 years (Andersen et al., 1999; Collier et al., 2005; Peters et al.,
1998). Also similar to humans, older NHPs are generally impaired on a variety of tasks that
9 require the retention of new information over delays of variable length including the delayed non-match to sample (DNMS) and delayed recognition (DR) tests (Herndon et al., 1997; Rapp and Amaral, 1991). Despite these many similarities with humans, aged monkeys do not develop
Alzheimer’s disease (Peters and Kemper, 2012; Peters, 2002; Peters et al., 1996), providing a useful model to study brain aging free from the effects of neurological disease.
1.3.1.1. Comparing targeted and systems approaches to studying the aged NHP brain
Targeted analysis of the aged NHP hippocampus using post-mortem approaches has revealed similarities to human aging. Just as older humans with cognitive impairment show evidence for synaptic alterations in the DG-OML, the aged monkey DG-OML shows greater numbers of synaptic boutons with no apparent post-synaptic element and fewer boutons that contact multiple post-synaptic targets, indicating a shift towards less efficient synapses, that reliably correlates with impaired DNMS performance (Hara et al., 2011). These changes to synaptic morphological also occur at the same time as a significant downregulation of post- synaptic receptors (specifically NMDARs, discussed in greater detail in section 1.4.2. and 1.5), although the relationship to cognition was not assessed in this study (Gazzaley et al., 1996).
Whereas older humans with cognitive impairment exhibit greater activity in the DG/CA3 network, the metabolic rate of the DG is lower in aged monkeys with cognitive impairment
(Eberling et al., 1997; Small et al., 2004). However, these data do not preclude the possibility that task-related DG/CA3 activation is greater in impaired NHPs because no studies have used electrode implanation or functional imaging approaches to investigate this parameter. The latter approach, while non-invasive, is not likely, as monkeys must be anesthestized for imaging, prohibiting concurrent task performance to recruit specific neural circuits.
10 As the monkey brain may be imaged using medical scanners designed for human neuroimaging, it is possible to conduct brain-wide analyses in aged monkeys. In stark contrast to human neuroimaging findings, MR imaging of the aged monkey brain reveals no volumetric decline with advancing age and not relationship with cognitive performance (Shamy et al.,
2006). Rather, MR imaging has revealed that the dorsolateral prefrontal cortex and striatum are significantly smaller in aged NHPs compared to younger adult NHPs (Alexander et al., 2008;
Shamy et al., 2011) and greater shrinkage was associated with worse performance on DNMS.
Apart from changes to grey matter, there are also reductions in frontal white matter, the fiber bundles that contain the axons conveying information to and from discrete cortical subdivisions
(Shamy et al., 2011; Wisco et al., 2008), although there is no consensus if this parameter associates with memory performance. However, white matter tracts may undergo changes to myelin integrity that compromise signal propagation without overt white matter reductions.
Accordingly, Makris and colleagues revealed that older monkeys exhibit decreased fractional anisotropy of frontal white matter tracts, indicating that myelin structure is compromised and may lead to impaired coordination between frontal cortical regions (Makris et al., 2007).
1.3.1.2. Considerations in the design and conclusions of aging NHP studies
The conclusions drawn from imaging-based studies of aged monkeys generally deemphasize a role for the hippocampus in the appearance of memory deficits leading one to question whether apparent age-effects evident in older humans are due to undetected or subclinical pathological processes affiliated with Alzheimer’s disease or whether the changes that occur with age in the hippocampus of non-human primates are not amenable to study given the obstacles associated with working with these animals. Issues to consider include imaging the smaller (relative to human) NHP hippocampus on MR equipment designed for humans or using
11 fewer animals per study given the expense of housing and maintaining monkeys. To illustrate,
Raz et al. (2004a) imaged the hippocampi of 200 human participants (ranging from late teenage years to early 80s) with an average hippocampal volume of 6.69 cm3 whereas Shamy et al.
(2006) imaged the hippocampus of 12 monkeys (9.5-29.2 years) with an average hippocampal volume of 4.15 cm3. As both studies used an MR scanner with a 1.5 Tesla magnet (a common field strength for clinical applications), it possible that subtle shrinkage of the smaller monkey hippocampus (approximately 60% of the volume of the human hippocampus) may not exceed the detection limit of the scanner. Furthermore, human participants in Raz et al. (2004a) outnumbered monkeys used in Shamy et al. (2006) by nearly 17-to-1, demonstrating a clear difference in sample size and therefore statistical power to detect changes. Thus, it is apparent that despite the close homologies between the human and monkey brain, other animal models may provide a better cost-benefit trade off to power more refined studies of comparatively cryptic molecular processes that underlie changes to hippocampal-dependent cognition in older animals.
1.3.2. Rat Model of Cognitive Aging
Although not of the same phylogenic order as humans and monkeys, rats are mammals and their brains exhibit grossly translatable neuroanatomical features, including similar organization of the hippocampus (Amaral and Witter, 1989). However, rats exhibit a dramatically shorter life-span ranging between 2 and 3 years depending on the specific strain selected for use (Holloszy, 1997; Turturro et al., 1999). Despite the evolutionary distance between humans and rats, carefully designed behavioral tasks can effectively assess rodent cognition in a translationally appropriate manner. Since its characterization 30 years ago, the
Morris water maze remains the gold standard for testing hippocampal-dependent, spatial learning
12 in rats (Morris, 1984). This task requires rats to learn to navigate to a hidden, submerged platform located in fixed position within a pool of water by making use of visual cues located around the perimeter of the maze to guide their search. Of translational note, the human hippocampus is also crucial for spatial navigation and memory of specific locations (Aradillas et al., 2011; Bohbot et al., 2000, 1998; Goodrich-Hunsaker and Hopkins, 2010; Goodrich-Hunsaker et al., 2010; Kim et al., 2011; Stepankova et al., 2004) and is activated during fMRI scanning of subjects performing virtual navigation tasks (Baumann and Mattingley, 2010; Hartley et al.,
2003; Iaria et al., 2007, 2003; Marsh et al., 2010; Ohnishi et al., 2006; Shelton and Gabrieli,
2002; Weniger et al., 2010; Wolbers and Büchel, 2005). Older, non-demented humans are impaired relative to young adults on virtual forms of the water maze that have been reversed- engineered from the original rodent task (Driscoll et al., 2005, 2003; Moffat and Resnick, 2002;
Moffat et al., 2007; Rodgers et al., 2012). Thus, spatial learning is a reliable behavioral approach to characterize the functional status of the hippocampus and offers excellent translational parallels between rodents and humans.
1.3.2.1. Spatial learning in young and aged rats
Water maze performance is assessed using a combination of three trial types: training trials, probe trials and cued trials. During training trials, the platform is hidden just beneath the surface of the water but, once found, the rat “escapes” from the water and rears up on the platform to observe the testing arena. Over the course of several training trials, cognitively intact rats learn to swim shorter, more direct paths to the platform, regardless of their relative starting location. On probe trials, the platform is removed, requiring the rat to search for the platform for a fixed duration but also encouraging the rat to dwell in the vicinity of the former platform location promoting the use of a localized search pattern for the platform. At the end of the probe
13 trial, the platform is covertly raised into position thereby allowing for escape and maintaining
normal response-reinforcement contingencies. Cued trials, measured separately from the “place-
learning” phase of the protocol, entail extending the platform to rise just above the surface of the
water so that it is readily visible. During cued trials, all of the same motor, sensory and
motivational requirements are needed to swim to the platform but cognitive demands are
eliminated, thereby providing a means of controlling for non-cognitive performance factors like
swimming ability, visual acuity and motivation to escape on to the platform
Using these approaches, several papers have determined that aged rats (i.e. 22-28 months of age depending on strain), including Long-Evans (LE), F344 and Sprague-Dawley (SD) strains, are impaired on spatial learning tasks compared to younger rats (Barnes et al., 1997;
Bizon et al., 2009; Frick et al., 1995; Gage et al., 1984; Gallagher et al., 1993; Lee et al., 1994;
Rowe et al., 1998; Tombaugh et al., 2005), but often the aged cohort includes a subset of animals
with obvious cognitive impairment (i.e. performance is outside of the range of young adults)
while other aged rats exhibit performance that is similar to young rats (discussed in Baxter and
Gallagher, 1996). This heterogeneous mixture of cognitive outcomes in aged rats is notable
because, as reported in Albert (1997), a subset of elderly humans will perform neuropsychological tasks as efficiently as younger adults. The qualitatively similar patterns of cognitive profiles between aged humans and rats support the notion that the rat is a suitable
model for the study of age-related changes to hippocampal-dependent cognition and associated biological alterations to the hippocampus itself.
1.3.2.2. Hippocampal correlates of cognitive aging in rats
Synaptic and functional alterations observed in older, memory impaired humans are also observed in aged-impaired rats. Similar to non-demented humans, aged rats do not lose principle
14 neurons of the hippocampus or entorhinal cortex (Rapp and Gallagher, 1996; Rapp et al., 2002).
In contrast to humans (Scheff et al., 2006), perforant path synapses are not lost outright in the
DG of aged rats with cognitive impairment, (Newton et al., 2008 but see Geinisman et al., 1992).
However, there is a significant decrease expression of vesicular markers per synapse, possibly
due to diminished vesicular content (Davies et al., 2003), and these reductions correlate with spatial learning impairment (Smith et al., 2000). Despite, or perhaps in response to, loss of
cortical input, DG granule neurons are more excitable in aged rats than young (Barnes and
McNaughton, 1980) and the DG/CA3 network is hyperactive, particularly in aged rats with
spatial learning impairment (Patrylo et al., 2007; Wilson et al., 2005).These findings have led to
a hypothesis that compromised cortical input to the DG elicits compensatory changes in the
DG/CA3 network in the form of greater neural activity that is enabled by decreased inhibitory
regulation and this pattern very similar to that observed in MCI (discussed in section 1.2.4.2.).
1.3.3. Summary of animal models of cognitive aging
Animal models of cognitive aging offer a viable path to study parameters of normal aging, free from confounds associated with Alzheimer’s disease and other uniquely human factors. Despite the importance of the hippocampus to normal learning and memory and its alterations in human aging and Alzheimer’s disease, monkey models of cognitive aging have largely focused on the integrity of the cerebral cortex and the white matter tracts that interconnect brain regions. Presently, aged rodent models combine sufficient translational value with technical feasibility to execute well-powered studies of hippocampal morphometry and physiology. Therefore, aged rats are an appropriate model for use in ongoing studies to examine more refined (i.e. biochemical and pharmacological) parameters that are not easily accomplished using human or monkey tissue.
15 1.4. G-Protein Coupled Receptors in the Hippocampus
Once a tractable model has been selected in which to investigate neurobiological changes
to the aged hippocampus, therapeutically pliable targets must be identified and characterized to
advance the development of the next generation of treatment strategies that prevent or reverse
age-related cognitive deficits. G-protein coupled receptors (GPCRs), also referred to as
metabotropic receptors, or more correctly, 7-transmembrane receptors, constitute the largest
family of neurotransmitter receptor proteins in the mammalian brain and, significantly, comprise
the most frequently targeted class of proteins by approved therapeutics (Drews, 2000;
Fredriksson et al., 2003). GPCRs natively respond to synaptically-released neurotransmitters and
recruit diverse signaling mechanisms that regulate a variety of intracellular processes. Exogenous
or synthetic agonists may stabilize unique conformations that select for specific signaling
pathways allowing for a high degree of control provided the structure-activity relationships are
well understood (Wood et al., 2011). Such compounds may bind to the same site as the
endogenous ligand (orthosteric) or bind to a site elsewhere on the receptor protein to modulate
the activity of the endogenous ligand (allosteric; Digby et al., 2010). Thus, the ability of GPCRs
to regulate neurotransmission and the potential to design novel, synthetic compounds to
therapeutically modulate select mechanisms of action make them attractive targets for the study of aging and cognition.
1.4.1. GPCR mechanism of action
Whereas ionotropic neurotransmitter receptors form ion channels that open in response to neurotransmitter binding, GPCRs organize into heteromeric signaling complexes with diverse mechanisms of actions and effects on neural activity. Broadly, all GPCRs are comprised of a 7- transmembrane receptor protein that contains an N-terminal extracellular domain of variable
16 length that participates in neurotransmitter/ligand-binding and a C-terminal domain that mediates interactions with a G-protein heterotrimer (Jingami et al., 2003; Strosberg, 1997). This heterotrimer is comprised of Gα, Gβ and Gγ subunits (Bünemann et al., 2003). Although
encoded on separate genes, the Gβ and Gγ subunits are structurally and functionally inseparable
once assembled at the receptor complex. While the identity of the receptor protein determines
what neurotransmitter will activate the receptor complex, the Gα-subunit determines the
coupling to specific intracellular signaling cascades that facilitate the transduction of
extracellular signals to intracellular responses (Fig. 1.3 and Table 1.1). Gαs-subunits catalyze the
conversion of adenosine-5'-triphosphate (ATP) to the second messenger cyclic adenosine
monophosphate (cAMP) via the enzyme adenylyl cyclase (AC; Brandt et al., 1983; Katada et al.,
1984). The production of cAMP activates a second effector, protein kinase A, which regulates
the activity of other proteins in a phosphorylation dependent fashion. The activation of the
cAMP pathway is opposed by subunits of the Gαi- and Gαo-subtypes which inhibit AC (Katada
et al., 1984; Sunahara et al., 1996). Another system, the phosphoinositol system, is activated by
Gαq- and Gα11-subunits that couple to phospholipase C (PLC), an enzyme that converts
phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol
triphosphate (IP3; Taylor et al., 1991). DAG activates protein kinase C (Mori et al., 1982; Takai
et al., 1982) and IP3 opens IP3 receptor-gated Ca2+ channels (IP3Rs) located on intracellular
(endoplasmic reticular) stores (Streb et al., 1983).
Despite these differences in their effector-coupling profiles, all Gα-subunits share a
common signaling mechanism. Under resting conditions, the Gα-subunit is bound to a guanosine
diphosphate (GDP) molecule but, in response to neurotransmitter binding by its coordinate
receptor, the Gα-subunit exchanges the GDP for a guanosine-5'-triphosphate (GTP) molecule
17 (Gilman, 1987). In its GTP-bound form, the Gα-subunit dissociates from the receptor and
modulates the activity of the appropriate effector enzyme (Arad et al., 1984; Klein et al., 2000).
Importantly, this activation is self-limiting as Gα-proteins also contain an intrinsic GTP
hydrolytic domain that cleaves the GTP back to GDP, returning the Gα-protein to its resting state
(Bourne et al., 1990). This state is also critical in modulating the affinity-state of the receptor
protein, as well; when coupled to the Gα-GDP complex, the receptor protein is stabilized in a
structural conformation that exhibits high affinity for its neurotransmitter or similar orthosteric
agonists, but following dissociation of the Gα-GTP complex the receptor shifts to a low-affinity
conformational arrangement (Hamblin and Creese, 1982). Unlike agonist binding, binding of
antagonists is insensitive to the receptor:Gα state of the complex (Hoffman et al., 1980).
1.4.2. Metabotropic glutamate receptors
Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous
system (Hollmann and Heinemann, 1994). Glutamate is produced and released by the principle
cortical and hippocampal neurons including Layer II neurons of the entorhinal cortex, granule
neurons of the DG and pyramidal neurons of the CA3 and CA1 regions. While the majority of
fast excitatory neurotransmission is achieved by ionotropic glutamate receptors of the 2-amino-
3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) subtype (Hollmann and
Heinemann, 1994), metabotropic glutamate receptors (mGluRs) are GPCRs that respond to
synaptic and extrasynaptic glutamate. There are 3 subclasses of mGluRs; Group I mGluRs
(mGluR1 and mGluR5) are predominantly found post-synaptically and couple to the Gαq/11 class of Gα-proteins where as Group II and Group III mGluRs are mainly found on pre-synaptic terminals and couple to the Gαi/o class of Gα-proteins (Bortolotto et al., 1999). Among Group I
mGluRs, mGluR5 is abundant in the synaptic layers of all 3 hippocampal subfields with greatest
18 expression in CA1 relative to CA3 and DG (Romano et al., 1995; Shigemoto et al., 1993) while
expression of mGluR1 is greatest in DG but almost absent in CA1 (Fotuhi et al., 1993;
Lavreysen et al., 2004). Despite differences in expression within the CA1, mGluR1 releases
intracellular Ca2+ in a PLC-dependent manner while mGluR5 potentiates Ca2+ entry via another
subtype of ionotropic glutamate receptor, the N-Methyl-D-aspartate (NMDA) receptor
(Mannaioni et al., 2001). Given these mechanisms of action, activation of Group I mGluRs generally increase excitability of post-synaptic neurons. Conversely, as Group II/III mGluRs are found presynaptically and coupled to Gαi/o, these receptors will act as autoreceptors that limit
further synaptic glutamate release and therefore serve a predominantly inhibitory function
(Shigemoto et al., 1997). Direct activation of Group I mGluRs with the agonist (S)-3,5-
dihydroxyphenylglycine (DHPG) will induce a form of long-term depression (LTD), a durable
decrease in the efficiency of neurotransmission (Nicoll et al., 1998; Watabe et al., 2002) whereas
activation of Group I mGluRs immediately prior to or during synaptic stimulation will result in
long-term potentiation (LTP), or an increase in the efficiency of synaptic transmission (Kwag
and Paulsen, 2012; Miura et al., 2002).
1.4.3. γ-Aminobutyric acid (GABA)B receptors
GABA is the predominant inhibitory neurotransmitter in the hippocampus and cerebral
cortex. GABA released within the hippocampus is produced by either local interneurons that
modulate the activity of principle neurons (McBain and Fisahn, 2001) or from a basal forebrain
projection pathway that selectively inhibits interneurons (Freund and Antal, 1988), thereby
producing disinhibition. These actions are achieved via binding of GABA to either ionotropic
GABAA receptors or metabotropic GABAB receptors. GABABRs are obligate heterodimers, that
is, the functional receptor complex is comprised of at least one GABABR1 subunit joined with a
19 GABABR2 subunit (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998). The
GABABR1 subunit contains the orthosteric binding site while the GABABR2 subunit interacts
with the G-protein which is always of the Gαi/o-subtype (Kaupmann et al., 1997; Robbins et al.,
2001). Furthermore, the GABABR1 protein is differentially expressed as the R1a or R1b isoform;
the R1a isoform contains an N-terminal extension which directs trafficking of R1a-containing
complexes to the distal axon while the R1b isoform lacks this extension leading to dendritic
targeting of R1b-containing complexes (Biermann et al., 2010; Kaupmann et al., 1997; Vigot et
al., 2006). In this way, GABABRs modulate both pre- and post-synaptic signaling, but its
ultimate actions at either location are generally inhibitory. GABABRs are found on GABAergic
and glutamatergic terminals, indicating that these receptors serve as both auto- and hetero-
receptors (Waldmeier et al., 2008). GABABRs decrease membrane excitability by opening
+ inwardly-rectifying K channels in a Gβγ-dependent fashion (Lüscher et al., 1997). GABABRs also to produce a form of heterosynaptic depression at mossy fiber synapses by limiting glutamate release, leading to decreased efficiency of synaptic transmission (Guetg et al., 2009).
1.4.4. Muscarinic acetylcholine receptors
Acetylcholine is an important modulatory neurotransmitter in the hippocampus; cholinergic neurons located in the medial septum/vertical diagonal band of Broca project to all layers of the hippocampus via the fornix (Amaral and Kurz, 1985) and release acetylcholine that binds to and activates ionotropic, nicotinic acetylcholine receptors (nAChRs; Levin, 2002) or G- protein coupled muscarinic acetylcholine receptors (mAChRs; Volpicelli and Levey, 2004).
There are five mAChR subtypes; M1, M3 and M5 couple to the Gαq/11 class of Gα-proteins, in
contrast to the M2 and M4 subtypes that couple to the Gαi/o class of Gα-proteins (Caulfield and
Birdsall, 1998). Within the hippocampus, M1 is predominant postsynaptic mAChR while M2 is
20 the predominant presynaptic mAChR (Volpicelli and Levey, 2004). Like Group I mGluRs,
activation of hippocampal M1 mAChRs with the agonist carbachol (CCh) will produce LTD in
CA1 (Scheiderer et al., 2006), but when application is accompanied by synaptic stimulation, an enhancement of LTP in CA1 is observed (Blitzer et al., 1990).
1.4.5. Summary of GPCRs in Hippocampus
GPCRs are well positioned to influence a multitude of key neural processes within the hippocampus. GPCRs are found presynaptically and postsynaptically on the major cell types of the hippocampus controlling processes such as neurotransmitter release and postsynaptic excitability (see Figure 3 and Table 1.1). Despite differences in their neurotransmitter-binding and G-protein coupling profiles, GTP-exchange is a biochemical mechanism common to all
GPCRs that can be used to make comparisons across systems. As this biochemical event is also the first step linking the binding of an extracellular neurotransmitter to intracellular signaling, it is a vital functional parameter of GPCRs with important implications to all subsequent transduction steps.
1.5. Intracellular Calcium and Synaptic Plasticity
Ca2+ is a vital intracellular signaling molecule within hippocampal neurons. Under resting
2+ 2+ conditions, the cytosolic, or intracellular, concentration of Ca ([Ca ]i) in CA1 neurons is
2+ 2+ generally 100 nM but stimulated Ca influx transiently increases [Ca ]i to 500-1000 nM; up to
2+ 2+ 300 nM [Ca ]i .stimulates further Ca influx, but greater values are generally elicit negative feedback (Bootman and Lipp, 1999). Ca2+ enters into the cytosol through Ca2+ channels located
on the plasma membrane which gate entry of extracellular Ca2+ or through channels found on
intracellular (endoplasmic reticular) stores. In the hippocampus, the primary mediators of Ca2+
21 entry from extracellular sources are the NMDAR (Dingledine et al., 1999) and the voltage-gated
Ca2+ channel (VGCC; Magee et al., 1998). The NMDAR will pass Ca2+ in response to binding of glutamate in the presence of membrane depolarization, which relaxes Mg2+ blockade of its Ca2+
pore (Dingledine et al., 1999). VGCCs, on the other hand, are not ligand gated, but open in
response to membrane depolarization (Magee et al., 1998). On the intracellular aspect, IP3Rs and
ryanodine receptors (RyRs), each gate a common pool of Ca2+ sequestered in the ER known as
the intracellular Ca2+ store (ICS; Bardo et al., 2006). IP3Rs open in response to binding of IP3
produced by the hydrolysis of PIP2 (Yang et al., 2002). RyR receptors, however, are opened by
Ca2+ itself (Bezprozvanny et al., 1991); as Ca2+ enters the cytsol via membrane-bound Ca2+ channels, RyRs are opened to release even more Ca2+ into the cytosol driving a process known as
Ca2+-induced Ca2+-release (CICR; Zucchi and Ronca-Testoni, 1997).
2+ Elevation of [Ca ]i supports changes to synaptic strength. Whereas high frequency
stimulation will increase synaptic efficiency, producing LTP (Collingridge et al., 1983),
sustained, low frequency stimulation will depress synaptic efficiency, or LTD (Collingridge et al., 2010). A variety of mechanisms are recruited to modulate synaptic strength and specific
forms of plasticity have implicated a role for NMDARs (Collingridge et al., 1988), VGCCs (Ito et al., 1995; Onuma et al., 1998) and IP3Rs (Fernández de Sevilla and Buño, 2010; Fernández de
Sevilla et al., 2008) as necessary for LTP where as NMDARs (Nicoll et al., 1998; Oliet et al.,
1997), M1 mAChRs (Scheiderer et al., 2006) and Group I mGluRs (Nicoll et al., 1998; Oliet et al., 1997; Watabe et al., 2002) are activated in the induction of LTD. Given that all of these targets facilitate Ca2+ flux into the cytosol, it is no surprise that elevated post-synaptic Ca2+ is also necessary to induce either LTP or LTD (Cavazzini et al., 2005), although a lower level is necessary to induce LTD compare to LTP (Hansel et al., 1996).
22 Ca2+ is important intracellular signaling molecule as it is dynamically modulated by
synaptic activation of neurotransmitter receptors and, in turn, induces changes at those very same
synapses. Given the therapeutic relevance of GPCRs (discussed in section 1.4), it is notable that
activation of M1 mAChRs and Group I mGluRs can either release Ca2+ from ICS or potentiate
2+ membrane Ca sources, thereby linking the actions of these receptors to changes in [Ca2+]i and from there to synaptic plasticity. It is also significant that the activity of GPCRs relative to concurrent synaptic function can change the direction of synaptic plasticity from LTD (Nicoll et al., 1998; Scheiderer et al., 2006; Watabe et al., 2002) to LTP (Blitzer et al., 1990; Kwag and
Paulsen, 2012; Miura et al., 2002). This latter observation suggests therapies that target these receptors could normalize the balance between synaptic enhancement and depression without grossly occluding endogenous neurotransmission.
1.6. Research Questions
Rat models of neurocognitive aging demonstrate changes to hippocampal innervation and activity that bear close resemblance to the human condition. However, the cellular and synaptic bases for the changes are not fully understood. Additionally, it is unclear to what extent differences between rat strains affect the emergence of age-related cognitive impairment and the co-emergence of neural changes. The following text reviews the current, relevant findings and pressing questions divided into four areas. First, behavioral parameters of cognitive aging are compared between strains and models. Second, cellular correlates of cognitive aging are discussed. Third, synaptic correlates of cognitive aging are presented with a specific focus on
GPCRs. Fourth, neurophysiological correlates of cognitive aging are reviewed connecting alterations to intracellular Ca2+ to impaired plasticity.
23 1.6.1. Cognitive aging among rat strains with differing lifespan and genetic background
Various research groups utilize different strains or methods to characterize age-related
changes to water maze performance. To illustrate, Rowe et al. (1998) selected aged LE rats that
performed either within 0.5 standard deviations of the mean latency of young or greater than 2.0
standard deviations from young to generate age-unimpaired and age-impaired cognitive groups,
respectively. However, this approach excludes the use of those aged rats whose performance
falls between 0.5 and 2 standard deviations of young, comprising approximately 40% of the aged population, and as such, eliminates an opportunity to examine rats of intermediate cognitive
status while artificially altering the naturally occurring variance of the aged population. In
contrast, Gallagher et al. (1993), also evaluating the spatial learning ability of aged LE rats,
describes the calculation of a learning index, a graded measure of spatial learning ability. Such a
measure takes note of individual variability and uses it as strength to model normal age-related
decline. When this measure is applied, aged rats span a wider range of scores that encompasses both unimpaired and impaired values relative to young, however as the index is a continuous measure it may be used as a covariate for correlation with neurobiological endpoints. Thus, the approach of Gallagher et al. (1993) allows for statistical evaluation of interrelated changes to behavior and biology as continuous outcomes, rather than comparing between artificially constructed, divergent cognitive categories.
Recently, methodology of Gallagher et al. (1993) has been applied to the F344 rat strain yielding a similar pattern of cognitive results (Bizon et al., 2009). However, aged F344 rats are prone to a number of pathologies that can complicate their use in aging studies (Maeda et al.,
1985; Markowska et al., 1990). Instead, the F344 × Brown Norway F1 (FBNF1) hybrid is a vigorous strain that exhibits robust health into older ages (Spangler et al., 1994) but the timing
24 and pattern of cognitive changes in this strain have not been evaluated to the same extent as those reported by Gallagher et al. (1993) and Bizon et al. (2009). Notably, while most studies have examined spatial learning in aged FBNF1 rats beginning at 27-28 months, Nieves-Martinez et al. (2012) reported that deficits may first emerge as early as 18 months (but see Bizon et al.,
2009). Therefore, it is unclear whether the onset of age-related impairment may occur sooner than has been generally assumed for this specific strain. Chapter II investigates the pattern of naturally occurring spatial learning impairment in aged FBNF1 rats, a strain specifically bred for health and longevity.
1.6.2. Neurocytological correlates of cognitive aging
Volumetric changes discussed in section 1.2.4.1 logically lead one to question whether cell loss is a mediator of morphological alterations to the brain. The outright loss of neurons is a clear factor in neurodegenerative disorders such as AD, but changes in normal aging are likely subtle. Even among neurons, it is possible that particular subtypes (i.e. those that produce and release specific neurotransmitters) may be more susceptible to aging than others. Also, it remains to be determined whether cells are actually lost or simply cease to express traditional markers that aid in their detection. The following sections examine evidence from behaviorally characterized rats that implicate changes to discrete classes of neurons as contributing to cognitive impairment in aged rats.
1.6.2.1. Hippocampal neurons are preserved while inhibitory markers are lost
As in humans, aged rats do not lose principle, excitatory neurons in any hippocampal subfield and this preservation is true regardless of cognitive status (Rapp and Gallagher, 1996;
Rasmussen et al., 1996). Similarly, there is no loss of layer II entorhinal neurons in aged- impaired rats (Rapp et al., 2002). These observations from aged rats and humans demonstrate
25 that principle neurons are highly resistant to the effects of aging. While aged humans show a
reduction in hilar volume, aged rats show no such shrinkage (Coleman et al., 1987; Shetty and
Turner, 1999). However, there is a decrease in the number of hilar neurons that express the 67
kDa isoform of glutamic acid decarboxylase (GAD-67+), the enzyme that synthesizes GABA for
synaptic release (Shetty and Turner, 1998; Stanley and Shetty, 2004), although the neurons
themselves are not lost, rather they cease to produce this inhibitory neurotransmitter. These hilar
neurons provide inhibitory modulation of DG granular neurons (Leranth et al., 1990). Although decreased GAD-67+ neuron number is apparent in other hippocampal subregions, emerging data
suggests that a decreased number of GAD-67+ hilar neurons is only observed in the hippocampus
of aged rats with spatial learning impairment (Stocker and Gallagher, 2011). Importantly, hilar
neurons co-express enzymes that produce inhibitory neuropeptides including neuropeptide Y
(NPY) and somatostatin (SST). Although NPY+ neuron number is similarly decreased in the
hilus of spatial learning unimpaired and impaired rats compared to young controls, only recently
has data demonstrated SST+ hilar neuron loss uniquely identified aged-impaired rats (Spiegel and
Gallagher, 2012). Thus, reduced capacity of interneurons to produce inhibitory molecules can
distinguish between impaired and unimpaired rats and possibly provide a cellular basis for
decreased inhibitory control over the DG/CA3 network (section 1.3.2.2)
1.6.2.2. Emerging evidence for changes to basal forebrain neurons
Degeneration of basal forebrain cholinergic neurons is well-established in AD patients
(Whitehouse et al., 1981) and some transgenic mouse models of AD (Belarbi et al., 2011),
although the relationship to normal aging and cognitive status has not been fully resolved. In
normal aging there is a discrepancy in findings where some papers, using profile-based methods,
claim there is decreased density of cholinergic neurons in the aged brain (Armstrong et al.,
26 September; Baskerville et al., 2006; Fischer et al., 1991a; Greferath et al., 2000) in contrast to more recent papers using precise stereological methods revealing either no loss of cholinergic neurons with age (Ypsilanti et al., 2008) or a modest loss that is not related to spatial learning impairment (Bañuelos et al., 2013). However, the former study did not include behavioral analysis to differentiate between unimpaired and impaired individuals, while the latter study did not differentiate between cholinergic neuron populations that innervate hippocampus from those that project to cortical regions. Thus, it remains unclear whether cholinergic neurons that specifically innervate the hippocampus are lost in the brain of aged rats with cognitive impairment. Chapter III investigates whether septohippocampal cholinergic neuron loss is a contributing factor cognitive decline in aged FBNF1 rats.
1.6.3. Synaptic correlates of cognitive aging
Aging, even in animals with learning impairment, is generally not associated with loss of principle neurons or widespread loss of synapses (section 1.3.2.2.). Thus, much attention has turned to examining whether an alteration to synapses at the molecular or anatomical level closely associates with spatial learning impairment in aged rats. One such alteration, decreased expression of synaptophysin at perforant path terminals (Smith et al., 2000), has already been discussed (section 1.3.2.2.). While the altered packaging and release of neurotransmitters in presynaptic vesicles is clearly one parameter that could deleteriously affect synaptic function, postsynaptic neurotransmitter-receptor interactions are another parameter of synaptic function necessary for normal information processing. To illustrate, there is decreased expression of ionotropic glutamate receptors in the DG, CA3 and CA1 of aged rats (Adams et al., 2008;
Newton et al., 2008; Shi et al., 2007; Wenk and Barnes, 2000), although the relationship to spatial learning is not universally evident (Nicolle et al., 1996; Smith et al., 2000).
27 GPCRs are also an attractive synaptic target as they are found pre- and post-synaptically.
It is unclear whether Group I mGluR expression associates with age-related cognitive impairment (Ménard and Quirion, 2012; Nicolle et al., 1999) and currently no data are published regarding Group II/III mGluRs in neurocognitive aging. There is no relationship between spatial learning impairment and nicotinic or muscarinic acetylcholine receptor levels in the aged brain
(Aubert et al., 1995; Chouinard et al., 1995; Smith et al., 1995; Zhang et al., 2007). However, combined pharmacologic and biochemical approaches revealed aging impairs receptor:G-protein coupling, a functional property of the GPCR signaling complex, without changes to receptor or
Gα-subunit protein expression (Zhang et al., 2007). This latter finding demonstrates that receptor binding and protein expression may not sufficiently characterize receptor integrity while functional measures are more relevant in determining relationships to cognitive status.
1.6.3.2. GABABRs in neurocogntive aging
GABA receptors have not been studied as intensively in the context of neurocognitive
aging as receptors for glutamate and ACh. Hippocampal GABAAR density does not change
between young adult (3 months) and aged (23 months) male FBNF1 rats (Turgeon and Albin,
1994). Similarly, levels of discrete GABAAR subunits are not changed in the hippocampus
between 6 and 18 months of age in Wistar rats nor is there any change in the relative compliment
of subunits; α2 and α5 are the predominant subunits followed by γ2, α1 and α3 (Yu et al., 2006),
although similar analyses have not been conducted on older rats (i.e. greater than 22 months).
Comparatively less known is regarding the status of GABABRs in normal aging; a lone study of
GABABRs demonstrated no change in receptor density between 3 month-old, 13 month-old or 23
month-old male, FBNF1 rats, suggesting stability of the system over the adult lifespan into older
ages (Turgeon and Albin, 1994). However, practically nothing is known regarding the
28 biochemistry of GABABRs in the aged brain, and this is a consequential issue given its unique
obligate heterodimeric nature and the need to interact with a G-protein heterotrimer to achieve
normal signaling. Significantly, GABABR antagonists were investigated for use in treating MCI following promising behavioral findings in animal models (Froestl et al., 2004; Helm et al.,
2005; Lasarge et al., 2009), but ultimately failed to reach significant clinical endpoints (Sabbagh,
2009) likely to due to a lack of receptor pharmacology and biochemistry data necessary to optimize the selection of specific compounds. What’s more, as systemically administered compounds will act at all GABABR binding sites, it becomes important to understand how
expression or pharmacological properties of GABABRs differ between brain regions in addition
to the effects of aging. Chapter IV tests the hypothesis that alterations to GABAB receptor
expression or G-protein coupling contribute to impaired spatial learning in aged F344 rats in
a brain-region dependent manner.
1.6.4.1. Pre- versus post-synaptic mAChRs
Unlike GABABRs, muscarinic receptors exhibit a heterogeneous G-protein coupling
profile; M1 receptors couple to PI hydrolysis via the Gαq/11 class of Gα-proteins while M2
receptors inhibit neurotransmitter release via Gαi/o. Importantly, the distribution of these
receptors is segregated, M1 receptors are postsynaptic while M2 receptors are presynaptic.
Therefore, as ACh is released into the synapse during cognitively demanding tasks, it will first
diffuse across the synaptic cleft to activated postsynaptic M1 receptors. After prolonged activity,
levels of synaptic ACh accumulate until eventually activating M2 presynaptic autoreceptors to
inhibit further ACh release. Cholinesterase inhibitors (ChIs), the mainline treatment for older
adults presenting with clinically evident memory impairment, block the enzymatic degradation
of ACh, to indirectly promote levels of synaptically available ACh (Gauthier, 2005; Pepeu and
29 Giovannini, 2009; Raschetti et al., 2007; Terry et al., 2011). When given to aged rats, ChIs restore spatial learning performance, providing indirect evidence of cholinergic insufficiency in aging (Riekkinen et al., 1996; Stemmelin et al., 1999; Yavich et al., 1996). However, this approach provides no means to bias neurotransmitter binding towards M1 or M2 activity and presently it is unclear whether age-related cognitive impairment is associated with deficient postsynaptic activation of M1 receptors or alterations to M2-mediated control of neurotransmitter release.
1.6.4.2. Postsynaptic mAChRs in the hippocampus of aged rats with cognitive impairments
Studies consistently observe no loss of M1 receptors in the hippocampus of rats, regardless of spatial learning ability (Aubert et al., 1995; Chouinard et al., 1995; Smith et al.,
1995; Zhang et al., 2007). However, a number of studies have measured changes in the ability of muscarinic receptors to stimulate PI turnover in aged rats. Chouinard et al (1995) reported that maximal IP formation stimulated by oxotremorine-M (a non-selective mAChR agonist) was blunted in the hippocampus of 28-29 month-old male LE rats compared to 7-8 month-old controls and the magnitude of this deficit was associated with worse spatial learning in the
MWM. These conclusion differs from that of Parent et al (1995), who reported that IP formation stimulated by carbachol (CCh, a nonselective cholinergic agonist) was greater in the hippocampus of aged (24-25 months) LE rats with spatial learning impairment compared to young (6 month old) controls. Similar disagreement is also noted between studies examining mAChR-stimulated production of DAG, Nicolle et al (2001) reported decreased oxotremorine-M stimulation of CDP-DAG in 25-26 month-old aged-impaired (25-26 months) while Parent et al
(1995) found no difference in CCh-stimulated CDP-DAG in 24-25 month-old aged-impaired rats, both studies examining male LE rats.
30 While differences in the properties of oxotremorine-M and CCh may, in part, explain
differences in receptor-mediated PI-hydrolysis, Chouinard et al (1995) found no difference in
basal PI-turnover but Parent et al (1995) saw an increase in basal activity. Basal PI-turnover is
not dependent on agonist-stimulation and other technical differences may impact this measure
(see also Ayyagari et al., 1998; Tandon et al., 1991). For example, discrepancies may relate to
the manner in which tissues were prepared for analysis. Parent et al (1995) utilized a cross-
chopped tissue preparation that retains dendrites, where GPCR complexes are located in close
proximity to other effectors such as IP3Rs (Fitzpatrick et al., 2009), while Chouinard et al.
(1995) prepared acutely dissociated cells, a process that shears dendrites, but minimizes
presynaptic influences (Cunha, 1998).
However, the blunted receptor-mediated PI-hydrolysis observed in Chouinard et al
(1995) may be due in part to reduced expression of PLCβ1 by this same study population
(Nicolle et al., 1999) whereas M1 receptor and Gαq/11 protein expression are stable with age
(Chouinard et al., 1995; Nicolle et al., 1999; Zhang et al., 2007). To determine whether signal
transduction mediated by mAChRs is impaired independent of PLCβ1 activity, Zhang et al
(2007) reported that oxotremorine-M stimulated GTP-Eu (a non-hydrolyzable GTP analogue)
binding was lower in the hippocampus of 26 month-old rats with spatial learning impairment
compared to 6 month-old controls. Although M1 receptors outnumber M2 receptors by a factor
of almost 5:1 [compare Tables 5 and 6 of Smith et al. (1995) and Tables 4 and 5 of Aubert et al.,
(1995)], GTP-binding assays more readily detect exchange by Gαi/o than by Gαq/11 or Gαs
(discussed in Milligan, 2003), so it these findings cannot be entirely ascribed to M1 receptor function. Thus, it remains unclear whether changes to mAChR-stimulated PI-hydrolysis are due to changes in receptor:Gαq/11-protein coupling.
31 1.6.4.3. Presynaptic mAChRs in the hippocampus of aged rats with cognitive impairments
Models of rodent neurocognitive aging broadly demonstrate that M2 receptor density and
function is conserved in the hippocampus of older rats. Smith et al (1995) found no difference in
[3H]AFDX-384 (an M2-preferring antagonist) binding between 6-7-months-old and 24-25-
months-old male LE rats and no relationship between density of radioligand binding in any hippocampal region examined and water maze performance. Aubert et al (1995) did observe
greater [3H]AFDX-384 binding unique to the molecular layer of the DG of 24-25-months-old
rats with spatial learning impairment. The functionality of M2 receptors, which control release of
ACh, was measured by Fischer et al. (1991) via in vivo microdialysis in the hippocampi of
young (3 months) and aged (24 months) female SD rats that were previously evaluated in the
MWM. Evoked ACh was statistically similar between young and aged rats, regardless of water
maze performance, whether this release was stimulated by KCl, to induce depolarization of the
presynaptic membrane, or by atropine, a non-selective muscarinic antagonist. Much later,
Birthelmer et al (2003) examined neurotransmitter release in hippocampal slices prepared from
young (3-5 months) and aged (25-27 months) female SD rats grouped according to spatial
learning ability. In this study, slices from aged rats, independent of cognitive status, did not
accumulate [3H]choline (which served as the precursor to form ACh) to the same degree as
young. Consequently, aged rats released lower absolute amounts of [3H]ACh following electrical
stimulation, but when expressed as a fraction of accumulated [3H]choline, similar proportional
values were observed in all slices. The magnitude of inhibition of [3H]ACh release by
oxotremorine-M was not different among groups, either. Thus, it seems likely that the
presynaptic activities of M2 receptors are conserved in the hippocampi of aged rats, regardless of
behavioral status. However, the preservation of presynaptic M2 mAChRs occurs in spite of
32 convincing evidence that cholinergic innervation of the hippocampus is diminished. Using
stereologic approaches, Ypsilanti et al (2008) found that the total length of cholinergic fibers was
significantly decreased in all 3 hippocampal subregions between 3 and 24 months of age in
female F344 rats. Interestingly, Lukoyanov et al. (1999) observed greater reductions in cholinergic fiber length within the DG of aged (23 months) male Wistar rats compared to age- matched females. Thus, the lack of statistically significant results reported in Fischer et al.
(1991) and Birthelmer et al (2003), which made use of aged female rats, may reflect sex-
differences in the severity of age-related changes to cholinergic input to the hippocampus,
although the work of Ypsilanti et al (2008) indicate that structural changes to the cholinergic
system are clearly evident in aged, female rats. Despite these unresolved discrepancies, a larger
question looms as to how the cholinergic system can undergo gross, circuit-level changes within
the aged hippocampus without causing commensurate changes to levels or functions of M2
receptors.
1.6.4.4. Comparing M1 and M2 mAChRs in the aged hippocampus
Given the lack of consensus regarding age and cognition-related changes to either M1 or
M2-dependent functions in the hippocampus, further experimentation is necessary to directly
compare whether one mAChR subtype is more affected than the other. Ideally, a therapeutic
agent that targets cholinergic transmission in the hippocampus would either directly potentiate
postsynaptic M1 function or indirectly facilitate ACh release by blocking presynaptic M2
receptors. However, it will be vital to determine whether agents acting at M1 fail to efficaciously
activate intracellular signals due to impaired M1 coupling with Gαq/11 in the aged brain.
Conversely, it is not known if M2 receptor function is potentiated in the aged hippocampus, or
whether reductions in fiber length lead to diminished M2-mediated functions. The first portion
33 of Chapter V directly tests the hypothesis that Gαq/11-coupled muscarinic receptors are
uniquely dysfunctional, compared to Gαo-coupled receptors, in aged FBNF1 rats with
learning impairment. Additionally, hippocampi were subdissected into DG, CA3 and CA1 to
test whether severity of age-effect was subregion-dependent.
1.6.5.1. Intracellular Ca2+ modulates a shift in plasticity in the aged hippocampus
Presently, it is hypothesized that learning observed in intact animals is associated with
changes in the efficiency of synaptic connections between hippocampal neurons (Bliss and
Collingridge, 1993). Cellular evidence of this phenomenon, long-term plasticity, has been investigated in aged rats, primarily at the Schaffer collaterals (SC) forming synapses in the CA1
region. When high frequency stimulation is delivered to the SC pathway, a comparable
magnitude of LTP is observed in slices prepared from either young or aged rats (Kumar et al.,
2007). However, the parameters used to induce maximal LTP differ greatly from the normal
patterns of synaptic activity of the hippocampus. When a burst of stimulation is delivered at 5
Hz, mimicking the theta frequency that characterizes hippocampal activity during normal
learning, slices produced from aged rats with spatial learning impairment show reduced LTP
compared to those slices made from young rats and aged rats without spatial learning impairment
(Tombaugh et al., 2002). Induction of LTP requires an increase in concentration of intracellular
2+ 2+ Ca ([Ca ]i)within the postsynaptic neuron; NMDARs, voltage-gated Ca2+ channels (VGCCs)
or IP3Rs all gate Ca2+ entry into the cytosol and various forms of LTP are known to recruit Ca2+
from each source (discussed in section 1.5). Boric et al. (2008) discovered that NMDA-LTP is similarly reduced in slices prepared from aged-unimpaired and aged-impaired rats, but VGCC-
LTP is only reduced in slices from aged-impaired rats. However, evidence for adaptive use of
VGCC-dependent plasticity remains controversial as Thibault et al. (2001) reported that
34 increased VGCC-channel conductance was inversely related to synaptic plasticity in aged rats
due to increased VGCC channel density (Thibault and Landfield, 1996). Importantly, Ca2+ influx via VGCCs releases Ca2+ from intracellular stores (ICS) by opening ryanodine receptors leading
2+ to dyshomeostatic regulation of [Ca ]i in aged CA1 neurons (Gant et al., 2006). Reversing this
process through ICS depletion or Ca2+ chelation improves synaptic transmission and reverses
spatial learning impairments in aged rats (Kumar and Foster, 2004; Tonkikh et al., 2006).
Just as the relationship among aging, spatial learning and specific mediators of LTP
remains unresolved, similarly there is no consensus as to how these processes relate to LTD.
Impaired spatial retention in aged rats is associated with a greater susceptibility to synaptically
evoked LTD (Foster and Kumar, 2007) and this enhanced LTD is associated with an increase
contribution from ICS (Kumar and Foster, 2005). However, Lee et al (2005) discovered a form
of LTD that requires activation of GPCRs linked to PLC and that is uniquely enhanced in
hippocampal slices prepared from aged-unimpaired rats. Further complicating matters, selective
pharmacological activation of M1 mAChRs (with CCh) or Group I mGluRs (DHPG) will also
trigger LTD and the magnitude of CCh-LTD and DHPG-LTD are greater in aged rats, although
the relationship between spatial learning and these specific GPCR-linked forms of plasticity is
not conclusively known (Kumar and Foster, 2007; Kumar, 2010).
Discrepancies in the conclusions from various studies of LTD may relate to differences in
the Ca2+/Mg2+ ratio of recording solutions which were differentially adjusted to alter the
2+ propensity to observe LTD. As [Ca ]i is central parameter in both LTP and LTD but results of
studies of plasticity differ widely based upon the method of induction, examination of changes to
2+ [Ca ]i will help to better the understanding of neural physiology in the aged CA1 area, separate
from LTP and LTD. The second portion of Chapter V will investigate whether cognitive aging
35 in FBNF1 rats is associated with altered modulation of intracellular calcium levels by
mAChRs and Group I mGluRs in the CA1 subregion, and whether these changes involve a
shift in the relative contribution of Ca2+ sources.
1.7. Summary
It is the purpose of this dissertation to present evidence validating place-learning
performance as a behavioral index of hippocampal-dependent cognition in aged, male rats and
then to discuss the application this behaviorally-defined model to investigate changes in neuron
2+ number, GPCR:Gα-protein coupling and GPCR modulation of [Ca ]i. The neurobiological
parameters in this study were selected to address shortcomings in knowledge regarding the
integrity of cellular and synaptic processes modulated by specific classes of GPCRs, with a
special emphasis on cholinergic signaling, because these receptors are ideal targets for
therapeutic intervention. Ultimately, these studies investigate an over-arching hypothesis that impairments in spatial learning observed in aged rats (1) are not caused by
neurodegeneration, rather they are associated with (2) selective deficits in hippocampal
GPCR:Gα-subunit coupling that (3) alter modulation of intracellular physiological processes.
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Figure 1.1. The interaction between chronological or biological aging and cognitive
function (“cognitive aging”). Cognitive function (shown on the left y-axis) generally declines
with age (shown on the x-axis), but age alone is not sufficient to predict cognitive performance
across all individuals of the same age because cognitive outcomes are more variable with
advancing age. Some older individuals exhibit little or no cognitive impairment relative to younger individuals. Other individuals will present with modest, but significant, deficits in some cognitive domain, usually memory. As cognitive deficits emerge in older individuals, there is
increased risk for further decline into dementia, most frequently Alzheimer’s disease, which is
associated with neuropathology including neuron loss and brain atrophy. However, such diseases
are distinct from normal aging, not an inevitable consequence. Even with improved clinical
evaluations (right y-axis), the boundary that distinguishes moderate impairment from progressive
neuropathology remains unclear (denoted by the color transitions of the background: green =
normal; orange = mild impairment/at risk for dementia; red = dementia). Modified from
McQuail and Nicolle (2012).
69
Figure 1.2. Schematic illustration of the hippocampus and adjacent structures. Dashed lines represent the approximate boundaries among the primary hippocampal subfields. The directionality of synaptic connections (axon→dendrite) is indicated by the arrows that encircle the diagram. GCL=granule cell layer; IML=inner molecular layer; MF=mossy fibers;
MML=middle molecular layer; OML=outer molecular layer; PCL=pyramidal cell layer;
PP=perforant path; SO=stratum oriens; SC=Schaffer collaterals; SL=stratum lucidum;
SLM=stratum lacunosum molecular; SR=stratum radiatum.
70
Figure 3. Key GPCRs in the Hippocampus. GPCRs differ in their neurotransmitter binding and effector coupling profiles. Major GPCRs in the hippocampus respond to glutamate (green), acetylcholine (blue) and GABA (red). One signal transduction pathway, the phosphoinositide
(PI) system (shown the the left), involves GPCR activation of a Gαq/11 subunit (orange) stimulates PLCβ1 (purple) to catalyze the conversion of PIP2 to DAG and IP3. Another pathway, the adenylyl cylcase pathway (shown to the right), is inhibited when activated Gαi/o prevents the formation of cAMP from ATP by the enzyme adenylyl cylcase. These two pathways produce largely opposing effects in hippcampal neurons; activation of the PI pathway increases neuronal excitation, kinase activity and ICS release while activation of Gαi/o decreases cell excitation
(post-synaptically) and release of neurotransmitters (pre-synaptically).
71 Table 1.1. Classification and Properties of GPCRs in the Hippocampus
GPCR Class Ligand Group Receptor Subtypes Gα-protein Synaptic Localization Function
C Glu Group I mGluR1 Gαq/11 Post PI-turnover, ICS release
mGluR5 Gαq/11 Post PI-turnover, NMDAR potentitation
Group II mGluR2 Gαi/o Pre Inhibition of AC, inhibition of Glu release
mGluR3 Gαi/o Pre
Group III mGluR4 Gαi/o Pre
mGluR6 Gαi/o Pre
mGluR7 Gαi/o Pre
mGluR8 Gαi/o Pre
GABA GABABR (heterodimer of R1 and R2) Gαi/o Pre and Post Inhibition of AC, inhibition of NT release
(Glu and GABA)
A ACh M1-like M1 Gαq/11 Post PI-turnover, ICS release
M3 Gαq/11 Post
M5 Gαq/11 Post
M2-like M2 Gαi/o Pre Inhibition of ACh release
M4 Gαi/o Pre
72
CHAPTER II
A MODEL OF COGNITIVE AGING IN THE F344 × BROWN NORWAY F1 HYBRID
RAT: RESULTS FROM A TRIAL-BASED ANALYSIS
Joseph A. McQuail and Michelle M. Nicolle
A portion of this behavioral dataset was published as Supplemental Materials in
Neuropharmcology 70:64–73, 2013. Stylistic variations are due to the requirements of the journal.
73
Abstract
Aging is generally associated with impairments in the ability to learn new information.
Consequently, aged rats are impaired in the acquisition of knowledge for spatial locations
(“place-learning”) suggesting they are a useful model for the study of cognitive aging. Using a
sensitive, distributed training, place-learning version of the Morris water maze, we report that 24 month-old male FBNF1 rats are impaired relative to 6 month-old controls. Examination of individual performance reveals that impairment is due to inferior spatial learning in a subset
(~55%) of aged rats (aged-impaired) while the remaining aged rats are behaviorally similar to young (aged-unimpaired). Despite this pronounced spatial learning impairment, aged-impaired rats retain performance over a 24 hour delay and are not slower at swimming within the training pool. Collectively, our data demonstrate that age-related cognitive impairment first emerges at 24 months of age in FBNF1 rats and this cognitive impairment is due to a slower rate to acquire spatial knowledge, not retention deficits or motor differences, that is evident in a subset of aged rats compared to young rats or aged rats with preserved spatial learning.
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2.1. Introduction
Normal aging is associated with a progressive decline in learning and memory, processes that depend on the integrity of the hippocampus (Albert, 1993; Petersen et al., 1992; Salthouse,
2003). As in humans, individual differences in age-related cognitive outcomes are apparent in humans and this naturally-occurring phenotype has been well established for Long-Evans (24-27 months) and F344 (22 months) rats (Bizon et al., 2009; Gallagher et al., 1993). The F344 ×
Brown Norway F1 (FBNF1) hybrid is an increasingly popular strain for use in aging studies for two reasons: (1) it ages with relatively low frequency of pathology (Spangler et al., 1994) and (2) this strain may be obtained from the National Institute on Aging’s Aging Rodent Colony
(Turturro et al., 1999). Thus, there is great interest within the field of neurocognitive aging to understand the timing and the pattern of cognitive changes in this strain. Many prior reports have claimed that age-related cognitive impairment emerges between middle-age (18 months) and old age (~28 months) in FBNF1 rats (Burgdorf et al., 2011; Fitting et al., 2008; Hasenöhrl et al.,
1999; Markowska and Savonenko, 2002; Shi et al., 2011; Thornton et al., 2000; VanGuilder et al., 2011a, 2011b; Wong et al., 2006; Zhang et al., 2012). However, few studies have specifically investigated the rate of spatial learning in these rats separate from procedural learning. Using the methods of Gallagher et al (1993) and Bizon et al (2009), which incorporate multiple probe trials interspersed throughout training to differentiate between procedural and spatial learning, Nieves-
Martinez et al (2012), determined that age-related cognitive decline is apparent as early as 18 months of age in this strain, although the distribution of individual differences was not discussed in this paper. Therefore, we present a series of trial-based analyses of young (6 months) and aged
(24 months) FBNF1 rats performing a distributed training, place-learning protocol in the Morris water maze. We selected 24 months as a possible critical point in the window of cognitive
75
change in this strain where examination of individual difference was likely to yield roughly equal numbers of aged rats that could be classified as either spatial-learning unimpaired or impaired relative to young controls.
2.2. Methods
2.2.1. Procedures
A description of the animals, apparatus and methods is given in full in Chapter V
(sections 5.2.1-5.2.2.). The following text specifically describes the results of trial-based comparisons that were not included in the main text of that published report (McQuail et al.,
2013).
2.2.2. Behavioral measures
Training trial performance was quantified using the “cumulative distance” measure, a proximity-based measure computed by sampling the rat’s distance from the platform location 10 times per second and cumulatively summing these values over the duration of the trial. For probe trials, the time spent in the training platform quadrant and the time spent in the opposite quadrant were recorded as was another proximity-based measure, “average distance”, which is computed in a similar manner to the aforementioned cumulative distance, but values are averaged, rather than summed, over the course of the fixed-duration probe trial. To summarize the spatial learning performance of individual rats, a “proximity score” was computed by summing the average distance measure recorded for each rat on probe trials 2, 3 and 4. Thus, larger values reflect a consistent search further from the platform location or worse performance. Additionally,
“discrimination indexes” were calculated for each probe trial using the formula: ((Time in
Platform Quadrant) – (Time in Opposite Quadrant))/((Time in Platform Quadrant) + (Time in
Opposite Quadrant)). This formula returns values between +1 and -1 with values greater than 0
76
indicating relatively greater time spent searching in the platform quadrant, values less than 0
indicate greater time spent searching the opposite quadrant and a value of 0 indicates no
preference for either quadrant. Although the training procedure used in the current study does not
incorporate retention probes (probe trials administered following a delay with no training trials
administered immediately prior to the probe), each training block includes a 24 hour delay
between the 3rd and 4th training trials. If deficits in retention, rather than acquisition, underlie
impaired, then one would expect impaired subjects would show poor savings across this 24 hour delay, that is, the first trial given post-delay will be significantly worse than the final trial given before the delay. To address this matter, “savings scores” were calculated by subtracting performance on trial 4 (post-delay) from trial 3 (pre-delay) in each block. If a performance is similar across this delay, the savings score will be 0, demonstrating retention of previously acquired performance. If performance is worse after the delay, this value will be a negative number; conversely, improved performance yields positive numbers. Swim speed was also recorded for all trials.
2.2.3. Statistical analysis
Performance on training and probe trials were tested using a repeated measures (RM) analysis of variance (ANOVA) with age as a between subjects factor and trial (separated by block when necessary) as a within subjects factor. Discrimination indexes were additionally tested using one-sample t-tests against a hypothetical value of 0. To demonstrate impaired performance can be specifically and reliably ascribed to a subset of aged animals, proximity scores were used to segregate the aged cohort by splitting between those that performed below
(n=21) or above the mean (n=26) of the aged group (i.e. a “split-mean”; Fig. 2.2B). Importantly, this procedure also separates the aged group into a subset that performs within the range of
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young (i.e. aged-unimpaired or AU) while the remainder fall outside of the range of young [i.e.
aged-impaired or AI; consistent with (Bizon et al., 2009; Gallagher et al., 1993)]. Analyses were
repeated using this cognitive grouping as a between subjects factor. Swim speed was compared
between cognitive groups using a one-way ANOVA.
2.3. Results
2.3.1. Training trial performance
Trial × age RMANOVAs were performed for individual training trials from blocks 1 and
2 (significant differences were not observed in blocks 3 and 4; see section 5.3.1 for results). In
the first block of training trials, there was a main effect of age (F(1,66)=33.71, p<0.001) and trial
(F(4,264)= 25.12, p<0.001) and a significant interaction between these factors (F(4,264)=3.71,
p<0.01). Post hoc tests revealed that while young and aged rats did not differ on the first training
trial, young rats performed better than aged on the second (p<0.001), third (p<0.001) and fourth
training trials (p<0.05), but no differences in performance were observed on the fifth training
trial (Fig. 2.1A). In the second block of training trials, there was a main effect of age
(F(1,66)=19.75, p<0.001) and trial (F(4,264)=2.87, p<0.05) but no significant interaction between these factors (F(4,264)= 1.90, p>0.10). Post hoc tests revealed that young and aged rats differed on
the first (p<0.01) and second (p<0.001) training trial of the block, but did not differ on trials 3-5
(Fig. 2.1B). Similar data from blocks 3 and 4 are also shown for comparison (Fig. 2.1C&D).
2.3.2. Probe trial performance
To further underscore that average distance measures obtained from probe trials and the
summary “proximity scores” sufficiently reflect an age-related deficit to acquire knowledge for
the training platform location, we cross-validated findings from primary, proximity-based
measures against a quadrant-derived measure. Similar to the results obtained for average distance
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measures (presented in section 5.3.1), an age × probe trial RMANOVA yielded a main effect of
age (F(1,66)=45.73 p<0.001) and a main effect of probe trial (F(3,198)= 5.52, p<0.01) with no significant interaction (F(3,198)=0.88, p>0.4; N.S.). Bonferroni post hoc tests determined that
young rats had higher discrimination scores on all probes compared to aged (p<0.05 for 1st and
4th probes, p<0.001 for 2nd and 3rd probes; Fig. 2.2A). Thus, young rats clearly discriminate, or
focus their search within, the training platform quadrant to a greater degree than aged rats, and
the ability to discriminate the training quadrant improves with continued testing. To further
examine this effect, one-sample t-tests (using 0 as a hypothetical test value) conducted separately
in each age group determined that young rats had a significant bias to search in the target
quadrant on all probes trials (p<0.001 for all probes) whereas aged rats only showed a preference
for the training quadrant on the 3rd and 4th probes (p<0.001 for both)
When discrimination index measures were tested with a cognitive group × probe trial
RMANOVA, there was a main effect of cognitive group (F(2,65)=52.60, p<0.001) and probe trial
(F(3,195)=7.48, p<0.001) but no interaction (F(6,195)=1.54, p>0.1; N.S.). Bonferroni post hoc tests
determined that young rats had higher discrimination scores on all probes compared to AI rats
(p<0.01 for 1st probe, p<0.001 for 2nd-4th probes; Fig. 2.2C). In contrast, young rats had higher
discrimination scores than AU rats on the 2nd probe trial (p<0.05) but not 1st, 3rd or 4th (p>0.05
for all) demonstrating this subset of aged rats can achieve performance comparable to young on
probe trials with sufficient training whereas the AI rats remained impaired throughout (p<0.01 on
probe 1 and p<0.001 for probes 2-4). In fact, one-sample t-tests (using 0 as a hypothetical test
value) conducted separately for AU and AI rats determined that AU rats had a significant bias to
search in the target quadrant on probes 2-4 whereas AI rats did not show a significant bias until
the final probe trial, although this bias is still significantly lower relative to performance of
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young (p<0.001) and AU (p<0.05) rats. Thus, conclusions derived from probe trial data are
identical when using either a proximity-based or quadrant-derived method to analyze
performance of young and aged rats in this training protocol.
2.3.3. Savings scores after a 24 hour delay
When savings scores were calculated for all animals for each block, savings scores were
generally not different from 0 in young, AU and AI, although in block 3, AI rats exhibited
savings scores that were significantly greater than 0, suggesting improvement after the delay
(t(25)=3.37, p<0.01; Fig. 2.3).
2.3.4. Swim speed of cognitive groups
A cognitive group × trial type (training trial or probe trial) RMANOVA revealed a main
effect of trial type (F(1,65)=229.31, p<0.001), but no effect of cognitive group (F(2,65)=1.03, p>0.3;
N.S.) or interaction (F(2,65)=0.57, p>0.5; N.S.). Thus, swim speed does not differ between rats of different ages or spatial learning abilities (Fig. 2.4).
2.4. Discussion
The training procedure employed in this study used a distributed training protocol that
incorporates repeated probe trials to identify reliable spatial learning deficits in a subset of aged
rats. Although the aged cohort achieves training trial performance comparable to young rats by
the end of the second training block, probe trials designed to specifically assess spatial
knowledge for the platform location indicate persistent deficits in the aged group. Thus, some
rats may utilize non-spatial strategies (e.g. circling) to rapidly locate the training platform when
available for escape, but this search strategy is not successful on probe trials where performance
is dependent on focused search at the training platform location. The proximity-based average
distance measure used in this current report is a highly sensitive indicator of the distribution of a
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rat’s search relative to the platform location (Maei et al., 2009) and the use of multiple probe trials interspersed throughout the training protocol permits one to observe the rate at which rats form a spatial bias for the training platform location, an advantage over the use of a single probe administered after the end of training. The proximity score used in this study is calculated from probe data obtained after both age groups have achieved asymptotic performance on training trials (and age groups did not differ on training trials administered immediately prior to these probe trials), thus excluding potentially confounding procedural learning differences that may occur during the first block of training. Proximity-based measurements provide identical results to traditional quadrant-derived measures and summary proximity scores can segregate the aged cohort into subgroups that show relatively spared learning ability as well as aged rats with robust, persistent learning deficits. Some researchers have noted that worse performance in the aged cohort may stem from greater within-subject variability from one trial to the next rather than reliable individual differences [Barnes et al (1997), but see Bizon et al (2009)]. However, the quadrant-derived measures reveal that impairment is evident throughout the training protocol specifically in the aged-impaired cohort, underscoring the reliability of individual differences in this study population and generally disputing the former hypothesis.
The selection of ages is critical when evaluating the significance of findings. The 50% survival age of FBFN1 rats is approximately 33 months in contrast to F344 rats where 24 months is the age of 50% survival (Turturro et al., 1999). Therefore, the use of aged F344 rats at 22 months [as in Bizon et al (2009)] approaches 90% of this time-point suggesting a similarly aged
FBFN1 rat would be ~29 months, approximately the age range used in many prior reports of
FBNF1 brain aging (Burgdorf et al., 2011; Fitting et al., 2008; Hasenöhrl et al., 1999;
Markowska and Savonenko, 2002; Shi et al., 2011; Thornton et al., 2000; VanGuilder et al.,
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2011a, 2011b; Wong et al., 2006; Zhang et al., 2012). While comparing survival curves is one
approach to compare between strains with different life-spans, we considered an alternative
approach that utilized existing evidence of cognitive change beginning in middle-age. Bizon et al
(2009) reported that spatial learning impairment is evident by 18 months in F344 rats, even
though most rats are not categorically impaired relative to young, but, at 22 months of age there
were roughly equal numbers of impaired and unimpaired aged. With this in mind, we used the
impairment reported by Nieves-Martinez et al. (2012) in 18 month-old FBNF1 rats as a critical
neuropsychological milestone in this strain’s aging to then reason that roughly equal numbers of
impaired and unimpaired aged individual FBNF1 rats may be found at 24 months of age, much
sooner than typically expected for this strain using less sensitive training protocols. This approach to scale cognitive aging relative to modest impairments that manifest at middle-age was successful, revealing that aged-impaired rats constitute ~55% of the cohort at 24 months of
age while the remaining aged rats are behaviorally similar to young (i.e. aged-unimpaired).
Spatial learning impairments in the aged-impaired group do not appear to be associated
with retention deficits. It has been previously argued that impairments observed in aged rats may
result from deficits in retention, rather than acquisition, of spatial information (Blalock et al.,
2003; Foster and Kumar, 2007; Foster et al., 2003; Mabry et al., 1996; Norris and Foster, 1999 and reviewed in Foster, 2012). Specifically, when rats are mass-trained, that is, when all training trials are given in a single day, aged rats will initially exhibit performance deficits but achieve levels comparable to young with additional training. Most, if not all, aged rats will demonstrate effective search for the training platform location when a probe is administered immediately subsequent to this massed training protocol. However, when another probe is administered 24 hours later, a considerable number of aged rats will exhibit below chance performance,
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suggesting that some aged rats fail to retain information as effectively as young rats, even after training to comparable levels of performance. There is a caveat to this task design in that all animals receive the same number of training trials. But, as trial-based results demonstrate, young rats achieve asymptotic performance sooner than aged rats. Therefore, superior retention may be a function of continued training beyond acquisition of optimal performance or over-training may protect against recall failure. Regardless, it is not possible to fully dissociate differences in the rate of acquisition from retention of spatial information as aged rats are impaired with respect to both processes. The protocol described here uses multiple days of training and probes to assess changes in retention and acquisition over the span of the learning curve. By adjusting for the level of performance achieved at any given point in the training protocol, we observe no evidence of age-related retention deficits, although acquisition of spatial search (determined by probe trial) is impaired, even after performance on platform trials is similar between age groups.
In general, this pattern of results indicates that retention deficits are not at work within this cognitive model, even among the subset of aged rats with pronounced learning deficits.
However, these findings are not conclusive as savings scores were calculated from training trial, not probe trial, data. Non-spatial strategies may yield successful performance on training trials, thus one cannot conclude that complete “spatial” retention is reflected in these savings scores, rather, whatever strategy each rat is using in a given block, that performance strategy is effectively recruited despite the delay.
While our data suggest that age-related deficits in spatial learning are not likely due to retention deficits, we also demonstrate that these cognitive differences cannot be explained by motoric or motivational differences. To examine whether aging or spatial impairment is associated with more global (i.e. non-cognitive) performance deficits during the hidden platform
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training procedure, swim speed was compared between cognitive groups on both training trials
and probe trials. Although rats tended to swim faster on probe trials relative to training trials,
there was no difference between cognitive groups. These data, when considered with comparable
performance on cued trials (see section 5.3.1), leads us to conclude that differences in
performance are genuinely due to differences in hippocampal-dependent cognitive strategies, not
non-specific impairments in task demands.
In summary, our findings reveal that cognitive aging does not equally affect all FBNF1
rats of a given age. Relative to 6 month-old controls, a 24 month-old cohort may be reliably
divided into aged-unimpaired and aged-impaired subgroups based upon probe trial performance.
The use of probe trials is critical as the aged group exhibits comparable training trial performance to young at phases of testing where aged rats remain impaired at using spatial- guided search strategies, revealing that improved training performance cannot simply be attributed to “spatial” learning. Despite the differences in spatial learning between young and aged rats, and even between unimpaired and impaired rats, there was no evidence for loss of previously acquired performance over the course of 24 hour delays presented throughout training, suggesting that learning, not retention, is impaired in aged rats. Finally, no differences were observed with respect to swim on any trial type, indicating that non-cognitive task requirements do not cofound the interpretation of spatial learning results.
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Figure 2.1. Training trial performance of young and aged rats. Young (6 months; n=21) and aged (24 months; n=47) rats were trained on a hidden-platform/place-learning version of the
Morris water maze arranged in 5 training trials per block; data from blocks 1 (A), 2 (B), 3 (C) and 4 (D) are illustrated. The dashed line between trials 4 and 5 of each block denotes a 24-hour delay between these trials; other trials within each block were separated by a 30 s intertrial- interval. *p<0.05, **p<0.01, ***p<0.001 vs young according to Bonferroni Post hoc test.
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Figure 2.2. Quadrant-derived measures characterizing probe trial performance in young and aged rats. Rats are initially grouped by chronological age to examine differences in ability to discriminate the training quadrant (A) but proximity scores (B) may be used to sort aged rats into aged-unimpaired (below dashed line; AU) and aged-impaired (above dashed line, AI) subgroups to reveal that deficits in probe trial performance are reliably associated with the AI subgroup (C). *p<0.05, **p<0.01, ***p<0.001 vs young according to Bonferroni Post hoc test.
#p<0.05, ###p<0.001 vs 0 according to one-sample t-test.
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Figure 2.3. Savings scores following 24 hour delay imposed in each training block in young, aged-unimpaired and aged-impaired rats. Y=young, AU=aged-unimpaired, AI=aged- impaired. ##p<0.01 vs 0 according to one-sample t-test.
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Figure 2.4. Swim speeds of young and aged rats during training trial and probe trial conditions. Y=young, AU=aged-unimpaired, AI=aged-impaired.
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CHAPTER III
NEUROINFLAMMATION NOT ASSOCIATED WITH CHOLINERGIC
DEGENERATION IN AGED-IMPAIRED BRAIN
Joseph A. McQuail, David R. Riddle, and Michelle M. Nicolle
The following manuscript was published in Neurobiology of Aging 32(12):2322.e1–2322.e 4,
2011. Stylistic variations are due to the requirements of the journal.
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Abstract
Degeneration of the cholinergic neurons in the basal forebrain and elevation of inflammatory markers are well-established hallmarks of Alzheimer’s disease; however, the interplay of these processes in normal aging is not extensively studied. Consequently, we conducted a neuroanatomical investigation to quantify cholinergic neurons and activated microglia in the medial septum/vertical diagonal band (MS/VDB) of young (6 months) and aged (28 months)
Fisher 344 × Brown Norway F1 rats. Aged rats in this study were impaired relative to the young
animals in spatial learning ability as assessed in the Morris water maze. Stereological analysis
revealed no difference between aged and young rats in the total numbers of cholinergic neurons,
demonstrating that loss of cholinergic neurons is not a necessary condition to observe impaired
spatial learning in aged rats. In this same region, the total number of activated microglia was
substantially greater in aged rats relative to young rats. Jointly, these data demonstrate that aging
is characterized by an increase in the basal inflammatory state within the MS/VDB, but this
inflammation is not associated with cholinergic neuron death.
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3.1. Introduction
The loss of cholinergic neurons in the basal forebrain is a reliable trait of Alzheimer’s disease (AD; Whitehouse et al., 1981). Additionally, the AD brain is characterized by activated microglia that release pro-inflammatory cytokines (reviewed in Schwab and McGeer, 2008), suggesting that neural inflammation and related glial responses may contribute to degeneration of cholinergic neurons (Willard et al., 1999). Aging remains the predominant risk factor for AD but the effect of chronological aging on the cholinergic and microglial populations of the basal forebrain is not fully understood. To address this matter, we utilized rigorous stereological techniques to estimate the total numbers of cholinergic neurons and activated microglia in the medial septum/vertical diagonal band (MS/VDB), the subdivision of the basal forebrain that innervates the hippocampus (Amaral and Kurz, 1985). Fisher 344 × Brown Norway F1
(F344×BN) rats were used in this study as this is a particularly vigorous hybrid strain that ages robustly albeit exhibiting a pattern of memory impairment similar to other strains including
Long-Evans and F344 rats (LaSarge and Nicolle, 2009). We hypothesized that aged rats with spatial learning impairment would exhibit a loss of cholinergic neurons and concurrent increases in activated microglia in the MS/VDB.
3.2. Methods and Materials
3.2.1. Subjects
Young (6 months, n = 9) and aged (28 months, n = 6) F344×BN hybrid male rats were obtained from the colony maintained by Harlan Sprague Dawley, Inc. (Indianapolis, IN) from the
National Institutes of Aging and housed at Wake Forest University School of Medicine in a facility accredited by the American Association for Accreditation of Laboratory Animal Care.
Animals were maintained on 12 hour light/dark cycle with ad libitum access to food and water.
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The Institutional Animal Care and Use Committee approved all protocols described in this report.
3.2.2. Spatial reference memory in the Morris water maze
Rats were trained according to the procedure of Gallagher (1993) and data were collected and analyzed using EthoVision software (Noldus, Leesburg, VA). The primary measures used to assess spatial-learning performance were path length and average distance from platform (Maei et al., 2009). Average distance values obtained from the second, third and fourth probe trials administered during the protocol were summed to generate individual “proximity scores.” This score reflects the development of an efficient search pattern near the escape platform location; lower scores indicate better performance and higher scores reflect search farther from the platform. Visible-platform training administered at the end of the protocol provided an assessment of sensorimotor and motivational factors that might influence performance in the spatial learning task.
3.2.3. Tissue processing and immunohistochemical staining
Rats were deeply anesthetized with ketamine/xylazine and perfused transcardially with phosphate buffered saline (pH 7.4) followed by 4% paraformaldehyde in phosphate buffer.
Brains were extracted and processed as described in Schindler et al (2008). Tissue sections containing the MS/VDB were sequentially immunolabelled with anti-choline acetyltransferase
(ChAT; a marker of cholinergic neurons; Chemicon; 1:150) and anti-CD68 (ED1 clone; a marker of activated microglia; AbD Serotec; 1:500). Labeling was detected with the appropriate biotinylated anti-IgG (Vector; 1:300) and peroxidase conjugated avidin-biotin complex (ABC;
Vector) using Vector SG substrate, forming a grey/black reaction product (ChAT), or diaminobenzidine, forming a brown reaction product (CD68). Sections were then counterstained
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with the nuclear binding dye 40,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma
Aldrich). Sections were mounted on charged slides, dehydrated, defatted and cover-slipped with
Cytoseal mounting medium (Richard Allan Scientific).
3.2.4. Quantitative analyses
A modified optical dissector technique was employed to estimate total cell numbers
(Kempermann et al., 1998). ChAT+ cells were counted in every 8th section and CD68+ cells in every 16th section of the basal forebrain in a systematically random series beginning rostrally at
the genu of the corpus callosum and ending caudally at the decussation of the anterior
commissure. Using an Olympus microscope equipped with a motorized stage controlled by a PC
running the Neurolucida software program (MicroBrightField), the MS/VDB was defined at low magnification according to (Paxinos and Watson, 1998); see Supplementary Figure S1A).
Immunopositive cells were observed through a 40× plan-apo objective lens, 0.75 N. A., and counted exhaustively within each section. In accordance with previously applied counting procedures, only ChAT+ cells with visible nuclei were counted (Baskerville et al., 2006) and
CD68+ cells in the top focal plane were excluded to avoid overestimation (Schindler et al., 2008;
see Supplementary Figure S1B and C). The sum of profiles counted was multiplied by the inverse of the section sampling fraction to achieve an estimate of final cell number.
3.2.5. Statistical analyses
Training trial performance was analyzed by repeated measures ANOVA with trial block as a within-subjects factor and age as a between-subjects factor. The proximity score and the
estimates of total cell numbers were compared between young and aged rats by means of
independent-samples t-tests. Cell density measures were analyzed by repeated measures
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ANOVA with section as a within-subjects factor and age as a between-subjects factor.
Significant effects were subsequently tested using a paired-samples t-test.
3.3. Results
3.3.1. Morris water maze performance
Aged rats demonstrated impairment relative to young rats during the course of the training protocol (F(1,13) = 15.45, p = 0.002; Figure 1A). Similarly, aged rats were characterized
by significantly higher proximity scores, reflecting a poor spatial bias for the platform location
(t(13) = 6.82, p < 0.001; Figure 1B). Despite the observed performance deficits on the hidden
platform version of the task, no differences were observed between young and aged rats in
finding a visible platform, indicating that performance deficits were not due to sensorimotor or
motivational differences (t(13) = 1.17, p = 0.27 NS; Figure 1C).
3.3.2. Quantitative measures of cholinergic neurons and activated microglia in MS/VDB
The total number of cholinergic neurons (ChAT+ cells) did not differ between young and
aged rats (t(13) = -0.12, p = 0.991 N.S.; Figure 2A). Similarly, there were no age-related
differences in density of neurons observed when considering the rostro-caudal extent of sections
counted (F(1,6) = 0.591, p = 0.736 N.S.; Figure S2). In contrast, there was a robust increase in the
+ total number of activated microglia (CD68 cells) in aged relative to young (t(13) = 18.29, p <
0.001; Figure 2B), indicating an increased basal inflammatory state in the aged MS/VDB. In
aged rats, there was a greater density of activated microglia in the rostral MS/VDB compared to
the medial and caudal divisions, suggesting that the neuroinflammatory response is not
homogeneous throughout this nucleus (F(2,10) = 7.262, p = 0.011; Figure 2C).
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3.4. Discussion
3.4.1. No loss of cholinergic neurons in aged-impaired MS/VDB
Our findings demonstrate that spatial learning impairment in aged F344×BN rats is not accompanied by loss of cholinergic neurons that project to the hippocampus. These results are in agreement with a recent stereological study in aged F344 rats and a non-stereological study in aged mice showing no changes in total numbers of MS/VDB cholinergic neurons (Ypsilanti et al., 2008;Hornberger et al., 1985). Our estimate of the total number of cholinergic neurons in the
MS/VDB in F344×BN rats is similar to those reported in F344 rats by Ypsilanti and colleagues
(2008) and also provides evidence indicating the stability of this neuronal population despite spatial learning impairment. These findings are in contrast to previous non-stereological studies in aged rats that describe a loss of septohippocampal cholinergic neurons in rats with cognitive impairment. This pattern of loss occurs across a number of rat strains including Sprague-Dawley
(Fischer et al., 1992), Fisher 344 (Armstrong et al., 1993), Dark Agouti (Greferath et al., 2000) and Long-Evans rats (Baskerville et al., 2006); but see also (Stemmelin et al., 2000). It is possible that caveats such as exact age, precise anatomical localization of the counting area or counting method contributes to the apparent discrepancies in the literature. Importantly, stereological evaluations, such as ours, yield precise estimates of total number that may be readily compared across studies, unlike profile-based counts that may be presented as a variety of statistics with or without correction factors.
3.4.2. Numbers of activated microglia are increased in aged MS/VDB
Importantly, a substantial increase in the number of activated microglia was observed with aging, indicating a considerable increase in the local inflammatory state. Previously, it was assumed that cholinergic neurons were uniquely susceptible to inflammatory insults. The
99
inflammatory agent lipopolysaccharide stimulates microglial activation and proliferation and subsequently induces degeneration of cholinergic neurons when infused intracerebrally into the basal forebrain (Willard et al., 1999) or applied to mixed neuronal/glial cultures (McMillian et al., 1995). In contrast, our data show that cholinergic neurons persist in spite of an age-dependent increase in microglial activity. Notably, a greater density of activated microglia was observed in the rostral MS/VDB of aged rats relative to medial and caudal divisions. The rostral MS/VDB mainly contains the ventral division of the cholinergic basal forebrain that preferentially innervates the septal, or dorsal, hippocampus (Amaral and Kurz, 1985). As the dorsal hippocampus is critical for spatial learning performance (Moser and Moser, 1998), these findings may be suggestive of biochemical alterations that explain observed memory impairments. While a link between neuroinflammation and cognitive impairment is far from definitive, the release of inflammatory cytokines by activated microglia alter a number of neurophysiological parameters including neurotransmitter release, maintenance of long-term potentiation and neurite outgrowth
(reviewed in Godbout and Johnson, 2009).
3.4.3. Conclusions
In summary, we propose that outright loss of cholinergic neurons comprising the septohippocampal system is not a requisite feature of cognitive aging. In the absence of overt neuron loss, the increased number of activated microglia indicates that the aged basal forebrain is characterized by increased inflammation, although the relationship between neuroinflammation and memory loss in older rats remains unclear. Although previous experiments have demonstrated that administration of proinflammatory agents activate microglia and subsequently trigger death of cholinergic neurons in young rats and in cell culture, our findings indicate that a
100
naturally occurring, proinflammatory environment in the MS/VDB of aged rats does not result in a loss of cholinergic neurons.
3.5. Acknowledgements
The authors thank Adam Wilson, Jennifer Sousa and Liz Forbes for technical assistance and advice. This work was supported by NIH Training Grant NS07422 (JAM), NIA Grant
AG11370 (DRR & MMN), NIA Grant AG020572 and Nestle Nutrition (MMN).
101
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Godbout JP, Johnson RW (2009) Age and neuroinflammation: a lifetime of psychoneuroimmune
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Greferath U, Bennie A, Kourakis A, Barrett GL (2000) Impaired spatial learning in aged rats is
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of Water Maze Probe Test Performance? Front Integr Neurosci 3:4.
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Selective killing of cholinergic neurons by microglial activation in basal forebrain mixed
neuronal/glial cultures. Biochem Biophys Res Commun 215:572-577.
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Stemmelin J, Lazarus C, Cassel S, Kelche C, Cassel JC (2000) Immunohistochemical and
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of acute and chronic neuroinflammation within the basal forebrain cholinergic system of
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104
Figure 3.1. Morris water maze performance in young and aged rats. Aged rats were impaired relative to young in a place-learning task in the Morris water maze, as evidenced by greater path length measures throughout the training protocol (A) and greater composite proximity scores derived from probe trials (B). The solid line denotes group mean. Aged rats were not impaired on a visible-platform version of the task (C).
105
Figure 3.2. Quantitative measures of cholinergic neurons and activated microglia in young and aged rats within the MS/VDB. No difference between stereological estimates of total number of ChAT+ cells (cholinergic neurons) in young and aged rats (A). Total number of
CD68+ cells (activated microglia) was substantially increased in aged relative to young (B).
When considering only aged rats, a greater density of activated microglia was observed in the
rostral MS/VDB of aged rats (C).
106
3.S. Supplementary Materials
Figure 3.S1. Bright-field photomicrographs depicting ChAT- and CD68-immunostaining in the MS/VDB of young and aged rats. No qualitative differences were observed in the patterns of staining between young (left) and aged (right) rats (A). MS: medial septum; VDB: vertical diagonal band; HDB: horizontal diagonal band; NAS: nucleus accumbens (shell); LV: lateral ventricle. Increased magnification demonstrates typical cholinergic neuron morphologies in young (B) and aged (C) rats (black-stained cell bodies and processes). Activated microglia
107
(DAB-brown puncta) were largely absent from the MS/VDB of young rats (B), but were abundant in the aged basal forebrain (C; see arrows).
108
Figure 3.S2. Density of ChAT+ cells thorough rostro-caudal sampling distribution. Similar
density of ChAT+ cells was observed between young and aged rats in sections along the rostro-
caudal axis of the basal forebrain. Given that no differences are seen in estimates of total number
or density within a particular rostro-caudal plane, aging is not associated with a reorganization of
MS/VDB cholinergic neurons.
109 CHAPTER IV
GABAB RECEPTOR GTP-BINDING IS DECREASED IN THE PREFRONTAL
CORTEX BUT NOT THE HIPPOCAMPUS OF AGED RATS
Joseph A. McQuail, Cristina Bañuelos, Candi L. LaSarge, Michelle M. Nicolle and Jennifer L.
Bizon
The following manuscript was published in Neurobiology of Aging 33(6):1124.e1–1124.e12,
2012. Stylistic variations are due to the requirements of the journal
110 Abstract
GABAB receptors (GABABRs) have been linked to a wide range of physiological and cognitive processes and are of interest for treating a number of neurodegenerative and psychiatric disorders. As many of these diseases are associated with advanced age, it is important to understand how the normal aging process impacts GABABR expression and signaling. Thus,
we investigated GABABR expression and function in the prefrontal cortex (PFC) and
hippocampus of young and aged rats characterized in a spatial learning task. Baclofen-stimulated
GTP-binding and GABABR1 and GABABR2 proteins were reduced in the PFC of aged rats but
these reductions were not associated with spatial learning abilities. In contrast, hippocampal
GTP-binding was comparable between young and aged rats but reduced hippocampal GABABR1 expression was observed in aged rats with spatial learning impairment. These data demonstrate marked regional differences in GABABR complexes in the adult and aged brain and could have
implications for both understanding the role of GABAergic processes in normal brain function
and the development of putative interventions that target this system.
111 4.1. Introduction
GABAB receptors (GABABRs) are G-protein coupled receptors (GPCRs) and modulation
of these receptors shows potential for treating a number of neurological and psychiatric disorders. In post-synaptic neurons, GABABRs bind γ-aminobutyric acid (GABA) and are
coupled to the Gi/o class of Gα-proteins that inhibit adenylyl cyclase to decrease intracellular
levels of cyclic AMP (cAMP; Odagaki et al., 2000; Odagaki and Koyama, 2001). Additionally,
the Gβγ-subunit activates the inward rectifying postassium current that modulates the late, or
slow, phase of the inhibitory post-synaptic potential (Luscher et al., 1997). GABABRs are also located on axon terminals where their activation decreases Ca2+ influx (Takahashi et al., 1998)
and inhibits neurotransmitter release (Waldmeier et al., 2008). GABABRs are unique among
GPCRs as they are obligate heterodimers comprised of at least one GABABR1 subunit with one
GABABR2 subunit (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998), although more complex arrangements are speculated (Pin et al., 2009). The R1 subunit contains the
orthosteric binding site and is expressed as one of two isoforms, GABABR1a or GABABR1b
(Kaupmann et al., 1997). While no ligands can distinguish between the two (Kaupmann et al.,
1997, 1998), molecular and biochemical evidence has identified distinct cellular distributions and functions for each isoform. GABABR1a contains a pair of short consensus repeats at the N-
terminal that act as an axonal targeting factor that trafficks GABABR complexes containing this
isoform to presynaptic terminals where they modulate neurotransmitter release (Biermann et al.,
2010). Conversely, GABABR1b lacks this N-terminal extension and is preferentially trafficked to
dendrites where it controls postsynaptic inhibition (Vigot et al., 2006). However, functionality of
the receptor is not observed until this R1 subunit associates with an R2 subunit; the R2 subunit
mediates interactions with the G-protein (Robbins et al., 2001) as well as facilitates expression of
112 the receptor complex at the plasma membrane (Margeta-Mitrovic et al., 2000).
Despite the significant functional implications, surprisingly little is known regarding the
normal composition of GABABR complexes across distinct brain regions and the extent to which
such complexes and their activity change with age. Such information is vital given the diversity
of signaling offered by unique receptor configurations and emerging evidence that GABAergic indices change with age. For example, in aged rats, hippocampal interneurons degenerate or
cease to express glutamic acid decarboxylase (GAD-67), the GABA-synthesizing enzyme
(Shetty and Turner, 1998; Stanley and Shetty, 2004) and aged canines demonstrate interneuron
loss in superficial and deep cortical layers of the PFC (Pugliese et al., 2004). Moreover, evoked
GABA release is decreased in the CA1 subregion of the aged rat hippocampus (Stanley et al.,
2011) .
Among the functional consequences that could stem from age-related alterations in
GABAergic signaling is a loss of cognitive abilities. Indeed, GABAergic signaling has been
implicated in cognitive processes supported by both medial temporal lobe and frontal cortical
systems, and these brain regions are particularly vulnerable to changes associated with advancing
age. Age-related frontal cortical dysfunction, reflected in a loss of behavioral flexibility, has been
detected in aged rodents using tasks such as attentional set-shifting (Barense et al., 2002;
Schoenbaum et al., 2002; Rodefer and Nguyen, 2008). Loss of declarative/spatial memory
supported by hippocampus is also a prominent feature of advanced age and such deficits can be
modeled in aged rats using spatial learning tasks such as the Morris water maze. A unique
feature of aged rat models characterized on spatial learning tasks is that reliable individual
differences in performance can be detected, such that aged rats can be subgrouped into those that
perform within the range of young rats and those that perform outside this range, demonstrating
113 hippocampal-dependent learning impairment. Such behavioral models have been used to
implicate a number of neurobiological alterations in age-related cognitive deficits, including
marked alterations of signaling downstream of muscarinic acetylcholine (mAChR) or Group I
metabotropic glutamate receptors (mGluRs; Chouinard et al., 1995; Nicolle et al., 1999; Zhang et
al., 2007). In contrast, the relationship between cognitive abilities in aged animals and GABABR
signaling has not been thoroughly investigated, although GABABR antagonists are known to
restore memory function in various animal models of aging (Froestl et al., 2004; Lasarge et al.,
2009) while GABABR agonists impair spatial learning in young rats (McNamara and Skelton,
1996). Consequently, we hypothesized that aging may modulate GABABR expression or
function in close association with cognition in a brain region-dependent manner. To test this
hypothesis, we performed parallel pharmacological and biochemical analyses of hippocampal
and PFC GABABRs in tissues obtained from young and aged rats previously tested for spatial
learning ability.
4.2. Materials and methods
4.2.1. Animals
Young adult (6 mo, n=8) and aged (22 mo, n=16) male Fischer 344 (F344) rats were
obtained from the National Institute on Aging colony (Harlan, IN, USA) and individually housed
in the AALAC-accredited Psychology Department vivarium at Texas A&M University for 2
weeks prior to the onset of behavioral testing. The vivarium was maintained at a constant 25°C with a regular 12:12h light/dark cycle (lights on at 08:00), and rats had free access to food and
water at all times. All rats were screened daily for health problems including but not limited to
cataracts, jaundice, food and water intake, and the appearance of tumors. Sentinel rats housed in
the same room were routinely screened and found to be negative for a range of pathogens. All
114 animal procedures were conducted in accordance with approved institutional animal care procedures and NIH guidelines.
4.2.3. Behavioral testing
Animals were tested for spatial learning ability according to the methods developed by
Gallagher and colleagues (Gallagher et al., 1993) with specific modifications for training F344 rats (Bizon et al., 2009). Rats were trained in a Morris water maze apparatus consisting of a circular tank (1.8 m diameter) filled with water (27°C) made opaque by the addition of nontoxic tempera paint. A retractable escape platform (12 cm diameter) was submerged 2 cm below the surface in the southwest quadrant of the maze. Each rat received 3 trials a day for 8 consecutive days to learn to swim to the submerged platform using white, geometric spatial cues affixed to a black curtain surrounding the maze. Rats were placed into the water facing the wall of the maze at one of four equally spaced start positions (north, south, east or west) in a pseudo randomized order such that all rats started from each of the locations the same number of times. Rats were allowed to swim for up to 90 s in order to locate the platform before they were guided to it by the experimenter. Rats remained on the platform for 30 s, and subsequently transferred to a holding cage for a 30 s inter trial interval (ITI). Every sixth trial (i.e. the third trial on days 2, 4, 6 and 8) was a probe trial during which the platform was lowered to the bottom of the tank and made unavailable for escape for the first 30 s of the trial, after which the platform was raised and subjects were allowed to escape. Data were acquired via a video camera mounted above the maze that was connected to a DVD recorder and computer with a video tracking system (HVS
Image, Buckingham, UK).
After the last day of spatial training, rats received one session with six trials of cue training to assess sensorimotor function and motivation. Here, rats were trained to escape to a
115 visible black platform extending 2 cm above the water surface, the position of which varied from trial to trial. On each trial, rats were given 90 s to reach the platform followed by a 30 s ITI.
4.2.4. Membrane preparation
Approximately 2 weeks following the completion of behavioral testing, animals were
decapitated, brains were removed from the skull and PFC and hippocampus were dissected on an
ice-cold plate and stored at -80°C until use. As cognitive task performance will elicit changes in
protein expression, this interval was selected to evaluate baseline, rather than behaviorally-
stimulated, protein levels and functions. Frozen tissue was weighed, thawed and homogenized in
10 volumes of an ice-cold buffer (50 mM HEPES, pH 7.4, 1 mM EDTA and 1 mM EGTA and
protease inhibitors; Roche, Mannheim, Germany) using a glass-teflon dounce homogenizer.
Homogenates were centrifuged at 14,000 RPM for 20 minutes at 4°C. The supernatant was
discarded and the pellet resuspended in 20 ml of the same buffer without protease inhibitors and
incubated on ice for 30 minutes followed by centrifugation at 16,500 RPM for 15 minutes at
4°C. This pellet was resuspended in 10 volumes of 50 mM HEPES, pH 7.4, and aliquots were
stored at -80°C until used for GTP-binding or Western blotting assays. Protein concentration was
determined using the Pierce BCA Kit according to the manufacturer’s protocol (Rockford, IL).
4.2.5. GTP-binding assay
All reactions were run in triplicate at room temperature. GTP-binding reactions were run
in a buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM MgCl2 and 50 µM GDP with the
appropriate concentration of baclofen (100 nM – 1 mM; Tocris, Ellisville, MO) or an equal
volume of water (basal activity) or 10 µM unlabelled GTPγS (non-specific binding) in each well of the 96-well filter plate. 10 µg of membrane was added per well and equilibrated for 20 minutes. GTP-binding was initiated by the addition of 10 nM GTP-Eu, a hydrolysis-resistant,
116 fluorescent GTP analogue (PerkinElmer, Waltham, MA) to a final total volume of 100 µl per
reaction. After 40 minutes, the reaction was terminated by filtration on a vacuum manifold and
washed four times with 200 µl of ice-cold 1× GTP wash buffer and read on a Victor3 fluorimeter
(PerkinElmer Life, Shelton, CT).
4.2.6. Western blotting
Unless otherwise noted, all reagents used for gel electrophoresis were purchased from
Bio-Rad (Hercules, CA) and all steps were performed at room temperature. Membrane proteins
were denatured and reduced in Laemmli sample buffer with 5% (v/v) β-mercaptoethanol (Fisher,
Pittsburgh, PA) and heated at 95°C for 5 minutes. 10 µg of protein per lane were
electrophoretically separated on a 4-15% Tris-HCl gel at 200 V for 55 minutes then transferred
to nitrocellulose membranes using a semi-dry transfer apparatus (iBlot; Invitrogen, Carlsbad,
CA) for 7 minutes at 20 V. Blots were washed 3 times with tris-buffered saline (TBS; pH 7.4)
then blocked for 1 hour in blocking buffer (Rockland, Gilbertsville, PA). Blots were then
incubated overnight at 4°C with anti-GABABR1 or anti-GABABR2 diluted 1:1000 (Cell
Signaling Technology, Beverly, MA) diluted in blocking buffer with 0.1% Tween-20. Blots were then washed three times with TBS and incubated with the appropriate AlexaFluor 680- conjugated anti-IgG (Invitrogen) diluted 1:20,000 in TBS with 0.1% Tween-20 (Bio-Rad) for 1 hour. Following three additional TBS washes, blots were scanned on an Odyssey imaging system
(LI-COR Biosciences, Lincoln, NE). A total of four experiments were conducted for each
subunit.
4.2.7. Data analysis
All data are presented as the mean ± standard error of the mean. The student’s t-test, one-
way or repeated measures ANOVAs were performed where appropriate. When necessary,
117 Fisher’s least-significant difference post-hoc tests were conducted to determine significant
differences between groups. In all statistical comparisons, values of p<0.05 were considered significant.
Behavioral data was analyzed using SPSS (Cary, NC). The Spatial Learning Index (SLI) was calculated by weighting and summing mean search error from the interpolated probe trials to provide an overall measure of spatial learning performance for each rat (Gallagher et al., 1993;
Bizon et al., 2009). The SLI was used to perform correlational analyses with neurobiological parameters (i.e. GTP-binding or protein levels); partial correlations correcting for the effects of age were used to test significant relationships between SLI and each neurobiological measure across all ages, while bivariate correlations were performed to test relationships separately for each age group.
GTP-binding parameters were analyzed using GraphPad Prism 5 (La Jolla, CA). Non- specific binding values were subtracted from each reaction and the specific stimulation by baclofen was normalized to young (e.g. basal=0% and EMAX of young=100%). These data were
log-transformed and curves were fitted using a sigmoidal, non-linear regression with variable
slope.
Digitized images of immunoblots were converted to gray scale and analyzed using
ImageJ software. Specific bands were identified by creating a threshold mask and excluding pixels that fell below threshold values. Integrated density, the product of mean optical density and area, was measured for each band and these values were normalized to young controls. To analyze R1 isoform protein, expression was normalized to the R1b isoform, as this is the most abundant isoform in the adult brain (Fritschy et al., 1999).
118 4.3. Results
4.3.1. Spatial learning performance
Young rats and aged rats perfomed similarly on the first training trial (F(1,22)=2.66, ns)
and while both groups improved over the course of training (F(3,66)=23.64, p<0.05), a two-way
repeated measures ANOVA (age × training trial block) revealed a significant main effect of age
(F(1,22)=8.96, p<0.05; Fig. 1A) indicating that aged rats were not as proficient in locating the
hidden platform compare to young rats. In agreement with training trial performance, a two-way
repeated measures ANOVA (age × probe trial) showed that search accuracy for the platform improved over the course of training (F(6,63)=30.93, p<0.0001) and that there was a main effect of
age on probe trial performance (F(1,22)=29.91, p<0.0001). In contrast to the effects of age on
spatial reference learning abilities, there was no impairment in the ability of aged rats to locate a
visible escape platform during cue (visible platform) training. A one-way ANOVA revealed no
significant difference between pathlength of young or aged rats (F(1,22)=1.95, ns) on the visible
cued task.
In order to relate biochemical measures to cognitive abilities, probe trial data were used
to calculate an overall “index” of spatial learning for each individual subject (Fig. 1B; described
in Gallagher et al., 1993 and Bizon et al., 2009). While the range of scores characterizing aged
performance spans a continuum, for some analyses, aged rats were sub-grouped based on their
spatial learning indices (SLI). The top 50% of aged rats (SLI<245) were classified “aged
cognitively-unimpaired” (AU) and the bottom 50% (SLI>245) were classified as “aged
cognitively-impaired” (AI; see also Baxter and Gallagher, 1996). Importantly this subdivision is
not arbitrary; rather it closely matches age-appropriate behavioral criteria for this strain of rat
previously reported in greater detail (Bizon et al., 2009). Furthermore, within this specific
119 experimental cohort, these AI rats are at least 2 standard deviations from the mean SLI of young
rats (Fig 1B compare left and right Y-axes) and fall outside the range of young range. To support such classification, a two-way repeated measures ANOVA (cognitive group × probe trial) was performed on sub-grouped data. While all cognitive groups improved over the course of training
(F(3,63)=30.93, p<0.0001), there was a main effect of cognitive group (F(2,21)=12.26, p<0.005; Fig.
1C). Post hoc analyses revealed that both young and AU groups were significantly different from
the AI group (young vs AI: F(1,14)=44.36, p<0.0001; AU vs. AI: F(1,14)=64.33, p<0.0001) but not
from each other (young vs. AU: F(1,14)=3.6, ns; Fig. 1C).
4.3.2. Baclofen-stimulated GTP-binding in hippocampus
To assess functionality of GABABRs in the hippocampus, GTP-binding was measured
following stimulation with baclofen, a selective GABABR agonist. Non-specific and basal GTP-
binding values were comparable between young and aged rats. Agonist stimulated parameters,
maximal GTP-binding (EMAX), agonist affinity (EC50) and Hill slope coefficients (NH), did not
differ between young and aged rats or between cognitive groups (Fs<0.75, p>0.05, Table 1 and
Fig. 2). Correlation analyses between maximal GTP-binding (determined by curve-fit) and SLI
confirmed that binding was stable across a range of cognitive abilities in young (r=-0.08, ns) and aged rats (r=0.04, ns). Furthermore, there were no differences between ages or cognitive groups at any dose of baclofen (Fs<1.0, p>0.05).
4.3.3. Baclofen-stimulated GTP-binding in prefrontal cortex
As in the hippocampus, non-specific and basal values obtained from PFC were not significantly different between young and aged rats. In contrast, while the fitted parameters did not differ (Table 2), two-way ANOVAs (age or cognitive group × concentration) comparing the mean GTP-binding values (i.e. not fitted data) obtained for each concentration of baclofen
120 revealed a significant interaction between age and baclofen concentration (F(7,154)=3.04, p<0.05)
and between cognitive group and baclofen concentration (F(14,147)= 1.76, p<0.05). Post hoc
analyses revealed that the interaction between age and concentration was attributable to a marked
28% decrease in baclofen-stimulated GTP binding in aged relative to young rats (p<0.05) at the
highest concentration of baclofen tested (1 mM; Fig. 3A). A post hoc one-way ANOVA
(cognitive group) demonstrated a similar trend towards decreased GTP binding in AU and AI
relative to young at the 1 mM baclofen dose but this change did not quite reach statistical
significance (F(2,21)= 2.89, p=0.08; Figure 3B). In agreement with the observation that binding
was reduced by a similar magnitude in both AU and AI rats (31% and 24%, respectively; Fig.
3B), no significant correlations were observed between GTP-binding stimulated by 1 mM
baclofen in PFC and SLI in young (r=-0.18, ns) or aged rats (r=0.20, ns).
4.3.4. Regional differences in cooperativity of binding
The discrepancy in the magnitude of the fitted and observed maximal values in the PFC may be a consequence of low slope factors (Hill coefficient; NH) apparent in this region. As
lower slope factors (i.e. NH <1) indicate decreased cooperativity or efficiency of receptor:G-
protein coupling, we compared the Hill slope coefficients in both the PFC and hippocampus.
Indeed, while the Hill slope coefficients (NH) did not differ with age in PFC, the slope
coefficients for both young and aged rats were significantly less than 1.0 (young: t(7)=2.45, p<0.05; aged: t(15)=4.63, p<0.001; Table 2). This is in contrast to hippocampus in which the slope
factors did not deviate from unity (NH=1.0) in either age group (young: t(7)=0.0003, ns; aged:
t(13)= 1.46, ns).
121 4.3.5. GABABR subunit expression in hippocampus
A two-way ANOVA (isoform × age) revealed no main effect of age (F(1,22)=0.54, ns; Fig.
4A) but a significant effect of isoform (F(1,22)=29.75, p<0.001) such that GABABR1b was modestly but significantly more abundant in hippocampus than was GABABR1a (~1.14-fold greater; p<0.001). These differences in isoform expression did not differentially interact with age
(F(1,22)=0.29, ns); however, a two-way isoform × cognitive group ANOVA revealed a main effect
of cognitive group (F(2,21)=5.72, p<0.05; Fig. 4B). Post-hoc analyses demonstrated significantly
lower protein levels in AI rats relative to young (p<0.05) and AU rats (p<0.01). One-way
ANOVAs comparing effects of cognitive group separately for each isoform demonstrated that both GABABR1a (F(2,21)=5.31, p<0.05) and GABABR1b (F(2,21)=5.48, p<0.05) were significantly
reduced in AI rats relative to young (-25% GABABR1a and -23% GABABR1b; p<0.05 for both) while levels of both isoforms were comparable between young and AU rats. Among aged rats, there was a trend towards lower GABABR1a protein levels being associated with greater SLI
scores (GABABR1a: r=-0.46, p=0.07; GABABR1b: r=-0.41, p>0.1).
In contrast to the GABABR1 subunits, levels of hippocampal GABABR2 did not reliably
differ as a function of age (t(22)=-1.31, ns; Fig. 4C) or cognitive status (F(2,21)=1.42, ns; Fig. 4D)
and no significant relationship between levels of this protein and SLI were observed (aged: r=-
0.20, ns; young: r=-0.10, ns).
4.3.6 GABABR subunit expression in prefrontal cortex
Comparisons between ages revealed main effects of GABABR1 isoform (F(1,22)=819.64, p<0.001) and age (F(1,22)=11.44, p<0.01; Fig. 5A). First, in contrast to hippocampus, GABABR1a was present in greater levels than GABABR1b (1.38-fold; p<0.001) in the PFC. In relation to age, both GABABR1a and GABABR1b protein levels were significantly reduced by a similar
122 magnitude in aged rats relative to young (-30% and -32%, respectively; p<0.01 for both). A two-
way isoform × cognitive group ANOVA also revealed main effects of isoform (F(2,21)=956.93,
p<0.001) and cognitive group (F(2,21)=5.48, p<0.05). Protein levels were significantly decreased
in both AU (p<0.05) and AI rats (p<0.01) relative to young, but were not significantly different
between these two aged groups. When isoforms were analyzed separately, each isoform was
significantly reduced in both AU (p<0.01 for alpha and p<0.05 for beta; Fig. 5B) and AI rats
(p<0.01 for both) relative to young, but neither isoform was significantly different when
comparing between the two aged groups. Futhermore, proteins levels of either isoform were not
associated with SLI (aged: r≤0.14, ns).
In PFC, GABABR2 protein levels were reduced in aged relative to young rats (- 32%);
t(22)= 3.65, p<0.01; Fig. 5C). There was also a main effect of cognitive group (F(2,21)=6.49,
p<0.01; Fig. 5D) but as with GABABR1, post-hoc comparisons revealed a similar magnitude
reduction of GABABR2 levels in AU and AI rats relative to young (-34% and -30%,
respectively, p<0.01 for both). GABABR2 protein levels did not differ between AU and AI rats
(p>0.05) nor was there a significant relationship between reduced GABABR2 protein levels and
SLI scores (aged: r=0.15, ns).
4.3.7. Regional differences in GABABR1 isoform expression
Main effects of isoform were observed, independent of age or cognitive status, demonstrating that the expression of each isoform was not equivalent within either region (Figs 4
and 5). The antibody used to detect GABABR1 was raised against an antigenic epitope common to both GABABR1a and GABABR1b isoforms thus enabling quantification of both isoforms within a single immunoblot due to their differing molecular weights. While direct comparisons
between isoform levels between brain regions are not possible in our current study, we computed
123 the ratios of alpha-to-beta for each animal for both regions to formally compare the relative
abundance of each isoform between the hippocampus and PFC. The ratio of R1a:R1b was
significantly different between the PFC and hippocampus (t(46)=16.60, p<0.001; Fig. 6A), and
these ratios were not significantly changed when dividing the experimental cohort between
young and aged rats (F(1,22)=1.187, ns; Fig. 6B) or among cognitive groups (F(2,21)=0.797, ns;
Fig. 6C). Futhermore, the PFC ratio was significantly greater than 1 (t(23)=15.547, p<0.001; Fig.
6A) whereas the hippocampus ratio was significantly less than 1 (t(23)=-6.638, p<0.001; Fig. 6A).
The results of these comparisons formally support the within-region conclusions: R1a is the
dominant GABABR1 isoform in PFC while R1b is dominant in hippocampus.
4.4. Discussion
An emerging concept in both human and rodent cognitive aging is that age-related declines in
hippocampal and frontal cortical systems occur somewhat independently and that consideration
of both systems is essential for a thorough understanding of cognitive dysfunction and the
development of effective interventions to promote successful cognitive aging. Findings from the
current study in which we report differential effects of age on GABABR expression and signaling
in hippocampus and PFC support this concept as signaling via this receptor is markedly
attenuated in PFC but largely unaltered in hippocampus.
4.4.1. Age-associated changes to GABAB receptors in the prefrontal cortex
Baclofen-stimulated GTP-binding as well as both GABABR subunits were significantly
decreased in the aged PFC. While decreased GABABR function and protein expression in the
aged PFC were not specifically associated with the severity of cognitive impairment, this result
is not entirely unexpected as the watermaze task used for behavioral characterization is
hippocampal-dependent and may not have been sufficiently sensitive to age-related alterations in
124 PFC function. While some investigations have revealed significant alterations to PFC GPCRs in
spatial learning-impaired aged rats (Parent et al., 1995; Zhang et al., 2007), impaired
performance on frontal cortical dependent tasks (e.g. working memory, attentional set-shifting) is
largely dissociable from hippocampal-dependent spatial reference memory deficits in aged rats
and mice (Schoenbaum et al., 2002; Barense et al., 2002; Lambert et al., 2005; Bizon et al.,
2009). Within this context, it is interesting to consider that the same signaling cascades
downstream of receptor:G-protein complexes can exert unique (and opposing) actions on
cognition supported by PFC and hippocampus. Protein kinase A (PKA) is a downstream effector
of the GABABR and reduced inhibitory drive by these receptors would be expected to result in a
failure to inhibit PKA activity. In agreement with this prediction, there is evidence that the
cAMP-PKA pathway becomes disinhibited in PFC at advanced ages, and moreover, that such
disinhibition contributes to loss of PFC-dependent cognition (Ramos et al., 2003). While drugs directed at increasing activation of the cAMP-PKA pathway generally enhance hippocampal- dependent plasticity, disinhibiting this signaling pathway and increasing PKA activity in PFC
can instead impair working memory in young adult rats (Taylor et al., 1999). These data,
together with the current findings, suggest that decreased modulation of this signaling pathway
within PFC in aging, but not hippocampus, could contribute to specific cognitive deficits.
Consequently, it will be important in the future to further explore the relationship between
behaviors that are mediated by frontal cortical nuclei and the status of this receptor system and
its effectors in frontal regions in young and aged rats, and specifically to determine the extent to
which alterations to the GABABR system described here contribute to age-related changes in
PFC-dependent cognition (Schoenbaum et al., 2002; Barense et al., 2002; Rodefer and Nguyen,
2008; Simon et al., 2010).
125 Although baclofen-stimulated efficacy was decreased in the aged PFC, affinity for the
receptor (EC50 values) did not change with age. In contrast to the binding of antagonists, agonists
preferentially bind to receptors in their G-protein coupled, or high affinity, state. Levels of Gαi or
Gαo, the G-proteins that couple to GABABRs, were not directly measured in the current study,
however, the lack of a change in EC50 suggests that sufficient quantities of Gαi and Gαo are present in aged PFC to facilitate normal agonist binding. In agreement, an autoradiographic study demonstrated decreased affinity of GABA for GABABRs in between 2 and 3 months of
age in F344 rat cortex, but no change between 3 and 23 months of age suggesting that early
development, but not advancing age, modulates GABABR:G-protein coupling (Turgeon and
Albin, 1994). Similarly, levels of Gαq/11, another G-protein subtype, are not changed by aging in
the PFC (Zhang et al., 2007) suggesting that G-protein expression is largely resistant to changes
by the normal aging process. Instead, a marked reduction in receptor proteins (-30%) was
associated with the attenuation in maximal receptor response, providing evidence that blunted
GTP-binding in aged PFC is largely mediated by loss of functional GABABR receptor proteins.
While the changes in GABABR signaling in PFC reported here adds to a growing body of
work indicating that GPCRs are altered in the normal aging process, unique mechanisms appear
to underlie changes in G-protein signaling across neurotransmitter systems. While mAChR GTP-
binding is also attenuated in the PFC of aged Long-Evans rats (Zhang et al., 2007), in contrast to the current findings, mAChRs in the aged PFC become functionally decoupled from their cognate G-proteins without outright loss of receptors. Other evidence indicates that mAChR- and mGluR-mediated production of inositol phosphates is actually elevated in spatially-impaired aged F344 rats relative to aged-matched spatially-unimpaired cohorts (Parent et al., 1995). While the reasons for discrepancies in mAChR status across studies are not entirely clear, the data to
126 date do support varied effects of age on GPCRs. Such differences can be likely attributed in part
to distinct G-proteins and downstream effectors that are activated by individual receptors. For
example, whereas a subset of mAChRs and mGluRs signal via activation of the effector
phospholipase C, GABABRs inhibit adenylyl cyclase. A better understanding of the specific
mechanisms that mediate age-related alterations in GPCR signaling in PFC across
receptor/effector systems is an important topic for future study, particularly with respect to the
development of pharmacotherapies targeting GPCRs.
4.4.2. GABABR1 subunit is selectively lost in the hippocampus of rats with cognitive impairment
In contrast to the PFC, no difference in baclofen-stimulated GTP-binding was observed
between the young and aged hippocampus. However, in this same tissue, expression of the
GABABR1 protein was selectively reduced in spatially-impaired aged rats while spatially-
unimpaired aged-matched controls expressed this protein at levels that were indistinguishable from young. The basis for the loss of this receptor protein may be attributable, in part, to the well-described phenotypic changes that are associated with GABAergic interneurons within the aged hippocampus. While the number of GAD-67-expressing cells declines with age, this change appears to be largely mediated by specific subclasses of interneurons such as somatostatin- and calbindin-immunopositive cells whereas those interneurons that express other molecular markers like calretinin and parvalbumin are relatively unaffected (Shetty and Turner, 1998; Stanley and
Shetty, 2004; Stanley et al., 2011). Notably, cytosolic expression of GABABR1 is observed
within these same susceptible somatostatin- and calbindin-immunopositive cells but generally is
not associated with resistant parvalbumin- or calretinin-expressing interneurons (Sloviter et al.,
1999). Thus, reduced GABABR1 protein may be secondary to functional loss of interneurons or,
127 alternatively, decreased GABABR1 expression may increase the vulnerability of these neurons to
the effects of aging.
Despite the significant, yet selective changes to GABABR1 in the aged hippocampus,
preserved receptor activity in this region may be of consequence to hippocampal-dependent
behaviors. Post-synaptic excitatory receptors or associated functions are depressed in the aged hippocampus, including NMDA receptor expression (Wenk and Barnes, 2000; Clayton and
Browning, 2001; Adams et al., 2001), NMDAR associated plasticity (Lee et al., 2005; Boric et al., 2008), and GPCR mediated activity dependent on M1 muscarinic receptors (Chouinard et al.,
1995; Nicolle et al., 2001; Zhang et al., 2007) and Group I metabotropic glutamate receptors
(Nicolle et al., 1999). Thus, the relative sparing of inhibitory GABABR signaling in hippocampus
suggests an imbalance between excitation and inhibition of hippocampal synaptic activity that
might in turn contribute to cognitive deficits. In fact, apart from the ability of GABABRs to regulate post-synaptic excitability directly or indirectly via inhibition of glutamate release,
GABABRs suppress NMDA receptor calcium signals in a G-protein/PKA dependent manner
(Chalifoux and Carter, 2010) and produce heterosynaptic depression at mossy fiber-CA3
synapses (Guetg et al., 2009) which are spared relative to perforant path inputs in the aged
hippocampus (Smith et al., 2000). If the contributions of the GABABR become more pronounced
with age in the hippocampus, moderate antagonism of the GABABR might be expected to restore
a more favorable ratio of excitatory-to-inhibitory neurotransmission and to facilitate normal
learning and memory. Indeed, GABAB antagonists have shown potential to enhance working and
reference memory in animal models and preliminary clinical studies (Froestl et al., 2004;
Lasarge et al., 2009), but no candidates have approved for use in patients (Sabbagh, 2009),
underscoring the need for additional basic science work to more clearly elucidate the role for this
128 system in the context of the aged brain and postulate a mechanism of action for GABAB-based pharmacotherapies.
4.4.3. Pharmacological characteristics and relative composition of GABABRs are region-specific
While the primary aim of our study was to assess age-related changes in the hippocampus and PFC, there are also notable differences in the pharmacology and relative abundance of
GABABR1 isoforms between the PFC and hippocampus. While binding parameters did not
deviate from unity in the hippocampus, slope factors in the PFC were significantly lower.
Negative cooperativity observed with baclofen-stimulated GTP-binding in PFC suggests these
GABABRs, but not those in hippocampus, form unique complexes possibly with other GPCRs,
effectors such as K+ channels, or allosteric modulators of G-protein activity (i.e. RGS4; David et
al., 2006; Fowler et al., 2007; Ciruela et al., 2010). The negative modulation of binding observed
in the PFC is consistent with emerging evidence that suggests that GABABR heterodimers can
form complexes with other GABAB heterodimers to negatively modulate ligand-binding:G-
protein coupling (Maurel et al., 2008).
Additionally, we compared the relative levels of GABABR1 isoforms and found that
GABABR1a is the dominant isoform in the PFC, while GABABR1b is the dominant isoform in the hippocampus. Our findings in the hippocampus closely match the observed ratios reported in a previous experiment using a similar approach to quantify relative levels of each isoform
(Fritschy et al., 1999). However, ours is the first study to specifically quantify relative levels of each isoform in the PFC, rather than the cerebral cortex as a whole. While homogenates prepared from whole brain or whole cortex demonstrate greater levels of GABABR1b relative to
GABABR1a, the PFC, a heterogeneous grouping of brain nuclei including the infralimbic/prelimbic, orbitofrontal, and frontal association areas, shows the reverse trend.
129 Collectively, these region-specific observations suggest that systemically administered compounds may activate GABABRs less efficiently in the PFC than the hippocampus and preferentially modulate presynaptic release in the PFC and post-synaptic excitation in the hippocampus. While it is not possible to infer how interactions between brain regions will ultimately influence complex behaviors in intact animals based upon our current data, these region-specific characteristics of GABABRs will help to better inform lead optimization when screening GABAB compounds and interpretation of behavioral endpoints in preclinical studies.
4.5. Conclusion
The present experiments investigated functions and protein levels of PFC and hippocampal GABABRs and revealed significant regionally-specific differences in expression of
GABABR complexes in adult and aged brains. Specifically, significant age-related reductions in
GABABR mediated GTP-binding were accompanied by decreased levels of GABABR proteins in the prefrontal cortex. Conversely, there was no change in GABABR GTP-binding in the hippocampus, but there was a selective loss of the GABABR1 subunit in aged rats with spatial learning impairment. Overall, these results indicate that normal aging differentially modulates the expression of GABABRs between the PFC and hippocampus and these changes have significant effects on signaling efficacy within the PFC.
4.6. Acknowledgements
This work was supported by the National Institute on Aging grants R01-AG020572 to
MMN and R01-AG029421 to JLB and the McKnight Brain Research Foundation. The authors also wish to thank Allyn C. Howlett for expert advice offered in the interpretation of the GTP- binding results.
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Figure 4.1. Spatial learning in young and aged rats.
While young and aged rats improved as a function of training, aged rats were impaired relative to
young at learning to swim to a hidden, submerged platform within the water maze [A]. Spatial learning index (SLI) scores were greater, on average (noted by horizontal line), for aged rats than
young indicating that aged rats were less proficient in searching for the platform location,
however, the distribution of individual index scores demonstrates that aged rat performance
spans a range which includes aged rats that are unimpaired relative to young (AU) and those
exhibiting impaired performance (AI) [B]. When subgrouped according to SLI score, AI rats
were significantly impaired on probe trial performance compared to young (Y) and age-matched
controls without spatial learning impairment (AU) [C].
138
Figure 4.2. Baclofen-stimulated GTP-binding in the hippocampus of young and aged rats.
Dose-response curves were not different between age groups [A] or cognitive groups [B]. The line of best-fit determined by non-linear, four-parameter fit is illustrated for young and aged rats.
139
Figure 4.3. Baclofen-stimulated GTP-binding in the prefrontal cortex of young and aged
rats. Aged rats exhibited a 28% decrease in GTP-binding elicited by 1 mM baclofen in the
prefrontal cortex [A] and this decrease was similar in aged-unimpaired (AU) and aged-impaired
(AI) rats [B]. The line of best-fit determined by non-linear, four-parameter fit is illustrated for young and aged rats. * p<0.05 young versus aged.
140
Figure 4.4. GABABR1 and GABABR2 protein levels in hippocampus of young and aged rats
Levels of hippocampal GABABR1 isoforms were similar between age groups [A] but aged- impaired rats expressed less of both isoforms than young [B]. Levels of GABABR2 were not changed by age [C] or cognitive group [D]. Insets of A-D demonstrate representative immunoreactive bands observed for young and aged PFC samples when incubated with the indicated antibody. *p<0.05 versus young.
141
Figure 4.5. GABABR1 and GABABR2 protein levels in the prefrontal cortex of young and
aged rats. Specific GABABR1 isoforms were similarly reduced by aging [A], but not
differentially changed among cognitive groups [B]. Levels of GABABR2 are decreased by 32% in the aged PFC [C] irrespective of cognitive group [D]. Insets of A-D demonstrate representative immunoreactive bands observed for young and aged hippocampal samples when incubated with the indicated antibody. ** p<0.01 versus young.* p<0.05, ** p<0.01 versus
young.
142
Figure 4.6. Ratios of GABABR1a:GABABR1b across PFC and hippocampus of young and aged rats. Relative levels of GABABR1a and GABABR1b were significantly different between the PFC and hippocampus (Hipp) when results from young and aged rats were pooled into a single analysis [A]. Relative expression ratios did not change as a function of age [B] or cognitive group [C]. Dashed line indicates a hypothetical value of 1 where the relative levels of expression of both isoforms are equivalent. *** p<0.001 PFC versus hippocampus.
143 Table 4.1. Parameters of baclofen-stimulated GTP-binding in the hippocampus of young and aged F344 rats
Parameter Young Aged-Total Aged-Unimpaired Aged-Impaired
EMAX (% of young) 100.00 ± 10.33 99.30 ± 11.35 89.48 ± 12.88 107.50 ± 16.42
Log EC50 (M) -4.78 ± 0.18 -5.01 ± 0.24 -4.89 ± 0.26 -5.14 ± 0.36
Hill Slope (NH) 1.00 ± 0.34 0.69 ± 0.21 0.96 ± 0.47 0.59 ± 0.23
144 Table 4.2. Parameters of baclofen-stimulated GTP-binding in the PFC of young and aged F344 rats
Parameter Young Aged-Total Aged-Unimpaired Aged-Impaired
EMAX (% of young) 100.00 ± 26.08 70.90 ± 8.13 62.81 ± 7.66 80.62 ± 16.58
Log EC50 (M) -4.44 ± 0.57 -4.73 ± 0.26 -4.86 ± 0.25 -4.53 ± 0.52 a Hill Slope (NH) 0.52 ± 0.20* 0.52 ± 0.10*** 0.68 ± 0.20 0.43 ± 0.12** a *p<0.05, **p<0.01, ***p<0.001 versus NH=1. p=0.15 versus NH=1.
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CHAPTER V
HIPPOCAMPAL Gαq/11 BUT NOT Gαo-COUPLED RECEPTORS ARE ALTERED IN
AGING
Joseph A. McQuail, Kathleen N. Davis, Frances Miller, Robert E. Hampson, Samuel A.
Deadwyler, Allyn C. Howlett and Michelle M. Nicolle
The following manuscript was published in Neuropharmacology 70:64–73, 2013. Stylistic variations are due to the requirements of the journal
146
Abstract
Normal aging may limit the signaling efficacy of certain GPCRs by disturbing the function of specific Gα-subunits and leading to deficient modulation of intracellular functions that subserve synaptic plasticity, learning and memory. Evidence suggests that Gαq/11 is more
sensitive to the effects of aging relative to other Gα-subunits, including Gαo. To test this hypothesis, the functionality of Gαq/11 and Gαo were compared in the hippocampus of young (6
months) and aged (24 months) F344×BNF1 hybrid rats assessed for spatial learning ability. Basal
GTPγS-binding to Gαq/11 was significantly elevated in aged rats relative to young and but not
reliably associated with spatial learning. mAChR stimulation of Gαq/11 with oxotremorine-M
produced equivocal GTPγS-binding between age groups although values tended to be lower in
the aged hippocampus and were inversely related to basal activity. Downstream Gαq/11 function
2+ was measured in hippocampal subregion CA1 by determining changes in [Ca ]i after mAChR
2+ and mGluR (DHPG) stimulation. mAChR-stimulated peak change in [Ca ]i was lower in aged
2+ CA1 relative to young while mGluR-mediated integrated [Ca ]i responses tended to be larger in
2+ aged. GPCR modulation of [Ca ]i was observed to depend on intracellular stores to a greater
degree in aged than young. In contrast, measures of Gαo-mediated GTPγS-binding were stable
across age, including basal, mAChR-, GABABR (baclofen)-stimulated levels. Overall, the data
indicate that aging selectively modulates the activity of Gαq/11 within the hippocampus leading to
2+ deficient modulation of [Ca ]i following stimulation of mAChRs but these changes are not
related to spatial learning.
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5.1. Introduction
G-protein coupled receptors (GPCRs) interact with a variety of Gα-subunits and effectors, giving rise to considerable diversity in signal transduction and resulting in the modulation of a variety of cellular processes including cell excitability, kinase activity,
2+ 2+ intracellular Ca concentration ([Ca ]i), neurotransmitter release and gene expression. GPCRs transduce extracellular signals via an associated G-protein heterotrimer that includes a Gα- subunit bound to a GDP molecule under resting conditions. In response to neurotransmitter binding, the transmembrane receptor protein undergoes a conformal change that drives a GTP- exchange reaction at the Gα-subunit. The active GTP-bound Gα-subunit can then modulate the activity of effector proteins until the GTP is hydrolyzed back to GDP by the Gα-subunit’s intrinsic enzymatic activity, thus terminating signaling action. Acetylcholine, glutamate and γ-
amino butyric acid (GABA), each interact with a subset of GPCRs, but the consequences for
neural activity are subtype-dependent. M1 muscarinic acetylcholine receptors (mAChR) and
Group I metabotropic glutamate receptors (mGluR), including mGluR1 and mGluR5, couple to
Gαq and Gα11 that stimulate phospholipase C (PLC) to catalyze the formation of inositol
phosphates (IP) and diacylglycerol (DAG) and subsequently releases intracellular Ca2+ stores
(ICS) via inositol triphosphate receptors (IP3Rs; reviewed in Caulfield and Birdsall, 1998; Bordi
and Ugolini, 1999). This signaling cascade is distinct from GPCRs, including M2 mAChRs and
GABAB receptors (GABABRs), that couple to Gαo and Gαi to inhibit adenylyl cyclase and limit
neurotransmitter release (reviewed in Caulfield and Birdsall, 1998, Chalifoux and Carter, 2011).
Aging is associated with progressive cognitive decline as well as increased risk for
neurodegenerative disorders such as Alzheimer’s disease (AD). Therapeutic interventions would
offer the greatest benefit if administered at the earliest indication of cognitive impairment, but
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the biological basis for this impairment must be sufficiently characterized to optimize therapeutic efficacy. Naturally occurring rodent models can assess the effects of normal aging on neural substrates and behavior without confounds stemming from neuropathological disease. Using these rodent models, mAChR-mediated phosphoinositide (PI) turnover has been reported as impaired (Ayyagari et al., 1998; Chouinard et al., 1995; Nicolle et al., 1999) or enhanced (Parent et al., 1995; Tandon et al., 1991) in the aged hippocampus. Similar disagreement is apparent in studies of Group I mGluR signal transduction (Nicolle et al., 1999; Parent et al., 1995).
Comparatively less is known about the integrity of M2 mAChR- or GABABR-stimulated
signaling in the aged hippocampus, but compounds that block either M2 mAChRs or GABABRs enhance learning and memory in aged rats (Froestl et al., 2004; Lasarge et al., 2009; Quirion et al., 1995). However, it is unclear if these benefits are derived from reversing age-related changes to GPCRs or indirectly promoting postsynaptic activity by facilitating neurotransmitter release.
Given the complex relationship between GPCRs and associated signal transduction mechanisms, this study presents findings from a series of comparative pharmacological analyses designed to determine whether aging selectively impairs receptor-stimulated activation of Gαq/11
leading to insufficient modulation of subsequent neural responses within the hippocampus of
young adult and aged rats that were characterized for spatial learning. First, this study used
35 mAChR and GABABR agonist-stimulated [ S]guanosine-5’-O-(3-thio)triphosphate (GTPγS)-
binding to assess functional coupling of these receptors to specific Gα-subunits that were
biochemically verified using an immunocapture scintillation proximity assay (SPA).
Subsequently, activity downstream of mAChRs or Group I mGluRs was examined by measuring
2+ agonist-stimulated changes to [Ca ]i.
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5.2. Materials and Methods
5.2. 1. Subjects
Male, Fischer 344 × Brown Norway F1 hybrid rats were obtained from the National
Institutes of Aging rodent colony maintained by Harlan-Sprague-Dawley, Inc., (Indianapolis, IN,
USA) and were 6 months of age (young; n = 21) or 24 months of age (aged; n = 47) at the time
of behavioral training. All animals were housed in a facility approved by the International
Association for the Assessment and Accreditation of Laboratory Animal Care at Wake Forest
University School of Medicine. The Institutional Animal Care and Use Committee of Wake
Forest University approved all protocols described in these studies.
5.2.2. Behavioral Testing in Morris Water Maze
Rats were behaviorally characterized using a standardized place-learning task developed to optimize detection of age-associated changes in spatial learning (Gallagher et al., 1993). Rats trained 3 trials a day for 8 days to navigate to a submerged platform using spatial cues surrounding the maze. Rats were placed into the water at one of four equally spaced start positions in a counterbalanced order and allowed 90 s to locate the platform after which time
they were guided to the platform. Rats remained on the platform for 30 s before transfer to a
holding cage for 30 s. Every sixth trial (i.e. the third trial on days 2, 4, 6 and 8) was a probe
where the platform was lowered and inaccessible during the first 30 s of the trial then
subsequently raised for escape. Following place-training, rats received a single session of 6 cued
trials, escaping to a visible black platform extending 2 cm above the water surface, to assess
sensorimotor function and motivation. Data were acquired via a video camera mounted above the
maze connected to a digital video recorder and computer running Ethovision software (Noldus,
Leesburg, VA, USA). Cumulative distance and average distance from platform assessed training
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and probe trial performance, respectively. Values from the second, third and fourth probe trials were summed to produce a “proximity score”, a graded measure summarizing individual performance (Gallagher et al., 1993; Bizon et al., 2009; Nieves-Martinez et al., 2012).
5.2.3. Hippocampal Microdissection and Membrane Preparation
Approximately 2 weeks after the completion of behavioral testing, rats were decapitated, the brain removed, and hippocampi dissected on an ice-cold plate. 1 mm-thick transverse sections were made through the septal-temporal axis using a tissue chopper and sub-dissected into dentate gyrus (DG; including the hilus), CA3 (including CA2) and CA1 regions. Regions from both hippocampi were pooled, frozen on dry ice and stored at -80°C until used for membrane preparation as described in (McQuail et al., 2012). Membrane protein content was measured using the Pierce bicinchoninic acid assay kit (Rockford, IL, USA) and aliquots were stored at -80°C until used for GTPγS-binding assays.
5.2.4. GTPγS-Binding and Anti-G-protein Scintillation Proximity Assay
All reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA), unless otherwise stated. GTPγS-binding reactions were conducted in triplicate in 96-well Opti-plates
(PerkinElmer, Waltham, MA, USA). The reaction buffer contained 100 mM NaCl, 5 mM MgCl2
in 50 mM HEPES (pH 7.4). To unmask Gαq/11 activity, membranes were pre-treated with 10 mM
N-ethylmaleimide for 30 minutes on ice (Salah-Uddin et al., 2008) and guanosine 5′-diphosphate
concentration was 0.1 mM (Delapp et al., 1999; Porter et al., 2002). For the Gαo assay, GDP
concentration was 50 mM (Delapp et al., 1999). Non-specific binding was determined in the
presence of 10 µM GTPγS. Basal GTPγS-binding was measured in the absence of any
experimental compounds. 100 µM oxotremorine-M or 300 µM baclofen (Tocris, Ellison, MO,
USA) stimulated mAChR and GABABRs, respectively. These concentrations were selected to
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produce maximal GTPγS-binding based upon prior studies examining total or Gα-subunit specific GTP-exchange in rodent brain (Delapp et al., 1999; McQuail et al., 2012; Porter et al.,
2002; Zhang et al., 2007). Ten micrograms of membrane protein was added to each well and equilibrated at room temperature for 30 minutes. GTPγS-binding was initiated by the addition of
500 pM [35S]GTPγS (PerkinElmer) to a final volume of 200 µl/reaction. After 60 minutes,
IGEPAL CA-630 was added to a final concentration of 0.3% (v/v) with agitation at +4°C for 30
minutes. Anti-Gαq/11 or anti-Gαo (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added
at a final dilution of 1:100 and incubated for 1 hour at +4°C. Anti-IgG-coated scintillation
proximity assay beads (PerkinElmer) were suspended in 25 ml of 50 mM HEPES and 50 µl
added per well then incubated for 30 minutes at +4°C. Plates were centrifuged and counted in a
TopCount scintillating microplate reader (PerkinElmer). Basal GTPγS-binding (counts per
minute; CPM) was determined by subtracting non-specific activity. Agonist-stimulated values were transformed to “percent over basal” [% = (stimulated-basal)/(basal)*100] to facilitate comparisons between age groups and subregions or “net CPM” values (i.e. basal subtracted from stimulated) for correlation with basal activity.
5.2.5. Hippocampal Slice Preparation and Calcium Imaging
Transverse hippocampal slices (250 µm) were prepared and loaded with Calcium Green-
AM (Molecular Probes, Eugene, OR, USA) as described in (Hampson et al., 2011). In contrast to intracellular injection, acetoxymethyl (AM) ester derivatives of fluorescent indicators allow for the labeling of multiple cells per slice/field of view. Imaging was performed on CA1 cells with an upright confocal microscope (Nikon, New York, NY, USA) equipped with a water-immersion objective, a Hamamatsu Orca-ER digital camera and an Ultraview spinning disc confocal system
(PerkinElmer). Calcium Green emission images (500-600 nm) were acquired by laser excitation
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at 488 nm and sampled at 0.3 s intervals. A piezoelectric “stepping” motor advanced the objective through the focal plane to acquire 40 vertical slices (2.5 µm) per field, producing a complete three-dimensional image every 12 s. Slices were perfused with artificial cerebrospinal fluid (ACSF; 126 mM NaCl, 20 mM NaHCO3, 5 mM KCl, 2 mM MgCl2, 2.5 mM CaCl2, 10 mM
glucose in 20 mM HEPES [pH 7.4]) for 2 min to determine baseline fluorescence and then
perfused with either 50 µM oxotremorine-M or 50 µM (S)-3,5-dihydroxyphenylglycine (DHPG;
Group I mGluR agonist; Tocris Bioscience) in ACSF for 3 minutes. Drug perfusion was
followed by 5 minute ACSF washout. Each slice was then incubated with 50 µM cyclopiazonic
acid (CPA; Tocris Bioscience) for 10 minutes to deplete intracellular calcium stores (Soler et al.,
1998) and the drug stimulation protocol was repeated. Calcium imaging results were expressed
as percentage change in cell soma fluorescence (ΔF) of baseline Calcium Green fluorescence (F0;
average of first 120 s of recording). This relatively quantitative approach is necessary because
uptake of the indicator dye is dependent upon infiltration of the cell plasma membrane followed
by cleavage of the dye from the conjugated acetoxymethyl group by endogenous esterases to
facilitate cytosolic localization and retention within the cell. Peak change in fluorescence (ΔF/F0;
i.e. the single greatest value observed during agonist administration) was obtained from each cell
under all conditions to determine maximal response and the cumulative effect of agonist-
2+ stimulated change to [Ca ]i was evaluated by calculating area under the curve (AUC) using the
integral of the response calculated in GraphPad Prism 5 software (LaJolla, CA, USA).
5.2.6. Statistical analyses
Data are presented as the mean ± standard error. Independent- or paired-samples t-tests
and repeated measures analysis of variance (RMANOVA) were performed using GraphPad
Prism 5 software. RMANOVAs were followed with Bonferroni post hoc tests to evaluate
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significant differences while correcting for multiple comparisons. In all statistical comparisons,
2+ p<0.05 was considered significant. GTPγS-binding and [Ca ]i results that were significantly different between young and aged rats were tested for correlation with proximity scores; bivariate correlations were performed for young and aged in a single analysis as well as separately for each age group.
5.3. Results
5.3.1. Spatial learning
While young and aged rats did not differ on the first training trial (see Section 2.3.1 and
Figure 2.1), there were main effects of age (F(1,66)=31.34, p<0.001) and training trial block
(F(3,198)=82.93, p<0.001) as well as an interaction between these two variables (F(3,90)=9.62, p<0.001). Post hoc comparisons revealed aged rats swam a greater cumulative distance from the platform on the first two training blocks (p<0.001 for both; Fig. 5.1A). Probe trial performance also differed between age groups (F(1,66)=65.81, p<0.001) and across trials (F(3,198)=9.39,
p<0.001) but these factors did not interact (F(3,198)=0.74, p>0.5 N.S.) demonstrating poorer spatial bias for the platform location on all probes in the aged group compared to young (Post hoc: p<0.01 on 1st probe and p<0.001 on 2nd-4th probes, Fig. 5.1B; also see Section 2.3.2. and
Figure 2.2.). However, age groups performed comparably on cued, visible-platform trials
(t(66)=1.36, p>0.1 N.S.), demonstrating that spatial impairment was not related to non-cognitive
factors such as swimming ability or motivation to escape to the platform (Fig. 5.1C; Section
2.3.4. and Figure 2.4.). Proximity scores were significantly higher in the aged group (t(66)=7.65,
p<0.001), consistent with searching further from the platform location, compared to young (Fig.
5.1D). Similar results were obtained comparing proximity scores between young and aged rats
2+ used for GTPγS-binding experiments (t(30)=4.62, p<0.001; Fig. 5.1E), oxotremorine-M [Ca ]i
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2+ imaging (t(19)=4.46, p<0.001; Fig. 5.1F) and DHPG [Ca ]i imaging (t(13)=4.77, p<0.001; Fig.
5.1G).
5.3.2. Muscarinic acetylcholine receptor-stimulated GTPγS-binding to Gαq/11
Non-specific binding was not different between age groups (F(1,30)=0.09, p>0.7 N.S.) or
hippocampal subregions (F(2,60)=1.63, p>0.2 N.S.; data not shown). Basal GTPγS-binding to
Gαq/11 was greater in aged rats relative to young rats; age × subregion RMANOVA revealed a main effect of age (F(1,30)=4.79, p<0.05), but no main effect of subregion (F(2,60)=0.95, p>0.3
N.S.) or interaction (F(2,60)=0.45, p>0.6 N.S.). However, when compared within each subregion,
basal GTPγS-binding to Gαq/11 did not significantly differ between young and aged in DG, CA3
or CA1 (Fig. 5.2A). When averaged across all three subregions (Fig. 5.2B), a trend towards a
positive correlation with proximity scores was observed for young and aged rats (r=0.31, p<0.1),
but no significant relationship was observed when aged rats were considered alone (r=0.12,
p>0.6 N.S.; Fig. 5.2C; but see supplemental materials, Table 5.S1 and Fig. 5.S1 for a specific
case). GTPγS-binding to Gαq/11 stimulated by oxotremorine-M did not differ as a function of age
(F(1,30)=1.81, p>0.1 N.S.) or subregion (F(2,60)=2.34, p>0.1 N.S.) nor did these factors interact
(F(2,60)=0.08, p>0.9 N.S.; Fig. 5.2D). Although no effect of age was observed for oxotremorine-
35 M-stimulated [ S]GTPγS-binding to Gαq/11, given previous findings in aged rats trained in an
identical manner (Chouinard et al., 1995; Zhang et al., 2007), we averaged data from all 3
subregions (Fig. 5.2E) for correlation with proximity scores, but observed no reliable association
across all rats (r=-0.25, p>0.3 N.S) or in aged rats alone (r=-0.20, p>0.1 N.S. .; Fig. 5.2F).
To determine whether greater basal GTPγS-binding to Gαq/11 is associated with lower
mAChR-stimulated GTPγS-binding to Gαq/11, correlations were performed between basal and
oxotremorine-M stimulated values (net CPMs). Basal and oxotremorine-M-stimulated GTPγS-
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binding to Gαq/11 were inversely related in the entire cohort when data from all 3 subregions were
averaged (r=-0.40, p<0.05; Fig. 5.3A), although when analyzed by age, young showed a
significant correlation (r=-0.69, p<0.05) whereas aged rats exhibited a trend (r= -0.37, p<0.1
N.S.). Within the DG, there was negative correlation between basal and stimulated GTPγS-
binding across the entire cohort (r=-0.17, p>0.3 N.S.; Fig. 5.3B), but when comparing between
age groups, young rats exhibited a negative correlation (r= -0.67, p<0.05) whereas aged did not
(r=0.09, p>0.6 N.S.). There was no significant correlation in the CA3 in the entire cohort (r=-
0.01, p>0.9; Fig. 5.3C) or in either age group (r(young)=-7.0e-4, p>0.9 N.S., r(aged)=- 0.01, p>0.9
N.S.). In the CA1, basal and oxotremorine-M-stimulated GTPγS-binding to Gαq/11 were
inversely correlated in the total cohort (r=-0.56, p<0.001; Fig. 5.3D) but when age groups were
analyzed separately, this relationship was only significant for aged rats (r=-0.63, p<0.01) and not
young (r=-0.49, p>0.1 N.S.).
5.3.3. Muscarinic acetylcholine and GABAB receptor-stimulated GTPγS-binding to Gαo.
Non-specific binding did not differ between age groups (F(1,30)=1.65, p>0.2, N.S.) or
among subregions (F(2,60)=1.00, p>0.3, N.S.) nor did these factors interact (F(2,60)=1.23, p>0.2,
N.S.; data not shown). There was a significant main effect of subregion on basal GTPγS-binding to Gαo (F(2,60)=38.28, p<0.001), but no main effect of age (F(1,30)=0.01, p>0.9, N.S.) or interaction (F(2,60)=0.60, p>0.5 N.S.; Fig. 5.4A). Subsequent t-test comparisons demonstrated
basal GTPγS-binding to Gαo was significantly different between all subregions; it was lowest in
CA3 and greatest in DG with intermediate values in the CA1 (p<0.05 for all comparisons).
GTPγS-binding to Gαo stimulated by oxotremorine-M was significantly different among
subregions (F(2,60)=82.46, p<0.001), but not different between age groups (F(1,30)=0.04, p>0.8,
N.S.) and there was no interaction between these two factors (F(2,60)=1.18, p>0.3 N.S.; Fig.
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5.4B). In contrast to basal activity, oxotremorine-M-stimulated GTPγS-binding to Gαo was
lowest in the DG and greater in the CA1 with intermediate values in the CA3 (p<0.05 for all
comparisons). Baclofen-stimulated GTPγS-binding to Gαo was also significantly different
between subregions (F(2,60)=8.54, p<0.001) but values were not different between age groups
(F(1,30)=1.38, p>0.2 N.S.) and there was no interaction between age and subregion (F(2,60)=1.36, p>0.2 N.S.; Fig. 5.4C). Subsequent t-tests indicated that baclofen-stimulated GTPγS-binding to
Gαo was lower in CA1 relative to CA3 (p<0.001) and DG (p<0.001) while binding was equivalent in CA3 and DG (p>0.5, N.S.).
5.3.4. Muscarinic receptor-stimulation of intracellular calcium in CA1
Representative Ca2+ imaging results are shown from slices prepared from young (Fig.
5.5A) and aged rats (Fig. 5.5B). When peak values from all cells were compared between age
2+ groups, oxotremorine-M-stimulated [Ca ]i was lower in aged cells relative to young (t(206)=2.19,
p<0.05; Fig. 5.4B), but the AUC was not different between ages (t(206)=0.82, p>0.4 N.S.; Fig.
5.5F). When oxotremorine-M stimulation was repeated following CPA treatment, peak values were significantly lower in aged (t(135)=3.41, p<0.001), but not young (t(71)=1.26, p>0.2 N.S.; Fig.
5.4C&D; Fig. 5.5C) cells. Similarly, CPA significantly attenuated AUC in aged (t(135)=4.09, p<0.001), but not young (t(71)=1.20, p>0.2 N.S.; Fig. 5.5F). When averaged by rat, a trend
2+ towards lower peak [Ca ]i was observed in the aged group (t(19)=1.77, p<0.1 N.S.; Fig. 5.5D)
and while decreased peak was associated with greater proximity scores in the entire sample (r=-
0.21, p<0.05), this relationship was not significant when aged rats were tested alone (r=-0.38,
p>0.1; Fig. 5.5E) There was no difference between young and aged rats when comparing AUC
(t(19)=0.81, p>0.4 N.S.; Fig. 5.5F).
5.3.5. Group I metabotropic glutamate receptor-stimulation of intracellular calcium in CA1
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Representative Ca2+ imaging results are shown from slices prepared from young (Fig.
5.6A) and aged rats (Fig. 5.6B). When peak values were compared between ages, DHPG-
2+ stimulated [Ca ]i was not different between young and aged cells (t(100)=0.75, p>0.4, NS; Fig.
5.6C), although aged cells showed a trend towards greater AUC relative to young (t(100)=1.90,
p<0.1; Fig. 5.6E). CPA treatment depressed peak DHPG-stimulated values in young (t(34)=2.34, p<0.05; Fig. 5.5C&D) and aged cells (t(66)=5.14, p<0.001; Fig. 5.6C) but only decreased AUC in
aged (t(66)=4.01, p<0.001) and not in young cells (t(34)=0.07, p>0.9; Fig. 5.6E). When averaged
by rat, there was no difference between age groups with respect to peak (t(13)=0.07, p>0.9; Fig.
2+ 5.6D) or AUC (t(13)=1.18, p>0.2; Fig. 5.6F) [Ca ]i response.
5.4. Discussion
The current data demonstrate that aging selectively alters Gαq/11-mediated GTPγS-
2+ binding as well as [Ca ]i after mAChR and mGluR stimulation in the hippocampus. In contrast,
GTPγS-exchange by Gαo stimulated with oxotremorine-M or baclofen was unchanged by age.
While stimulation of GTPγS-exchange by Gαq/11 via mAChRs was lower, though not
2+ significantly so, in all subregions of aged hippocampus, peak [Ca ]i stimulated by this same
receptor was significantly depressed in the aged CA1. Interestingly, peak Group I mGluR-
2+ stimulation of [Ca ]i was comparable between young and aged CA1 cells but there was a trend
2+ towards greater integrated [Ca ]i response in aged cells. Whether stimulated with oxotremorine-
M or DHPG, aged cells also demonstrated a greater dependence on intracellular calcium stores
compared to young. Collectively, the data suggest that modest changes to Gαq/11 may impair
downstream signal amplification, but additional intracellular signaling pathways may
compensate in a system-specific manner.
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Data indicate that aging selectively alters hippocampal receptor-mediated PI turnover due to modified receptor:G-protein coupling, but not protein expression (Aubert et al., 1995;
Chouinard et al., 1995; Nicolle et al., 1999; Smith et al., 1995; Zhang et al., 2007). But evidence obtained via the PI hydrolysis assay has yielded equivocal results. Chouinard and colleagues reported that mAChR production of IP elicited by oxotremorine-M was decreased in the hippocampus of aged Long-Evans rats and this decrease was associated with spatial learning impairment (Chouinard et al., 1995). However, Parent and colleagues, also examining PI hydrolysis in Long-Evans rats, reported elevated IP production in aged, spatial learning-impaired rats when stimulated with the cholinergic agonist carbachol (CCh Parent et al., 1995). Slight differences in the pharmacology of oxotremorine-M versus CCh may account for these discrepancies as additional studies examining oxotremorine-M or CCh-stimulation of CDP-
DAG report either a decrease (Nicolle et al., 2001) or no effect of age (Parent et al., 1995) in the hippocampus of aged Long Evans rats, respectively. Additionally, there is also no consensus on the effect of age on basal PI turnover in the hippocampus (Ayyagari et al., 1998; Nicolle et al.,
1999; Parent et al., 1995; Tandon et al., 1991).
While PI turnover assays probe only those GPCRs that signal via Gαq/11, insight from
traditional GTPγS-binding assays is limited as many agonists exhibit poor subtype selectivity
and, without modifications to detect binding to Gαq/11, results reflect a disproportionate
contribution from Gαi/Gαo (reviewed in (Milligan, 2003). However, the current data obtained via
a Gα-specific SPA, may bridge the disparate PI hydrolysis findings as elevated basal activity of
Gαq/11 lends credence to those studies reporting elevated basal PI hydrolysis, while modestly
depressed mAChR:Gαq/11 coupling (presumably M1, e.g. Porter et al., 2002) may translate to
significantly lower PI turnover downstream through deficits in signal amplification, especially
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given evidence for an age-associated decrease in PLCβ1 protein levels (Nicolle et al., 1999).
This assumption is strengthened by the reliable, negative association between basal and mAChR- stimulated GTP-binding in the CA1 hippocampal subregion and when values were averaged across subregions. Resolving the status of M1 activity in normal aging is imperative because mAChR-stimulated PI hydrolysis is decreased in post-mortem AD cortical samples (Greenwood et al., 1995; Jope et al., 1997), but stimulation of M1 inhibits amyloid-β formation (Caccamo et al., 2006; Jones et al., 2008). Thus, deteriorating M1 function expedites AD pathology and this receptor may be a key disease-modifying target if appropriate therapies can be administered at the earliest sign of cognitive changes.
In contrast to Gαq/11, Gαo activity remained stable with age. Detailed subregion analyses,
however, showed agonist-dependent differences in the relative activities between DG, CA3 and
CA1, potentially reflecting differences in M2 receptor densities and GABABR1 expression
profiles (Aubert et al., 1995; Fritschy et al., 1999; Quirion et al., 1995; Smith et al., 1995).
Although M2 autoreceptor density is reported as stable or modestly elevated with age (Aubert et
al., 1995; Quirion et al., 1995; Smith et al., 1995), cholinergic fiber length is decreased in the aged hippocampus (Ypsilanti et al., 2008). Similarly, expression of GABABR2, the subunit that
mediates GABABR:G-protein coupling (Robbins et al., 2001), is unchanged in the aged
hippocampus (McQuail et al., 2012) but there are fewer glutamic acid decarboxylase-67-
expressing (GAD-67+) interneurons (Stanley and Shetty, 2004) and possibly greater numbers of
basal forebrain GAD-67+ projection neurons innervating the hippocampus in cognitively
impaired aged rats (Bañuelos et al., 2013). Thus, prior findings indicate that circuit-level changes
may alter cholinergic and GABAergic modulation of the aged hippocampus while the current
data indicate the activity of M2 mAChRs and GABABRs is preserved with age.
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Given the tendency towards decreased mAChR-stimulation of Gαq/11 in the current study and prior evidence of decreased post-synaptic mAChR function in aged rats (Chouinard et al.,
1995; Nicolle et al., 2001; Zhang et al., 2007), it was hypothesized that oxotremorine-M would be less effective at stimulating downstream ICS release. Accordingly, there was a significant
2+ age-dependent decrease in peak oxotremorine-M-stimulated [Ca ]i of CA1 cells. As Group I
2+ 2+ mGluRs also release Ca from ICS, it was surprising to find that peak DHPG-stimulated [Ca ]i
was not blunted in aged CA1 cells. CPA did not completely block mAChR or mGluR-stimulated
2+ 2+ [Ca ]i, consistent with a role for cell membrane Ca channels including N-Methyl-D-aspartate
receptors (NMDARs) and voltage-gated Ca2+ channels (VGCCs). Aging is associated with
decreased expression of NMDARs (Shi et al., 2007; Wenk and Barnes, 2000 but see Nicolle et
al., 1996), diminished NMDAR function (Barnes et al., 1997; Potier et al., 2000; Bodhinathan et
al., 2010) and increased VGCC currents and channel density (Campbell et al., 1996; Thibault and
Landfield, 1996) in CA1 that may alter the balance of Ca2+ homeostasis and signaling within
aged neurons. When considered together, the data indicate a selective deficit in mAChR
2+ 2+ modulation of [Ca ]i, although a diversity of Ca entry, sequestration and release processes are
at work and changing with age in the CA1 area.
2+ Post-synaptic elevation of [Ca ]i is necessary to induce synaptic plasticity, including
long-term potentiation (LTP) and long-term depression (LTD). Electrophysiological and
pharmacological studies have shown that aging modulates the magnitude and mechanisms of
synaptic plasticity triggered by M1 mAChR or Group I mGluR-stimulation. Relative to young
rats and aged rats with impaired spatial learning, aged Long-Evans rats with preserved spatial
learning exhibit increased expression of a form of synaptically evoked, non-NMDAR LTD
dependent on the activation of PLC via Gαq/11-coupled GPCRs (Lee et al., 2005). This phenotype
161
was highly specific as NMDA-dependent LTD was depressed in aged animals with no relation to spatial learning and CCh-LTD was not enhanced in aged-unimpaired rats, rather it was lower in aged-impaired rats. In this same study population, NMDA-dependent LTP induced by theta-burst stimulation is depressed in all aged rats, regardless of cognitive status, but unimpaired aged rats exhibit greater VGCC-dependent LTP relative to either young or age-matched rats with impaired spatial learning (Boric et al., 2008). Collectively, these data argue a switch from NMDA- dependent to NMDA-independent mechanisms of plasticity is neuroprotective and beneficial to cognitive aging. This is consistent with biochemical evidence demonstrating that excessive Ca2+
influx via NMDARs is neurotoxic (Attucci et al., 2002), and memantine, a NMDAR antagonist
prescribed to AD patients (Reisberg et al., 2003), protects cultured hippocampal neurons against
excitotoxic insult (Volbracht et al., 2006). However, evidence for an adaptive switch in plasticity is not universal. In contrast to those findings obtained in aged Long-Evans rats, increased susceptibility to NMDAR-LTD in the CA1 of aged F344 rats is associated with worse spatial retention in the water maze (Foster and Kumar, 2007), possibly due to an enhanced contribution from ICS (Kumar and Foster, 2005). In this same study population CCh-LTD and DHPG-LTD are enhanced owing to an age-dependent shift towards greater contributions from NMDARs and
VGCCs (Kumar and Foster, 2007; Kumar, 2010). These latter studies implicate the age- associated shift in the induction thresholds for LTP versus LTD are largely dependent on
2+ alterations to [Ca ]i and, when activated, will recruit somewhat different mechanisms between
young and aged rats. Thus it remains to be resolved whether or not GPCRs provide an alternate
mechanism to support synaptic plasticity in aging and under what circumstances GPCRs will
facilitate LTP rather than LTD in the aged CA1 area.
162
When attempting to reconcile divergent conclusions such as beneficial versus maladaptive changes to GPCR modulation of plasticity, an increased Ca2+/Mg2+ ratio in recording solutions used in some studies (e.g. Lee et al., 2005) relative to others (e.g. Foster and
Kumar, 2007) will facilitate the induction of LTD over LTP (reviewed in Burke and Barnes,
2+ 2010), thus underscoring an important need to evaluate receptor-mediated changes to [Ca ]i to
2+ better inform studies of synaptic plasticity. Emphasizing specific evaluation of [Ca ]i following
application of selective GPCR agonists, the data show aged cells significantly depend on ICS to
2+ elevate [Ca ]i following mAChR-stimulation whereas young cells do not, consistent with an
age-dependent shift towards ICS that facilitates LTD. Furthermore, peak mAChR-stimulated
2+ 2+ [Ca ]i was lower in aged CA1, possibly consistent with the modest rise in Ca needed to induce
LTD relative to LTP, explaining enhanced LTD following mAChR activation. However, the
2+ relationship between this parameter and learning is unclear because peak [Ca ]i was not
different between ages when conducting a subject-based analysis, although a trend towards lower
2+ 2+ [Ca ]i was observed. Peak Group I mGluR-stimulated [Ca ]i was not different between young
and aged groups. However, aged cells tended to demonstrate a greater, CPA-sensitive integrated
2+ [Ca ]i response relative to young leading to speculation that this exaggerated response is a factor
in age-dependent changes to mGluR-mediated plasticity. DHPG application elicits both rapid
ICS release via mGluR1 and delayed potentiation of NMDARs via mGluR5 (Mannaioni et al.,
2+ 2001). Therefore, preserved peak followed by sustained elevation of [Ca ]i in aged cells is
consistent with increased contributions from mGluR1 and NMDARs (via mGluR5), respectively, that enhance DHPG-LTD in the aged CA1 (Kumar and Foster, 2007). However, such a mechanistic shift is likely maladaptive. NMDAR-stimulated release of ICS via ryanodine receptors (RyRs) is implicated in the progression of pathology in transgenic AD mice
163
(Goussakov et al., 2010; Oulès et al., 2012) and levetiracetam, an anticonvulsant that blocks
IP3R and RyR-mediated ICS release (Nagarkatti et al., 2008), reverses cognitive impairments in aged rats (Koh et al., 2010) and older humans with mild cognitive impairment (Bakker et al.,
2012).
2+ Robust stimulation will trigger significant increases in somal [Ca ]i in young and aged
CA1 neurons, either via electrical stimulation, as reported previously (Gant et al., 2006; Thibault et al., 2001), or using pharmacological stimulation, as in the present study. Although resting
2+ [Ca ]i is not significantly changed with advancing age (Gant et al., 2006; Thibault et al., 2001),
2+ repetitive synaptic stimulation, elicits a larger peak and integrated [Ca ]i response in middle-
aged (12-14 months) and aged (23 months) relative to younger adult rats (4-10 months) and this age effect is reversed by blocking RyRs, preventing the release ICS in response to Ca2+ entering
via membrane bound sources (Gant et al., 2006). What’s more, this larger Ca2+ response is
inversely related to frequency facilitation, a form of short-term synaptic plasticity (Thibault et
2+ al., 2001). Dysregulation of [Ca ]i is implicated in another aging biomarker, enhancement of the slow after hyperpolarziation potential (sAHP). Following hippocampal-dependent eye-blink
conditioning in rabbits, this Ca2+-dependent, K+-mediated potential, which normally holds the
cell in a hyperpolarized state following a burst of action potentials, is relaxed to allow enhanced
neural activity; but this potential remains greater in CA1 neurons of aged rabbits (i.e. older than
24 months) following conditioning and limits cell excitability relative to younger animals
(Disterhoft et al., 1996). More relevant to the rodent model used in the current study, elevated
sAHP amplitude is associated with worse spatial learning in the Morris water maze in aged F344
rats (Tombaugh et al., 2005). Appropriately, inhibition of ICS release by RyR blockade or
depletion of ICS by CPA or thapsigargin leads to the selective induction of LTP following peri-
164
threshold stimulation in slices from aged, but not young, F344 rats by lowering the AHP and enhancing synaptic transmission via NMDARs (Kumar and Foster, 2004). Lastly, systemic administration of BAPTA-AM, a cell permeant Ca2+ chelator, will improve spatial learning in
aged F344 rats performing a distributed training version of the Morris water maze whereas
BAPTA-AM has no effect on the performance of young rats (Tonkikh et al., 2006).
Collectively, these studies demonstrate that dysregulation of evoked intracellular Ca2+ is a
reliable marker of aging in CA1 neurons that is detrimental to neural modifications necessary for
learning and memory.
The use of selective compounds that signal via receptors linked to PI-hydrolysis is critical
when evaluating the significance the Ca2+ imaging results reported in this study. Prior studies
that examined the application of IP3 to rat hippocampal microsomes (Burnett et al., 1990) or
mouse cortical slices (Stutzmann et al., 2006) did not reveal differences in Ca2+ release between
samples prepared from young adult and older subjects. However, the current data support a hypothesis that changes to upstream signaling, such as altered receptor:G-protein coupling, will limit the ability of PLCβ1 to form IP3 in response to GPCR activation. Thus, while synaptic stimulation induces a larger Ca2+ response in aged rats (Gant et al., 2006), the current study
shows decreased peak oxotremorine-M stimulated Ca2+ and similar peak Ca2+ after DHPG
application, although the integrated response in the latter experiment tended to be greater in aged
cells, suggesting differential recruitment of Ca2+ sources in a pattern distinct from that elicited by
repetitive synaptic stimulation. Even though M1 and Group I mGluRs couple to the Gαq/11 subclass of G-proteins, application of oxotremorine-M and DHPG produced different results within the present study, underscoring the importance of post-synaptic scaffolds and other protein:protein interactions that confer unique, receptor-specific signaling pathways that support
165
synaptic plasticity (Dickinson et al., 2009; Jo et al., 2010; Ménard and Quirion, 2012; Volk et al.,
2007). However, the design of the current study did not pharmacologically isolate the GPCR-
Gαq/11-PLCβ1-IP3R pathway; synaptic release and post-synaptic membrane depolarization certainly activated membrane-bound Ca2+ channels and RyRs. Future studies will need to
employ additional manipulations to block basal synaptic transmission, membrane-bound Ca2+ sources and RyRs to achieve better isolation of this pathway while delivering compounds that more precisely localize impaired signaling steps. In addition, techniques to examine changes in dendrites would be appropriate as IP3R density is greatest in this cellular compartment
(Fitzpatrick et al., 2009) and responses may differ between dendrites and soma.
Although implicated in this study, it is unclear whether or how the increased basal activity of Gαq/11 observed in the aged hippocampus may account for any of the other changes
seen in this and similar studies of brain aging (i.e. changes to downstream effectors and Ca2+
dyshomeostasis). Notably, virally induced expression of a constitutively active Gαq mutant
protein, Gαq(Q209L), in LβT2 pituitary cells leads to a specific decrease in PLCβ1 expression
but increased stimulated calcium influx through VGCCs without altering endogenous receptor or
G-protein expression (Liu et al., 2005). As mentioned previously, aging also decreases
hippocampal PLCβ1 expression without similar changes to GPCR or G-protein levels
(Chouinard et al., 1995; Nicolle et al., 1999; Zhang et al., 2007) while potentiating responses
from VGCCs (Campbell et al., 1996; Thibault and Landfield, 1996; Thibault et al., 2001).
Importantly, Gαq(Q209L)-enhanced calcium influx via VGCCs was associated with decreased
downstream cellular responses due to impaired activation via the mitogen-activated protein
kinase pathway (Liu et al., 2005), a phenotype also observed in the aged hippocampus that is
tightly linked with normal learning and memory (Blum et al., 1999; Ménard and Quirion, 2012).
166
While highly speculative, it is provocative to hypothesize that elevated basal activity of this specific Gα-protein is a central parameter that links maladaptive changes to neural biochemistry and physiology and ultimately impaired cognition.
5.5. Conclusion
The current data demonstrate that aging selectively modulates the functions of a particular subtype of Gα-protein with implications for downstream signaling. Normal aging is associated with increased basal activity of Gαq/11, the G-protein subtype that links the activation of GPCRs, including M1 mAChRs and Group I mGluRs, to PI-turnover and Ca2+ release but
even within this subclass of receptors, compensatory signaling may arise in a system-specific
fashion. However, aging is also broadly associated with a shift in the relative contributions of
Ca2+ sources following pharmacologic stimulation. Thus, additional experimentation is necessary
to outline a role for constitutive activity of Gαq/11 in the modulation of neural signaling as well as to determine how an upstream gain-in-function will elicit paradoxical decreases in downstream responses following receptor stimulation. As much interest already focuses on the therapeutic potential of this class of receptors, such information will be vital to develop novel therapies that can beneficially modulate specific GPCR-initiated signaling pathways to favor the induction of
LTP over LTD while correcting Ca2+ dyshomeostasis in aged neurons.
5.7. Acknowledgements
This work was supported by NIH grants F31-AG038266 to JAM, R01-AG020572 to MMN,
R01-DA03690 to ACH and R01-DA007625 to SAD. The authors thank Dennis O. Rookwood,
Jr., for technical assistance in the behavioral training of rats used in this study.
167
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Figure 5.1. Performance of young and aged rats in the Morris water maze. Young (6 months; n=21) and aged (24 months; n=47) rats were trained on a hidden-platform/place- learning version of the Morris water maze organized into 4 blocks that each included 5 training trials (A) and a single probe trial (B) administered at the end of each training block. After the end of place training, rats received a single block of six visible-platform/cue-training trials (C). Probe trial measures were summed to compute “proximity scores” to characterize the range of individual performance differences within this study population (D; horizontal line denotes mean of each group). Proximity scores of each cohort used for GTPγS-binding (n=10 young, n=22
2+ aged; E), oxotremorine-M-stimulated [Ca ]i (n=6 young, n=15 aged; F) or DHPG-stimulated
2+ [Ca ]i (n=5 young, n=10 aged; G). **p<0.01 and ***p<0.001 vs. young according to
Bonferroni post hoc test (A&B). ***p<0.001 vs. young by independent samples t-test (D-G).
178
Figure 5.2. GTPγS-binding to Gαq/11 in the hippocampus of young and aged rats.
[35S]GTPγS-binding assay was combined with an antibody-capture scintillation proximity
counting approach to measure Gαq/11-specific GTPγS-binding in the 3 major hippocampal subregions (n=10 young, n=22 aged; A and D). Basal (agonist-free) GTPγS-binding to Gαq/11
(A) was averaged for all 3 subregions (B; *p<0.05 vs. young by independent samples t-test) and
subsequently tested for association with proximity scores (C; solid line denotes significant trend
line; INSET: r and p-values for aged group). GTPγS-binding to Gαq/11 stimulated by 100 µM oxotremorine-M (D) was similarly averaged across all 3 subregions (E) and tested for association with proximity scores (F; dashed line denotes non-significant trend line; INSET: r and p-values for aged group).
179
Figure 5.3. Basal and oxotremorine-M stimulated GTPγS-binding to Gαq/11 are inversely
correlated in the hippocampus of young and aged rats. Basal and oxotremorine-M
[35S]GTPγS-binding were measured for correlation in the average of 3 hippocampal subregions
(A), DG (B), CA3 (C) and CA1 (D). Solid lines denote significant trend lines and dashed lines
denote non-significant trend line; INSET: r and p-values for young (n=10) and aged (n=22)
group tested together.
180
Figure 5.4. GTPγS-binding to Gαo
in the hippocampus of young and
aged rats. [35S]GTPγS-binding assay
was combined with an antibody-
capture scintillation proximity
counting approach to measure basal
activity (A) as well as GTPγS-
binding to Gαo stimulated by 100 µM
oxotremorine-M (B) and 300 µM
baclofen (C) in the 3 major
hippocampal subregions of young
and aged rats (n=10 young, n=22
aged; A-C).
181
Figure 5.5. Oxotremorine-M-stimulated changes to intracellular Ca2+ concentration in CA1
of young and aged rats. Representative results of Ca2+ imaging in hippocampal slices showing
time-course of changes to intracellular Ca2+ concentration stimulated by 50 µM oxotremorine-M
(Oxo-M) in young (A) and aged CA1 cells (B); INSET: number of cells and mean peak and area
under curve (AUC), range of values is given in parentheses. Peak change in intracellular Ca2+
182
concentration of young and aged cells (C; n=72 cells from young, n=136 cells from aged), averaged by rat (D; n=6 young, n=15 aged) and tested for association with proximity scores (E; dashed line denotes non-significant trend line; INSET: r and p-values for aged group). Area under curve of intracellular Ca2+ response of young and aged cells (F) and averaged by rat (G).
Black bar in A and B denotes time period of agonist application. *p<0.05 vs. young according to independent-samples t-test; ###p<0.001 vs. oxotremorine-M control according to paired-samples t-test.
183
Figure 5.6. DHPG-stimulated changes to intracellular Ca2+ concentration in CA1 of young
and aged rats. Representative results of Ca2+ imaging showing time-course of changes to
intracellular Ca2+ concentration in hippocampal slices stimulated by 50 µM DHPG in young (A)
and aged CA1 cells (B); INSET: number of cells and mean peak and area under curve (AUC),
range of values is given in parentheses. Peak change in intracellular Ca2+ concentration of young
184
and aged cells (C; n=35 cells from aged, n=67 cells from young) and averaged by rat (D; n=5 young, n=10 aged). Area under curve of intracellular Ca2+ response of young and aged cells (E)
and averaged by rat (F). Black bar in A and B denotes time period of agonist application.
*p<0.05 vs. young according to independent-samples t-test; #p<0.05, ###p<0.001 vs. DHPG
control according to paired-samples t-test.
185
5.S. Supplemental Materials
Analyses of neurobiological parameters versus spatial learning
As this study utilizes a naturally occurring animal model comprised of rats of differing ages and behavioral characteristics, correlations between proximity scores and neurobiological parameters are generally performed following significant effects of age revealed by t-test or
ANOVA to constrain the total number of correlation analyses. Given the parameters of this model and this statistical approach, investigations seek to reveal differences between age groups that are reliably correlated with spatial learning within the aged cohort to demonstrate that such changes can discriminate between aged rats with conserved learning abilities and those with behaviorally confirmed learning impairments (relative to young). However, other relationships may exist, specifically, some positive correlations may be observed in the absence of main effects of age. To allow for this possibility, the results of all potential correlation analyses are presented in Table 5.S1. Correlations were performed separately for young and aged groups as well as all animals within a single analysis. Correlations evident in the combined cohort (i.e. young and aged), but not in either age group alone, could indicate a genuine relationship with spatial learning independent of age, but additional procedures must correct for the effects of age to avoid confounding results. While partial correlations can test for relationships between two variables while controlling for a third variable, age is a categorical, not a continuous, variable in this model. Thus, to correct for the effect of age, proximity scores and neurobiological measures were transformed to Z-scores within each age group, thus maintaining the within-group distribution of data while eliminating between-group differences. These age-corrected correlations were applied to data that revealed a significant relationship between proximity score and neurobiological measures in the entire cohort. Specifically, the significant association
186
between proximity score and basal GTPγS-binding to Gαq/11 in DG (r=0.35, p<0.05; Fig. 5.S5A)
observed across all rats survived this correction (r=0.37, p<0.05; Fig. 5.S5B) while a similar
2+ relationship between proximity score and peak oxotremorine-M-stimulated [Ca ]i in CA1 (r=-
0.46, p<0.05; see Fig.5E and Table 5.S1) was eliminated by this correction procedure (r=-0.30, p<0.18, N.S.), suggesting that the former parameter is genuinely associated with spatial learning independent of age while the latter is not.
187
Figure. 5.S1. Relationship between spatial learning and basal GTPγS-binding to Gαq/11 in
DG after correcting for differences between age groups. Basal GTPγS-binding to Gαq/11 in
DG is plotted as a function of proximity score across young and aged rats before (A) and after
(B) correcting for the effects of age. Solid line denotes the significant trend-line obtained when young and aged rats are tested for correlation in a single analysis (A&B), dashed line denotes the non-significant trend-line obtained when aged rats are tested as a separate group (A only).
INSET: r and p-values for young (n=10) and aged (n=22) group tested together or aged rats tested separately.
188
189 CHAPTER VI
DISCUSSION
190 6.1. Overview of Findings
Broadly, this thesis refines the understanding of age-related changes to behavior and
neural substrates. Age-related variability in spatial learning was detected in FBNF1 rats by 24
months of age (Chapter II) and tissue from these rats was used for subsequent neurobiological
assessments. Initial stereological analysis determined the neurological basis for age-related
spatial learning deficits was not due to a loss of cholinergic neurons innervating the
hippocampus, as is widely proposed in existing literature (Chapter III). The preservation of cell
number led to the hypothesis that synaptic parameters within the hippocampus are reliable
correlates of spatial learning in aged rats. GPCRs are widely distributed throughout the
hippocampus at both pre- and post-synaptic locations and are, therefore, well-positioned to
modulate synaptic activity. Using receptor-mediated GTP-Eu exchange as a surrogate to assess
GPCR functionality, data in Chapter IV show that maximal GABABR-mediated GTP-Eu
binding was preserved in the aged hippocampus, but depressed in the aged PFC with no relation to spatial learning. Western blot analysis verified that attenuated function in the PFC was due to loss of both GABABR1 and GABABR2 subunits, the constituent GABAB receptor proteins.
Interestingly, expression of GABABR1, but not GABABR2, was lower in the hippocampus of aged rats with spatial learning impairment, suggesting a change in GABABR subunit
composition may play a role in cognitive aging. Muscarinic AChRs are also positioned pre- and post-synaptically, but exhibit differential Gα-protein-coupling profiles at each site. The results in
Chapter V distinguish between Gα-protein subtypes and show that basal GTPγS-binding by
Gαq/11 is elevated in the aged hippocampus, with no relationship to spatial learning, while mAChR activation of Gαq/11 and Gαo were similar between age groups. Altered activity of Gαq/11 suggests that post-synaptic signaling is likely altered following GPCR activation, and
191 subsequently confocal Ca2+ imaging data also presented in Chapter V revealed an age-
2+ dependent blunting of mAChR-stimulated elevation of [Ca ]i of CA1 cells. In contrast, Group I
mGluRs, another post-synaptic GPCR, tended to elicit a greater integrated response in aged cells.
However, both mAChRs and mGluRs relied to a greater extent on ICS in aged cells compared to young. While these findings provide a better understanding of the relationship between the
emergence of behavioral deficits and manifestation of neural changes in aged rats, they also
bring to the forefront new questions that require further investigation.
6.2. Implications of the Current Research
The data presented in this thesis touch on a variety of important parameters of neural
function that are the focus of ongoing research in this and many other labs in the neurocognitive
aging field. The discussion section of each chapter reviews, in detail, previously published
findings of relevance and then relates my own conclusions back to that literature to emphasize
the way in which these new findings expand upon, clarify, or sometimes refute, prior theories for
the neural basis of cognitive aging. The following sections, however, extend beyond those
matters already addressed in the discussion within each chapter to further clarify the validity of
findings, propose outstanding alternative hypotheses as well as offer reasoned advice on the
design of next series of studies to advance specific lines of research.
6.2.1. Timing and characteristics of age-related spatial learning impairment in FBNF1 rats
The training protocol used in the current collection of reports has been applied
extensively to study hippocampal functioning in aged Long-Evans (LE) rats (Gallagher and
Rapp, 1997; Gallagher et al., 2003; Wilson et al., 2006). However, the National Institutes of
Aging (NIA) has selected the Fisher 344 × Brown Norway F1 (FBNF1) hybrid along with inbred
192 F344 and Brown Norway rat strains for inclusion in their Aged Rodent Colony (ARC), making
these specific strains available for use to NIA-funded researchers. The data presented in Chapter
II demonstrate that spatial learning impairment is evident in 24 month-old FBNF1 rats due to
prominent deficits apparent in approximately 50% of individuals in that age group, while the
remaining aged rats are cognitively similar to young. This pattern is strikingly similar to that
seen in 25-27 month-old Long-Evans rats (Gallagher et al., 1993) and 22 month-old F344 rats
(Bizon et al., 2009), thus, demonstrating the reliability of this approach to measure age-related
changes in behavior in 3 strains of rats with differing genetic backgrounds, including 2 strains
that are available from the NIA-ARC. Other behavioral studies investigating water maze
performance across the life-span of FBNF1 rats determined that cognitive decline proceeds in a
non-linear fashion; spatial learning is generally stable between young adults and middle-aged
rats (18 months), but there is a significant deterioration in spatial learning ability between
middle-aged and aged rats (27-35 months of age; Adams et al., 2008; Markowska and
Savonenko, 2002). Even at this advanced age, researchers argue whether there are clear impaired
and unimpaired cognitive subgroups (VanGuilder et al., 2011a, 2011b; Wong et al., 2006).
However, the behavioral data from Chapter II clearly demonstrates that spatial learning
impairments are apparent in a substantial subset of 24 month-old FBNF1 rats, several months
earlier in the lifespan of this strain than has been previously assumed.
The majority of published reports presume that significant impairments are not apparent
in the FBNF1 strain until at least 27 months (Burgdorf et al., 2011; Fitting et al., 2008;
Hasenöhrl et al., 1999; Markowska and Savonenko, 2002; Shi et al., 2011; Thornton et al., 2000;
VanGuilder et al., 2011a, 2011b; Wong et al., 2006; Zhang et al., 2012). Notably, many prior studies either used no probe trials or only a single probe delivered at the end of training to assess
193 performance. Probe trials are a necessary tool to determine whether animals are using a spatially- guided search (i.e. hippocampal-dependent strategy) to find the platform location and the use of multiple probes interspersed throughout the training protocol can reveal the rate at which an efficient search strategy is formed. Using the same procedure as described in this thesis, Nieves-
Martinez et al (2012) found that 18 month-old FBNF1 had significantly higher proximity scores than 8 month-old controls, suggesting that a measure that includes results from multiple probe trials is sensitive to the earliest signs of cognitive change. Although individual differences were not discussed in Nieves-Martinez et al (2012), Bizon et al. (2009) also determined that middle- aged (18 months) F344 rats are impaired relative to young according to a similar spatial learning measure, but the pattern of individual differences were qualitatively distinct from the older (22 months) group. At 18 months, there was a subtle shift towards higher scores, but most of these rats remained within the range of young. Conversely, the aged cohort could be parsed into aged rats that were cognitively similar to young (aged-unimpaired) and those that were performing outside the range of scores normally seen in young rats (aged-impaired). This latter observation stresses the value of examining the pattern of individual performance as age-related changes to cognition may be quantitative as well as qualitative.
Using Nieves-Martinez et al (2012) and Bizon et al. (2009) as starting points, the current study examined place learning in somewhat “younger” aged FBNF1 rats. At 24 months of age, not only is spatial learning impairment evident, but the aged cohort can be split into aged- unimpaired and aged-impaired subgroups, with roughly even numbers of rats in each group (e.g.
Section 2.2.3 and Figure 2.2). This conclusion is significant as investigations of neurocognitive aging should seek to identify neurobiological changes that co-merge with the earliest signs of memory impairment; examining neurobiological changes 3-8 months after the onset of memory
194 impairment leaves open the possibility that associated neural alterations may be secondary (or
compensatory) changes that have no role in the initial manifestation of cognitive deficits.
Therefore, earlier changes to neural substrates must be examined to aid in the identification of
the primary mediators of age-related learning impairments in FBNF1 rats.
6.2.2. Septohippocampal cholinergic neurons in older rats with cognitive impairment
Age-related losses to key neural substrates may render the aged brain unresponsive to
therapies that interact with those lost substrates; that is, therapeutic efficacy is limited because
there is insufficient target to modulate. Currently, cholinesterase inhibitors are the mainline
treatment given to older adults with memory deficits, although it is well understood that this
approach does not reverse underlying neurodegenerative processes. Therefore, the utility of this
and similar procholinergic therapies are limited by ongoing deterioration of the cholinergic system (Gauthier, 2005; Pepeu and Giovannini, 2009; Raschetti et al., 2007; Terry et al., 2011).
Thus, it is critical to evaluate the status of the brain’s septohippocampal cholinergic system in the
context of normal aging to establish whether a sufficient number of acetylcholine-producing
neurons remain to merit the use of agents that modulate cholinergic neurotransmission. In
Chapter III, stereological approaches determined that age-related deficits in spatial learning
are not caused by outright degeneration of the basal forebrain cholinergic neurons that
provide input to the hippocampus, although local neuroinflammation may disrupt cholinergic
cell function in the aged brain.
The findings presented in Chapter III agree with those of Ypsilanti et al. (2008) who found no loss of cholinergic neurons in 22 month old F344 rats, although the cognitive status of these rats was not evaluated. Interestingly, Bañuelos et al (2013) observed a subtle loss of cholinergic neurons in similarly aged F344 rats that was not related to spatial learning, however
195 this study analyzed a larger portion of the basal forebrain, including the horizontal diagonal band, which innervates ventral cortical regions, whereas Ypsilanti et al. (2008) and Chapter III
[and McQuail et al (2011)] examined the medial septum and vertical diagonal band, which exclusively innervate the hippocampus. Returning to Bañuelos et al (2013), despite the loss of
cholinergic neurons, there was no change in total neuron number. The basal forebrain contains a
heterogenenous mixture of cell types including cholinergic, GABAergic and glutamatergic
neurons, so estimating total neuron number may not be sensitive enough to detect the subtle loss of one class of neurons, or the neurons themselves may not degenerate but merely cease to express the marker used to visualize those neurons, in this case, ChAT. Loss of ChAT expression within these neurons would presumably render them neurochemically silent in a manner similar to that already described for interneurons that cease to express GAD-67 in the hippocampus of aged rats (Stanley and Shetty, 2004). If this is the case, then therapies aimed at restoring synaptic cholinergic transmission would be limited due to decreased neurotransmitter production, but a novel therapy that could re-activate expression of neurotransmitter-synthesizing enzymes in neurochemically quiescent cells would aid in the restoration in normal synaptic communication.
However, this point is largely moot with respect to the MS/VDB, as the results of my study demonstrate that memory impairment is not coupled to cholinergic cell loss.
6.2.3. Biochemistry and pharmacology of GABABRs in the hippocampus and prefrontal cortex of
aged rats
While previous investigations using models of rat neurocognitive aging have evaluated
the activity of GPCRs that couple to PI-hydrolysis, it is unknown whether GPCRs that couple to
other signaling cascades are similarly affected. Zhang et al (2007) also revealed that mAChR-
stimulated GTP-Eu binding exhibits interrelated decline between the hippocampus and PFC,
196 broadly suggesting that deficits are not exclusively evident in a single brain region. This latter
observation is critical from a therapeutic perspective as systemically administered agents will act
at sites across many brain regions, so a clear understanding of age-related changes to regionally-
defined pools of receptors can help form a better understanding of behavioral outcomes. In
Chapter IV, the data show that GABABR-stimulated GTP-Eu binding is preserved in the aged
hippocampus although aged-impaired rats express lower levels of the GABABR1 subunit
receptor protein. Interestingly, GABABR-stimulated GTP-Eu binding is significantly depressed
in the aged PFC due to decreased expression of GABABR proteins, although these reductions
are unrelated to spatial learning.
Chapter IV (and McQuail et al., 2012) is, apparently, the first and, to date, only
investigation of functional GABABR:G-protein coupling in a model of neurocognitive aging.
This observation is surprising given the interest in applying GABABR antagonists as therapeutic
agents to treat cognitive decline in aged rats, monkeys and humans (Froestl et al., 2004; Lasarge
et al., 2009). However, these compounds have not met with sufficient success in clinical trials
(Sabbagh, 2009), possibly because basic data needed to optimize the selection of ideal GABABR antagonists is lacking. The dynamic modulation of the GABABR complex is an important and
underappreciated issue. As the study presented in Chapter IV made use of the agonist baclofen,
this data is not necessarily representative of data obtained using GABA, the endogenous
neurotransmitter. Olianas et al (2005) reported that Ca2+ enhances the potency (i.e. a leftward
shift in the dose-response curve) of GABA, but not baclofen, to stimulate GTPγS-binding. From this one can assume that baclofen and GABA interact somewhat differently with the GABABR complex. Also of note, Ca2+ enhances CGP55845 antagonism of GABA-stimulated GTPγS- binding [i.e. a rightward shift in the dose-response curve; Olianas et al., (2005)]. These
197 observations are extremely interesting because, as mentioned in the introduction, neurons in the
aged hippocampus exhibited pronounced Ca2+ dysregulation (Gant et al., 2006; Thibault et al.,
2001), and CGP55845 can reverse odor-learning impairments in aged rats without disturbing performance of cognitively-intacted rats, young or aged (Lasarge et al., 2009). When considered together, a more complex model that examines interactions between GABA, Ca2+ and
CGP55845 may determine that Ca2+ dysregulation in hippocampal neurons of aged, cognitively
impaired rats aberrantly sensitizes GABABRs to activation by GABA and this mechanism is
selectively normalized by the application CGP55845. If this is the case, it may be that reduced
expression of GABABRs is a compensatory mechanism to decrease inhibitory signaling via this
pathway. A future study could utilize a hippocampal slice preparation to measure synaptically
evoked Ca2+ (using an indicator dye such as Calcium Green, see Chapter V) as well as the
evoked inward rectifying K+ current, which produces the slow inhibitory post-synaptic potential
(sIPSP) an electrophysiological parameter that is mediated by GABABRs (Lüscher et al., 1997).
Here, one would hypothesize that (1) an sIPSP produced prior to robust synaptic stimulation
would be much smaller than one produced after stimulation and that (2) neurons from aged-
impaired rats will exhibit greater Ca2+ dysregulation leading to more robust post-stimulation
sIPSPs but (3) these exaggerated responses should be reversed by application of CGP55845.
Subsequent receptor pharmacology studies could verify this effect by measuring the interactions
of GABA, Ca2+ and CGP55845 in a GTP-binding assay. While this model seems complex, such
a study design underscores the value of identifying and incorporating critical modulators of
GPCR function into assays that will reveal the mechanisms by which putative therapies manifest their beneficial effects.
198 6.2.4. Coupling of mAChRs to Gαq/11 and Gαo in the aged hippocampus
Chapter V (and McQuail et al., 2013) provides unique insight into the status of distinct
GPCRs and affiliated Gα-proteins. While a variety of studies have examined the effects of aging
on the ability of GPCRs to modulate PI turnover in the rat hippocampus (Ayyagari et al., 1998;
Chouinard et al., 1995; Nicolle et al., 1999; Parent et al., 1995; Tandon et al., 1991), few studies
have directly assessed whether impaired GPCR:Gα-protein coupling is the basis for this
functional deficit. Zhang et al. (2007) reported that age-related spatial learning impairment was
associated with lower oxotremorine-M-stimulated GTP-Eu binding in the hippocampus,
providing evidence that age-related deficiencies in signal transduction occur at the level of the
GPCR:Gα-protein complex. While this study offered a clear progression in our understanding of
deficits to GPCR-mediated signal transduction, it also begged new questions. Muscarinic AChR
and mGluRs present subclasses with different Gα-protein coupling (and therefore effector
coupling) profiles. Are deficits only apparent in the subset of receptors that couple to PI-
hydrolysis (i.e. Gαq/11)? As previous studies relied on the PI-hydrolysis assay, it was not possible to question whether Gαi/o receptors were altered with age or and whether any changes might
share a common basis. In Chapter V, data obtained via a modified version of the GTPγS-
binding assay determined that aging is associated with greater basal activity of Gαq/11,
although mAChR-mediated activation of Gαq/11 is not significantly changed by age , and no
differences in inhibitory G-protein (Gαo) activity were observed in the aged hippocampus.
The GTPγS-binding study presented in Chapter V was originally conceived to understand why maximal receptor-mediated PI-hydrolysis is lower in the hippocampi of aged-impaired rats whether stimulated via mAChRs or mGluRs. A simple hypothesis is that the effector proteins shared among various GPCRs are the culprit(s), leaving changes to the G-protein (Gαq/11) or its
199 downstream target, PLCβ1, to explain the deficit. However, there is no loss of Gαq/11 in the aged
hippocampus (Nicolle et al., 1999; Zhang et al., 2012) and while there is an age-related reduction
in PLCβ1 expression, it is not reliably associated with spatial learning (Nicolle et al., 1999),
therefore, protein levels are not sufficient to explain the relationship between blunted PI turnover
and spatial learning impairment. While Zhang et al (2007) reported a decrease in mAChR-
stimulated GTP-Eu binding in aged-impaired rats, suggesting that deficits associated with the G-
protein likely play a role, it was not possible to differentiate between mAChR coupling to Gαq/11
(M1-like) versus Gαo or Gαi (M2-like). However, when the activity of Gαq/11 is isolated, basal
GTPγS-binding to Gαq/11 is specifically increased with age while mAChR-stimulated binding
was similar between age groups. Importantly, this finding does not entirely undermine the initial
hypothesis. As levels of synaptic acetylcholine, and therefore stimulation of post-synaptic
mAChRs, fluctuate in response to synaptic activity associated with encoding, increased basal
activity of Gαq/11 may constrict the dynamic range of this window between basal and learning-
associated changes in mAChR activity. This assertion is strengthened by my data demonstrating an inverse correlation between basal and oxotremorine-M stimulated GTPγS-binding to Gαq/11; as basal activity increases, the maximal response decreases. However, as a number of receptors signal via Gαq/11, greater basal, or constitutive, activity would alter the signaling parameters of a
variety GPCRs, not just mAChRs. It is possible that the downregulation of PLCβ1 reported by
Nicolle et al (1999) is a compensatory mechanism to desensitize this pathway against persistent,
receptor-independent, activation by constitutively active Gαq/11. As the rats used in Nicolle et al
(1999) were somewhat older (26-27 month old LE rats) than those used in the current study, it
may be that increased constitutive activity of Gαq/11 preceeds a downregulation of PLCβ1 that
ultimately translates to impairments in signaling via GPCR-mediated PI-hydrolysis. This
200 progression is consistent with the observation of increased basal PI turnover in slightly
“younger” aged rats (24-25 month old LE rats) by Parent et al. (1995). Therefore, it may be that
some process affiliated with Gαq/11 is a manifesting event that elicits robust, but ultimately
maladaptive, changes in PI signaling. Future studies may seek to examine the basis for greater
constitutive activity Gαq/11. For example, it is understood that mutations to the α-chain of G-
proteins can impair intrinsic GTPase function leading to persistent, aberrant activity (Landis et
al., 1989). Once such mutant form of Gαq (Gαq(Q206L)) can increase basal GTP-binding while
lowering PLCβ1 expression in LβT2 pituitary cells (Liu et al., 2005). It would be useful to
explore the role of this mutant Gαq/11 in neuronal cell culture in vitro or use recombinant adeno -
associated virus delivery to enable the expression of this protein in hippocampal neurons in vivo
to assess effects on learning and memory.
6.2.5. GPCR-stimulated and store-related changes to intracellular Ca2+
Ca2+ is a critical intracellular signaling molecule that supports changes to synaptic
2+ strength and postsynaptic GPCRs can modulate [Ca ]i either via release of ICS from IP3R or by facilitating calcium-induced Ca2+ release (CICR), a process that involves membrane bound Ca2+
channels (NMDARs and VGCCs) that release ICS via RyRs. However, not all GPCRs modulate
all Ca2+ sources equally. Specifically, M1 mAChRs and mGluR1 will liberate ICS via IP3Rs
whereas mGluR5 potentiates NMDAR responses (Fernández de Sevilla et al., 2008; Mannaioni
et al., 2001). M1 does not directly interact with NMDARs, but can modulate AMPA receptors
which may in turn interact with NMDA signaling (Fernández de Sevilla and Buño, 2010).
2+ Therefore, the latter portion of Chapter V investigated age-related changes to [Ca ]i in response
to mAChR or Group I mGluR activation in the hippocampal CA1 region. The role of ICS was
tested by measuring receptor-elicited responses following depletion of ICS with cyclopiazonic
201 acid. The results of Chapter V demonstrate aging is associated with lower maximal elevation
2+ 2+ of [Ca ]i via mAChRs whereas Group I mGluR-stimulated [Ca ]i may be dysregulated,
2+ although both receptor systems demonstrate greater dependence on ICS to modulate [Ca ]i in
aged compared to young.
As mentioned at the end of Chapter V, there is currently a debate whether forms of plasticity that recruit ICS, including some that involve activation of GPCRs, are associated with preservation or deterioration of cognitive abilities in older rats (Boric et al., 2008; Kumar and
Foster, 2007, 2005; Kumar, 2010; Lee et al., 2005). Studies of plasticity can implicate a role for specific Ca2+ sources, but not necessarily address their functionality, and experimentally
2+ manipulated levels of divalent cations can occlude natural contributions of [Ca ]i to synaptic plasticity (reviewed in Burke and Barnes, 2010). However, the study design used in Chapter V
2+ did not pharmacologically isolate a specific signaling pathway; therefore changes to [Ca ]i likely reflected varying contributions of ICS released via IP3Rs and RyRs as well as Ca2+ influx
through NMDARs and VGCCs. If impaired PI signaling is a true correlate of cognitive aging
(Chouinard et al., 1995; Nicolle et al., 1999) and necessary to support adaptive plasticity in aged
2+ animals (Lee et al., 2005), then isolation of IP3R mediated changes to [Ca ]i should reveal a deficit in ICS release when stimulated with M1 mAChR and Group I mGluR agonists in the presence of compounds that block synaptic release (i.e. tetrodotoxin), NMDARs (APV) and
VGCCs (nifedipine). As the present hypothesis argues that deficits are upstream from PLC, then one should expect no difference between age or cognitive groups when IP3Rs are directly stimulated with IP3, consistent with the results obtained by Burnett et al (1990) showing preserved efficacy of Ca2+ release elicited by IP3 in hippocampal microsomes prepared from rats
ranging between 3 and 28 months of age. As mechanisms of plasticity shift with age (Boric et al.,
202 2008; Kumar and Foster, 2007; Kumar, 2010; Lee et al., 2005), it will be vital to demonstrate the
2+ 2+ Ca source, not just absolute [Ca ]i, is a key factor in the preservation of normal neural
plasticity.
A corollary to the previous question would involve assessing the relationship between
CICR and spatial learning impairment. While Thibault et al (2001) and Gant et al (2006) argue
that increased VGCC channel density leads to unchecked Ca2+ release via RyRs, Boric et al
(2008) revealed that VGCC-dependent LTP is enhanced in the brain of aged-unimpaired rats.
Reconciling these opposing interpretations of the consequences VGCC activation is complex. A key first step would be to determine whether aged-unimpaired rats exhibit the same degree of
CICR elicted by VGCC activation, or whether these rats express a mechanism to uncouple
VGCC Ca2+ entry from subsequent RyR activation, preserving synaptic transmission without triggering Ca2+ dyshomeostsis. Dysregulation of intracellular Ca2+ release is clearly detrimental
to plasticity; when aged slices are pretreated with compounds to block ICS release, LTP is readily induced, demonstrating that the age-related shift towards use of ICS ultimately occludes normal LTP (Kumar and Foster, 2004). However, this latter study did not take into the account the behavioral status of the aged rats or whether there was a more pronounced contribution of
VGCCs relative to NMDARS in LTP. Future studies will need to compare the contributions of
2+ NMDARs and VGCCs in the presence and absence of ICS-dependent changes to [Ca ]i across a
spectrum of cognitive abilities to determine how VGCCs may support beneficial plasticity
unaccompanied by dysregulation of intracellular Ca2+.
6.2.6. Parameters related to spatial learning
A major strength of the methods used in the current series of experiments was the initial evaluation of spatial learning for all rats used in neurobiological assessments. Not only does this
203 approach confirm that age-related learning impairments are evident in the experimental cohort,
but the computation of the “proximity score” provides summary measure of individual learning
ability that can be used to subdivide the aged cohort into unimpaired and impaired groups
relative to young or to perform correlational analyses to evaluate associations between
behaviorial and neurobiological data. Despite a number of significant differences between the
age groups, very few parameters were found to specifically associate with age-related cognitive
impairment.
In Chapter III, only aged-impaired rats were used to evaluate total number of ChAT+
neurons. This approach was employed to resolve a discrepancy between an earlier non-
stereological study that reported lower density of ChAT+ neurons in aged-impaired rats
(Baskerville et al., 2006) and another stereological investigation that found no loss of ChAT+
neurons in uncharacterized aged rats (Ypsilanti et al., 2008). Thus, in Chapter III, the use of only
an age-impaired cohort was justified to specifically dispute the notion that aged rats with
cognitive impairment lose septohippocampal projecting cholinergic neurons. However, this set of
specimens precluded the use of follow-up experiments that could have sought to measure other
cellular markers that could distinguish between aged-impaired and unimpaired rats.
In Chapter IV, expression of GABABR1 was selectively reduced in the hippocampi of aged rats with cognitive impairment relative to both young and cognitively unimpaired aged rats.
Interestingly, this reduction in receptor expression was not accompanied by a decrease in baclofen-stimulated GTP-Eu binding. Therefore, the mechanism by which decreased GABABR1
expression translates to altered hippocampal function and behavioral impairment is unclear. It is
possible that GABABR complexes undergo a change in the relative subunit composition which alters their intracellular binding parts, as GABABR1 and GABABR2 are known to differentially
204 interact with specific effectors (Guetg et al., 2010; Park et al., 2010). However, these findings
demonstrate the importance of combining traditional biochemical approaches, such as Western
blotting, with other functional analyses, like GTP-binding, to determine that protein expression is
not a sufficient surrogate for function. Importantly, the identification of the subgroup of aged-
impaired rats was vital to discover that GABABR1 expression is selectively reduced among these
rats, but not in the entire aged cohort.
In Chapter V, the relationship between the large number of neurobiological endpoints and
proximity score are presented in Table 5.S1. The associated discussion (Section 5.9) describes
how relationships between learning and memory may be assessed separate from the effects of
aging in this model. To illustrate, aged rats exhibited a trend (p<0.07) towards a significant
positive relationship between basal GTPγS-binding to Gαq/11 in DG and proximity score while
the full cohort showed a similar, significant relationship (greater basal activity was associated
with worse spatial learning). The inclusion of more animals lent greater power to the analysis,
especially the inclusion of animals with lower proximity scores (namely young rats). However, as age groups significantly differed with respect to proximity score and the main RMANOVA determined a significant difference between age groups with respect to basal GTPγS-binding to
Gαq/11, it is possible that this positive correlation is a statistical artifact of the main effects of age on both of the parameters being tested for association. To overcome this potential confound, the effect of age was partialled and the significant correlation survived this correction, demonstrating
that greater basal GTPγS-binding to Gαq/11 in DG is associated with worse spatial learning ability, albeit independent of age. However, this correction procedure does not artificially elevate the significance of all correlations that were evident in the combined cohort. To illustrate, a
2+ significant, negative correlation between peak oxotremorine-M stimulated [Ca ]i and proximity
205 score in the young and aged groups tested together was reduced below the level of the
significance after correcting for the effects of age. Thus, these examples demonstrate that
significant effects of learning and memory may exist independent of, or after correcting for, the
effects of age.
In conclusion, a primary strength of a model of neurocognitve aging is the use of
behavioral data to provide enhanced analyses of neurobiological endpoints beyond simple effects
of age (Baxter and Gallagher, 1996). The number of parameters that were reliably associated
with spatial learning impairment, either by subgrouping the aged cohort by cognitive status (in
Chapter IV) or through correlational analyses (in Chapter V), represents a small subset of the
total number of biological endpoints examined in this thesis. However, the reasoned use of
appropriate statistics can still reveal important relationships between brain and behavior.
Importantly, the data here demonstrate that aging is not process of widespread deterioration of
neurological functions; rather, behavioral impairments are the consequence of subtle alterations
to specific signaling molecules within the aged hippocampus.
6.3. Future Studies and Unresolved Questions
The studies presented in this thesis were designed to determine whether age-related
deficits in neurotransmission are due to loss of cells, changes to GPCRs or impaired modulation
of intracellular Ca2+. Broadly, the data indicated that activity at the level of the mAChR:G-
2+ protein complex or its modulation of [Ca ]i are significantly changed in the age brain, while
cholinergic cell number is not. The preceding discussion (Section 6.2) of the implications of
these findings also provided additional recommendations for the next logical steps to refine data collection to further these specific lines of research. However, there remain a number of larger
206 questions that could form entirely new lines of research that would provide vital complementary
data to enhance the understanding of the multitude of processes that interact with the parameters
studied in this thesis (behavior, morphology, pharmacology and physiology). The following
sections address broader questions or issues that are inspired by the current series of experiments
but would, if implemented, comprise largely divergent research projects.
6.3.1. Clarifying the sequence of neurobiological and behavioral changes
It is tacitly understood that neural changes drive, and therefore, precede, cognitive
deficits. Thus, the present approach to characterizing neurocognitive aging can only identify the
first signs of memory impairment, but not predict impending changes. Therefore, it would be most beneficial to examine changes that occur in middle-age, prior to the onset of behavioral
impairment, that lead to future cognitive decline. To illustrate, Murchison et al (2009) discovered
that basal forebrain cholinergic neurons of 18 month-old F344 rats exhibit enhanced Ca2+ buffering and then by 22 months of age, this enhancement is evident in aged-impaired rats, but not age-unimpaired rats. This leads one to speculate about the relationship between changes to the physiology of these neurons and subsequent changes to behavior. Do all middle-aged rats exhibit changes to Ca2+ buffering with a subset of rats recruiting an adaptive mechanism to
normalize Ca2+ buffering and maintain cognitive function? Conversely, are differences seen at
middle-age driven by a subset of rats that will become impaired because they are already
exhibiting failures to appropriately modulate neuronal physiology? Similarly, Gant et al. (2006) detected Ca2+ dyshomeostasis in CA1 neurons of 12 month-old F344 rats, a time-point that precedes the emergence of any spatial learning impairment in this strain (Bizon et al., 2009).
Thus, alterations to Ca2+ handling likely represent an early phase of neuron aging, but new
methods are needed to establish the relationship between these changes and future impairments,
207 emphasizing a need for longitudinal behavioral assessments. The water maze is not an ideal
apparatus for repeated, longitudinal testing; prior experience will inform or promote the guidance of spatial strategies, even if novel cues and platform locations are used. However, performance in an odor-learning task used in LaSarge et al (2007) was found to reliably correlate with spatial
learning ability in young and aged F344 rats, but, unlike the water maze, performance on the
odor-learning task did not improve across repeated administration of novel odor problems.
Therefore, this odor-learning task may serve as a surrogate marker of memory function in rats
that will undergo longitudinal assessment and enhance our understanding of behavioral changes
across the lifespan.
A truly longitudinal assessment of behavioral and neural changes in rodent models will
require either the use of non-terminal, non-invasive procedures that permit repeated sampling,
such as neuroimaging, that can assess a biomarker of interest (e.g. grey matter volume or
metabolites measured via proton nuclear magnetic resonance spectroscopy) or the development
of cross-correlated markers that serve as reasonable surrogates of measures that are traditionally
obtained via ex vivo approaches (e.g. fractional anisotropy of white matter tracts in vivo versus
osmium tetroxide staining of myelin fibers in histological sections). With improvements in brain
imaging technology (i.e. 7T MRI) or other innovative approaches to observe changes to the aged
brain in intact rats, it will be possible to form a precise understanding of how neural changes
precede behavioral impairment and select the appropriate animals to receive implement
preventative therapies.
208 6.3.2. Biochemical alterations and impaired cellular viability/functionality of cholinergic
neurons
Although Ypsilanti et al. (2008) and Chapter III (and McQuail et al., 2011), rule out loss of septohippocampal cholinergic neurons as mediators of cognitive decline, these studies do not exclude the possibility that other markers of cholinergic neurons change with age. To illustrate,
Greferath et al. (2000) found decreased numbers of p75 low-affinity neurotrophin receptor- immunopositive neurons in the MS/VDB. It is very interesting that this study used a marker of neurotrophic signaling specific to cholinergic neurons, as it is possible that these neurons continue to produce acetylcholine (i.e. are ChAT+), but are impaired with respect to trophic
signaling. As neurotrophin signaling is necessary to form and support connections between neurons, particularly across brain regions, age-related loss of neurotrophic support may be an initiating factor that destabilizes synapses, leading to neurochemical silencing and eventually
neuron loss. Therefore, enhancing neurotrophic signaling in cholinergic neurons may halt or
even reverse age-related changes that impair cholinergic neuron physiology and synaptic
function. Accordingly, Pelleymounter et al (1996) determined that intrahippocampal infusion of
nerve growth factor (NGF), but not brain-derived neurotrophic factor, improved spatial learning
in aged-impaired LE rats and increased high affinity choline uptake in the hippocampus. Thus,
further examination of NGF-mediated restoration of cholinergic function in aged-impaired rats
may provide a means to profile beneficial changes to cholinergic neurophysiology and allow for
the targeted development of more selective agents that mimic those effects.
Chapter III (and McQuail et al., 2011) also presents data demonstrating increased
expression of the CD68 marker of microglial activation, suggesting that the local
neuroinflammatory environment is elevated within the basal forebrain. Increased, or possibly
209 dysregulated, inflammation within basal forebrain is similar to that observed in the aged
hippocampus (Hua et al., 2012), but the relationship between inflammatory processes and spatial
learning in aged rats is complicated and unresolved (Nicolle et al., 2001; Sugaya et al., 1996;
VanGuilder et al., 2011a). However, it is likely that neuroinflammatory changes within the basal forebrain elicit changes in cholinergic neurons associated with increased metabolism leading to differential stress responses (Baskerville et al., 2008, 2006; McKinney et al., 2004; Personett et al., 2007). As neuroinflammatory processes occur throughout the aged brain, it is difficult assign a specific mechanism of action within a single brain region, let alone a specific cell type, suggesting that much more basic research is necessary to characterize the connection between age-related inflammation and subsequent responses by particular cell types in the basal forebrain.
Some work has focused on comparing cholinergic neurons within the basal forebrain to those in the brainstem, which are far less affected by aging and express a variety of different signaling pathways (Baskerville et al., 2008, 2006; McKinney et al., 2004; Personett et al., 2007).
However, these cell populations reside in vastly different brain regions and differential profiling of these two populations has not revealed new avenues for therapeutic modulation. As Bañuelos et al (2013) has identified differential effects of age and cognitive status on cholinergic and
GABAergic neurons in the basal forebrain, a more productive approach may entail complex molecular characterization of MS/VDB cholinergic neurons compared to adjacent GABAergic neurons. Such an approach could identify the differences that are most critical to the maintenance of normal neuron viability and function among these diverse populations of basal forebrain neurons that innervate the hippocampus. Once these basic neurobiological problems are more directly addressed, then it becomes possible to develop reasonable, mechanism-based approaches to appropriately modulate particular cell types.
210 6.3.3. Synaptic terminals and regulation by M2 mAChRs
Examining changes at the level of the cell body is not sufficient to characterize the
integrity of the cholinergic septohippocampal projection system. Ypsilanti et al. (2008) detemined that, despite preserved number of cholinergic cell bodies, there was a substantial reduction in the total length of cholinergic fibers in all 3 major subdivisions the of aged dorsal hippocampus. Although these animals were not assessed for spatial learning ability, it was presumed by those authors that reduced fiber length could drive impairments in hippocampal function. The status of presynaptic cholinergic markers in the aged hippocampus remains unresolved. Baxter et al (1999) determined that worse spatial learning is associated with greater hemicholinium-3 (HC-3) binding, a presynaptic cholinergic marker, in the dorsal CA2/3 of aged
F344 rats (but potentially greater in aged CA1 and unchanged in DG). However, Aubert et al
(1995) found no changes to HC-3 binding in any portion of the hippocampus between young, aged-unimpaired and aged-impaired rats although this report and related study (Quirion et al.,
1995) claimed there was an increase in M2 mAChR density in the DG-ML of aged-impaired rats.
This diverse assortment of outcomes suggests that there are both structural and biochemical changes to cholinergic synapses that differentially interact with cognitive function in the hippocampus of aged rats. Thus, it would be useful to develop a specific marker of cholinergic synapses that can be evaluated using a combination of biochemical and neuroanatomical approaches. Just as Smith et al (2000) used immunofluorescent staining for synaptophysin, a ubiquitous vesicular protein, to examine synapses within the hippocampus of aged rats, it would be useful to apply such a method to examine a vesicular marker that is specific to cholinergic synapses. Vesicular acetylcholine transporter (VAChT) is the protein that loads acetylcholine into vesicles for synaptic release (Weihe et al., 1996). A multi-level study that examines VAChT
211 protein levels using Western blot could then be extended into immunofluorescently labeled tissue
sections that are evaluated using either high resolution fluorometric scanning or stereological
analysis using confocal microscopy to measure changes to VAChT+ fibers. Thus, it would be possible to use this combination of approaches to examine a specific marker of cholinergic synapses in the hippocampus of young and aged rats to determine whether cognitive aging is associated with reduced hippocampal innervation by cholinergic synapses and/or reduced expression of synaptic markers necessary for normal cholinergic signaling. Furthermore, this approach may be combined with other markers, such as M2 mAChR immunolabeling, to determine whether cholinergic synapses are subject to greater muscarinic modulation. Such studies will provide vital information regarding the status of presynaptic cholinergic elements in the hippocampus to validate the appropriateness of therapies that rely on endogenous acetylcholine, including cholinesterase inhibitors and positive allosteric modulators.
Resolving the status of cholinergic synapses may provide additional information to enhance interpretation of the data presented in Chapter V detailing preserved mAChR coupling to Gαo in the aged hippocampus. Until now, investigations of neurocognitive aging have only been able to differentiate between M1 and M2 mAChRs at the level of ligand-binding, where
specific antagonists afford a greater degree of subtype selectivity. Such studies generally report
no change to M1 or M2 mAChR densities (Aubert et al., 1995; Smith et al., 1995), but at least
one study implicated greater M2 mAChR density in the DG-ML of aged-impaired rats and
improved spatial learning in rats treated with an M2 mAChR antagonist (Quirion et al., 1995).
However, it remains unclear whether such antagonists merely promote synaptic release of ACh
or reverse an M2 mAChR-related enhancement of neurotransmitter-limited synaptic release.
Data from Chapter V showed that mAChR-stimulation of Gαo, a G-protein associated with ion
212 channel regulation, is not changed with age or cognitive status in any of the major hippocampal
subdivisions. Therefore, it seems most likely that behavioral benefits observed in aged rats
receiving M2 antagonists are associated with greater release of ACh which then acts primarily
via postsynaptic mechanisms, not by correcting age-related changes to M2 receptors directly.
However, the current study could not evaluate the functional status of specific synapses that are
controlled by M2 mAChRs. Importantly, mAChRs also act as heteroreceptors that modulate the release of glutamate from CA3 neurons, limiting release of SC terminals on CA1 but also controlling excitation within the CA3 (Kremin and Hasselmo, 2007). Parsing the contributions of mAChR modulation to cholinergic and glutamatergic synapses in aging presents a challenge as one cannot readily differentiate between these synapses in traditional assays of receptor pharmacology. However, simultaneous recording of CA3 and CA1 neuron activity either during electrically evoked stimulation of BF afferents or behavioral activity in the presence of specific mAChR compounds, may reveal the dynamics of hippocampal activity that are selectively modulated by cholinergic function. As reviewed, in Wilson et al (2006), neurocognitve aging is associated with a shift in the highly non-linear input-output relationship of the CA3 leading to inappropriate recall of irrelevant information, while the CA1, which exhibits a more linear, graded input-output curve, remains normal. Using this pattern as template, a beneficial compound should, theoretically, suppress activity in the CA3 while simultaneously promoting synaptic transmission to the CA1. Whether this pattern may be achieved via M2 mAChR activation remains to be determined.
213 6.7 Conclusion
Aging is a complex biological process with variable consequences for neural function leading to a wide range of individual differences in learning and memory among older individuals. Using a translationally relevant rat model of neurocognitive aging, the present series of experiments sought to demonstrate that age-related changes to hippocampal function (1) are
not caused by neurodegeneration, rather they are associated with (2) selective changes in
hippocampal post-synaptic GPCR:Gα-subunit coupling that (3) alter modulation of
intracellular physiological processes. Using a combination of neuroanatomical, pharmacological
and physiological methods that focused predominantly on the cholinergic system there is now
sufficient evidence to propose the following mechanisms (illustrated in Fig 6.1 and 6.2) to
explain, in part, age-related changes in hippocampal function:
1. Cholinergic neuron number is not a relevant parameter to assess age-related changes
to hippocampal-dependent cognition; rather changes at cholinergic synapses (markers
of cholinergic innervation, pre- and post-synaptic cholinergic receptor
expression/functionality) within the hippocampus are more relevant to cognition in
normal aging
2. Age-related impairments in GPCR-linked PI hydrolysis are due, in part, to greater
constitutive activity of Gαq/11 subunits whereas the activity of mAChR-Gαo linked signal
transduction is unchanged within the hippocampus
3. Down-regulation of mAChR signal transduction via PLCβ1 leads to insufficient
release of Ca2+ from IP3R-gated intracellular stores and maladaptive recruitment of
other Ca2+ sources (NMDARs and VGCCs) triggering Ca2+ dyshomeostasis that
disrupts synaptic plasticity
214 While many differences were found between young and aged rats, fewer measures were reliably associated with impaired spatial learning. However, the age and strain selected in these studies emphasizes the earliest detection of age-related changes to hippocampal-dependent cognition, so it is possible that change is ongoing in these animals and small neurobiological effects interact in more a complex manner to produce impairments in neural functioning that underlie behavioral impairment. However, as many of the processes investigated in this report rely on activation of GPCRs, it will be possible to apply these findings in the optimization of the next generation of pharmacotherapies that operate via this diverse group of targets to restore cognitive function in older individuals.
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225
2+ Figure 6.1. Summaryr of changees to GPCRs and modulation of [Ca ]i at ageed hippocampal synapses.
226
Figure 6.2. Proposed model linking current findings to changes in synaptic plasticity.
Increased basal activity of Gαq/11 in the aged hippocampus is a biochemical parameter that may link deterioration in direct, GPCR-stimulated control of intracellular Ca2+ stores (ICS; via
2+ 2+ PLCβ1 and IP3Rs) with dysregulated Ca -induced Ca release (via VGCCs and RyRs). This shift in pathways that stimulate particular sources of ICS-release may underlie the age-dependent shift towards increased long-term depression (LTD) and impaired long-term potentiation (LTP)
227 APPENDIX
1. McQuail JA, Nicolle MM (2012) Animal Models of Aging and Cognition. Current
Translational Geriatrics and Experimental Gerontology Reports. 1:21-28
2. License Agreement for reproduction of Neurobiology of Aging 32(12):2322.e1–2322.e 4,
2011
3. License Agreements for reproduction of Neurobiology of Aging 33(6):1124.e1–1124.e12,
2012
4. License Agreements for reproduction of Neuropharmacology 70:64–73, 2013
5. License Agreements for reproduction of Current Translational Geriatrics and
Experimental Gerontology Reports. 1:21-28, 2012
228 Curr Tran Geriatr Gerontol Rep DOI 10.1007/s13670-011-0002-1
COGNITIVE FUNCTION AND AGING (DAVE MORGAN, SECTION EDITOR)
Animal Models of Aging and Cognition
Joseph A. McQuail & Michelle M. Nicolle
# Springer Science+Business Media, LLC 2012
Abstract Human aging is associated with variable rates of Hippocampus . Medial temporal lobe . Entorhinal cortex . cognitive decline. Aged laboratory animals, specifically rats Basal forebrain . Prefrontal cortex . Striatum . Reelin . and monkeys, show similar cognitive profiles when tested Acetylcholine . Calcium buffering . Dendritic spine . on species-appropriate behavioral tasks. Using these cogni- Afterhyperpolarization . Magnetic resonance imaging tively defined animal models, we now understand that nor- mal aging is associated with disconnections between functionally related brain regions that support specific Introduction behaviors. We review recent work describing molecular and cellular mechanisms that lead to decreased connections With improvements in the standard of living and medical between the aged hippocampus and associated cortical and innovation extending the human lifespan, more individuals basal forebrain neurons required for normal memory. We will live to older ages. Consequently, the number of Amer- also discuss behavioral evidence that aging leads to im- icans over the age of 65 years will grow from about 40 paired executive functions with concurrent changes to key million today to almost 90 million by 2050, and will make neural substrates within the frontostriatal network. Collec- up a larger fraction of our population, growing from 13% to tively, these studies demonstrate that cognitive deficits in 20% [1]. These demographic projections have important older individuals are related to modest, circuit-specific alter- ramifications to the neuroscience research community be- ations and that data obtained from such animal models can cause aging is associated with decreases in a number of help to better inform the design of future clinical studies and cognitive domains, most notably memory [2]. However, improve neuropsychiatric outcomes for older patients. age alone does not accurately predict cognitive ability, as older individuals exhibit a broad range of performance on Keywords Brain . Aging . Cognitive function . Cognitive neuropsychological tests. Some present with no or minimal aging . Cognition . Animal model . Memory . Spatial decline; others demonstrate moderate, but selective, deficits; learning . Morris water maze . Executive function . Reversal and the remainder display severe loss of function [3]. It is the learning . Delay discounting . Working memory . interaction of chronological (or biological) aging with cogni- tive function that gives rise to the concept of “cognitive aging” J. A. McQuail (Fig. 1). Furthermore, subtle losses in cognitive function may Program in Neuroscience, Wake Forest University, predispose individuals to more severe outcomes. About 15% 1 Medical Center Boulevard, of those over the age of 70 years are diagnosed with mild Winston-Salem, NC 27157, USA cognitive impairment (MCI), presenting with memory deficits M. M. Nicolle (*) that are worse than expected even after correcting for age and Department of Internal Medicine, Section on Gerontology level of education [4], and these patients are at increased risk and Department of Physiology & Pharmacology, for decline into dementia, most commonly Alzheimer’sdisease Wake Forest University School of Medicine, (AD) [5]. 1 Medical Center Boulevard, Winston-Salem, NC 27157, USA This article reviews the findings of several recent reports e-mail: [email protected] that examine alterations to neurobiological substrates of
229 Curr Tran Geriatr Gerontol Rep
model the effects of aging on both cognition and brain substrates to address research questions of translational rel- evance to humans. The Morris water maze is a well- described apparatus for testing learning and memory in rats. The classical place-learning, or “hidden-platform,” task is hippocampal-dependent, and aged rats (ie, 22–28 months of age, depending on the strain used) are impaired on this task relative to young controls (usually 4–6 months old) [6–9]. Importantly, when comparing performance of individual young and aged rats, there is greater variability in aged individuals; some aged rats exhibit performance similar to young rats while other aged rats are significantly worse than young rats [6]. This pattern of results closely approximates the cognitive aging phenotype conceptualized for aging Fig. 1 The interaction between chronological or biological aging and humans (Fig. 1) and is apparent across the three strains of cognitive function (“cognitive aging”). Cognitive function (shown on the rats most commonly used in aging research: Long-Evans, left y-axis) generally declines with age (shown on the x-axis), but age Fischer 344 (F344), and F344×Brown Norway (FBN) F1 alone is not sufficient to predict cognitive performance across all individ- Hybrids [10]. By using this translationally meaningful meth- uals of the same age because cognitive outcomes are more variable with advancing age. Some older individuals exhibit little or no cognitive od to sort rats based upon behavioral criteria, rodent models impairment relative to younger individuals. Other individuals will present of cognitive aging can more effectively identify changes to with modest, but significant, deficits in some cognitive domain, usually the aged brain that have a significant relationship to cognition, memory. Generally, these deficits are associated with selective changes to not merely age. specific circuits. As cognitive deficits emerge in older individuals, there is increased risk for further decline into dementia, most frequently Alzheimer’s disease, which is associated with neuropathology including Entorhinal Cortical Neurons and Memory Impairment neuron loss and brain atrophy. However, such diseases are distinct from normal aging, not an inevitable consequence. Even with improved clin- The entorhinal cortex (EC) provides most cortical input to ical evaluations (right y-axis), the boundary that distinguishes moderate impairment from progressive neuropathology remains unclear (denoted the hippocampus [11] and is an early locus of neuronal loss by the color transitions of the background: green 0 normal; orange 0 mild in patients with AD [12]. In a rodent model of cognitive impairment/at risk for dementia; red 0 probable dementia) aging, there is a loss of perforant path inputs to the hippo- campus formed by layer II EC neurons specifically in aged rats with cognitive impairment [13]. The rodent pattern of cognitive aging in animal models. We first focus on the denervation is similar to that observed in MCI patients [14] medial temporal lobe network, discussing changes to corti- and provides an excellent model for identification of factors cal and basal forebrain neurons that innervate the hippocam- that render this projection vulnerable in aging. A current pus to support normal spatial learning. We then transition to hypothesis stipulates that a loss of synaptic viability and a discussion of the frontostriatal system that supports be- plasticity leads to subsequent denervation. Reelin, a glyco- havioral flexibility and decision making. Finally, findings protein, is involved in synaptogenesis [15] and neuroplas- obtained from aged rodents will be extended by reviewing ticity [16] and is ideally positioned to influence perforant neurobiological assessments in aged monkeys obtained via path connections because layer II EC neurons are among the traditional postmortem techniques, as well as in vivo brain few excitatory cell populations to express this protein [17]. imaging studies that bear closer resemblance to clinical Recently, Stranahan and colleagues [18••] reported that lay- approaches used in humans. er II of the EC of aged rats with cognitive impairment contains significantly fewer reelinergic neurons than young or aged rats with preserved spatial learning. In addition to Neurobiological Changes in Aged Rats with Spatial hippocampal projections, these neurons form synapses with- Learning Impairment in the EC and these connections are also lost in aged- impaired rats. Therefore, not only does the aged-impaired Assessing Reference Memory in Aged Rats hippocampus receive less highly processed cortical input via the perforant path but remaining projections are likely less Reference (or episodic) memory is largely dependent upon regulated as cortical synaptic integrity is compromised. the integrity of the hippocampus and associated medial The work of Stranahan and colleagues [18••, 19••]isan temporal lobe (MTL) structures, which are present in all excellent example of a descriptive study that used an animal mammalian species, indicating that rodents can be used to model of cognitive aging to inform further hypothesis-
230 Curr Tran Geriatr Gerontol Rep driven studies. Using insight from the cognitive aging mod- [27•] reported reduced cholinergic fiber length in the aged el, the importance of reelin to normal learning and memory hippocampus, suggesting a loss of hippocampal cholinergic was validated in a follow-up study by the same research synapses. Decreased connectivity between BF cholinergic group, demonstrating that intra-entorhinal infusion of re- neurons and hippocampus is reminiscent of lost perforant combinant receptor–associated protein (RAP), which blocks path connections, and similarly invites future investigations reelin from binding to its cognate receptor, produces signif- to identify the mechanisms that mediate this effect. Previ- icant spatial learning impairments in young rats [19••]. ously, it was reported that memory-impaired aged rats Furthermore, RAP infusion decreased phosphorylation of expressed fewer p75-containing neurons in the BF [29]. disabled-1, reelin’s intracellular target, and decreased ex- This observation is significant because p75 is a nerve pression of the synaptic marker synaptophysin. Collectively, growth factor receptor exclusive to cholinergic neurons these studies demonstrate that reelin is a key molecular within the BF, demonstrating selective loss of trophic sup- component of normal MTL cortical function, and its loss, port [30]. Although p75 expression was lowest in impaired either age-associated or experimentally induced, triggers aged rats, the relationship between the BF cholinergic sys- profound synaptic loss and spatial-learning deficits. tem and spatial learning is complicated because selectively removing cholinergic fibers from the hippocampus does not Cholinergic Basal Forebrain System and Memory induce spatial impairment in young rats [31, 32]. Impairment The failure to recapitulate cognitive impairment via cho- linergic lesion suggests that additional factors impact cho- Acetylcholine-producing neurons located in the basal fore- linergic function in the aged BF. Calcium dyshomeostasis brain (BF) give rise to fibers traveling via the fornix that forms a central theme in cognitive aging, especially consid- terminate throughout the hippocampus [20]. Importantly, ering that calcium regulates a variety of signaling cascades, the release of acetylcholine controls switching between modulates membrane excitability, and is necessary for syn- memory formation and retrieval processes in the hippocam- aptic plasticity [33]. With this in mind, calcium buffering is pus [21] and acetylcholine release is phase-locked with significantly increased in BF cholinergic neurons from aged hippocampal theta oscillations [22], suggesting that normal learning-impaired F344 rats, whereas aged rats without im- cholinergic activity is closely linked to plasticity and learn- pairment have lower calcium buffering values similar to ing. The loss of cholinergic cell bodies in the BF is well young rats [34••]. Perhaps most interesting is that these established in AD [23]. Initially, it was presumed that a changes in calcium buffering can be observed in middle- similar loss was associated with normal aging; however, aged rats (13 months) that do not yet exhibit behavioral these early studies that reported a loss of BF cholinergic impairments. This suggests that changes to calcium buffer- neurons in aging utilized profile-based counting methods ing within cholinergic neurons precede the manifestation of [24–26]. Profile-based methods rely on a number of geo- cognitive deficits, or that some middle-aged rats recruit metric assumptions that may confound comparisons be- adaptive mechanisms to regulate intracellular calcium and tween age groups. In contrast, stereological approaches protect against subsequent cognitive decline. Collectively, make no such assumptions, but incorporate rigorous sam- these studies demonstrate that cholinergic degeneration is pling methodologies to achieve reliable estimates of total not a requisite feature of cognitive aging, but these neurons cell number. Recently, stereological investigations have may form fewer connections with the hippocampus and challenged the notion of BF cholinergic neuron loss in exhibit selective changes in calcium buffering that may, in normal aging. Ypsilanti and colleagues [27•] reported that part, explain age-related cognitive deficits. there was no change in total numbers of cholinergic neurons in the BF in 24-month-old versus 6-month-old F344 rats. Subsequently, McQuail and colleagues [28••] reported that Evaluating Age-Related Changes to Executive Function this same cell population is also preserved in 28-month-old and Associated Brain Substrates in Aged Rats FBN rats with spatial learning impairment. Importantly, because stereological techniques were employed in both Evaluating Executive Function in Aged Rats studies, it is significant that each reported strikingly similar numbers of cholinergic cells, thus validating both the same Executive function encompasses a number of cognitive pattern of results as well as the total estimates between these processes dependent on the prefrontal cortex (PFC) that two strains. involve behavioral control, organization, and adaptation, Although the stereological evidence indicates that BF including working memory, decision making, and behavior- cholinergic neurons do not degenerate with age, ongoing al flexibility. While not as widely studied in humans as work suggests that other parameters of this cell population memory deficits, neuropsychological testing has revealed may contribute to cognitive aging. Ypsilanti and colleagues executive function diminishes with increasing age [2, 35].
231 Curr Tran Geriatr Gerontol Rep
Although not without debate, rodent models of executive functional alterations to muscarinic receptors, further studies function have been described [36, 37] and are being applied are necessary to better elucidate the mechanisms that disrupt to the study of aging in rats [38, 39••, 40, 41••]. Because normal frontostriatal activity and associated behaviors, such there is no clear consensus on task parameters or specific as set shifting in aged rats. components of executive function within the context of rodent cognitive aging, we will review the novel behavioral Decision Making in Aged Rats results reported in two studies examining reversal learning and decision making, respectively. Temporal considerations often play a prominent role in decision making, particularly when one must choose be- Reversal Learning and Muscarinic Function in Aged Rats tween a very large reward that is not received until after a lengthy delay and an immediate smaller reward. From a Nieves-Martinez and colleagues [39••] examined reversal behavioral economics perspective, the subjective value of learning in young (12 months), middle-aged (21 months), the larger reward is diminished as the delay to receive the and aged (29 months) FBN rats using an attentional set- reward increases. This phenomenon is termed “delay dis- shifting task where rats must dynamically monitor and mod- counting.” While children and adolescents are more likely to ify their response strategy in light of changing task rules. In select the immediate smaller reward than adults as the delay all stages, rats obtained food rewards hidden in pots scented is increased [43], less is known with respect to how aged with specific odorants (first dimension) and filled with dis- adults would behave in these tasks. Furthermore, it is not tinct digging media (second dimension). In the first stage of known if differences between younger and older adults are this task, rats learned to discriminate between two odors (eg, due to differences in subjective life experiences that may lemon vs clove), where one (lemon) consistently identified impact decision making or if aging modulates the neurolog- the baited pot while the other (clove) was consistently ical circuitry that controls decision making. The aged rat is unbaited. The digging medium is irrelevant and variable an ideal model to test the latter hypothesis because it is across testing. All rats regardless of age can reliably learn possible to control for life experiences, even between age this rule; however, when the rule is reversed and the clove groups, to an extent that is not possible in humans. To assess scent signaled food reward, aged rats required more trials the effects of age on delay discounting, Simon and col- than young or middle-aged rats to learn this rule reversal. In leagues [41••] presented young and aged F344 rats with a the last phase, the digging medium (eg, shredded paper or choice between a small (one pellet) but immediate food Easter grass), which was never previously associated with reward or a large (four pellets) food reward presented after the food reward, became the salient signal for reward and a delay of up to 60 s. Interestingly, aged rats did not odor was made irrelevant. Similar to the odor reversal, aged “discount” the value of the larger food reward to the same rats also were impaired at shifting between stimulus dimen- extent as young rats even as the delay increased; aged rats sions (ie, odor to digging medium); aged rats again required consistently showed a preference to select the larger food significantly more trials to reestablish performance criteria reward up to the very longest delay, while younger rats than young or middle-aged rats. began to favor smaller rewards even after the introduction Following the completion of their behavioral studies, of relatively brief delays (10–20 s). These rodent data sug- Nieves-Martinez and colleagues [39••]analyzedmuscarinic gest that aging likely modulates the neural circuitry neces- receptors within the striatum, which interconnects with PFC, sary for decision making because rats, particularly those fed and found decreased receptor activity, without changes to ad libitum within commercial and laboratory colonies, have receptor density, in the 24-month-old group. The timing and minimal prior experience that would shape performance in localization of these neurobiological changes suggest that such a task. However, Simon and colleagues [41••] did not deterioration in muscarinic striatal signaling is a factor in analyze tissue from the rats used in this study, leaving age-related reversal deficits. Other neurobiological alterations interested readers to speculate as to the specific mechanisms have been observed in frontostriatal circuitry in aged rats that drive this shift in decision making in older rats. assessed in a similar manner, including changes in glutamate receptor binding [42]. Specifically, while both kainate and N- methyl-D-aspartate (NMDA)–receptor densities are decreased Changes in Cortical Function and Connectivity in the striatum of aged rats with impaired set-shifting ability, in Aged Monkeys with Working Memory Deficits the relationship between receptor density and set shifting was unique for each subtype; higher kainite binding was associat- Measuring Working Memory in Aged Monkeys ed with fewer errors while higher NMDA binding was asso- ciated with more errors. Given the divergent results observed While investigations of frontal cortex have become more for these two glutamate receptor subtypes as well as the tractable in rodents with the development of more refined
232 Curr Tran Geriatr Gerontol Rep behavioral testing methodologies, nonhuman primates, spe- were, on average, larger in the aged dlPFC than those found in cifically rhesus macaques, are translationally valuable to the the young dlPFC. Most significantly, the lower density, as study of cognitive aging owing to the greater homologies well as larger size, of “thin” spines in the aged dlPFC was between human and monkey brains, especially with respect highly correlated with poorer acquisition of the DNMS task. to cortical size and organization. The primate dorsolateral Thus, neurons in the aged-impaired dlPFC contain fewer prefrontal cortex (dlPFC) is critical for normal working modifiable spines and the loss of these most plastic substrates memory. Working memory in monkeys can be measured limits the efficacy of working memory processes. using the delayed response (DR) test, where monkeys must Plasticity is dependent on not only spine morphology, but remember the location of a cue over the course of a brief also neuronal activity. After normal learning, the afterhyper- delay before response selection. In contrast, recognition polarization (AHP), a calcium-dependent current that brings memory is often assessed in monkeys using a delayed the membrane potential to a hyperpolarized state after firing non-matching to sample (DNMS) task. In this task, an action potential and modulates spiking frequency, is re- monkeys view a sample object, then, after a short delay, duced to allow enhanced cell activity [51]. Traditional elec- the monkey must select a novel object presented alongside trophysiological approaches have revealed that the AHP is the sample object to complete the trial and receive a food increased in the hippocampus of aged rats with cognitive reward. While DR and DNMS may be respectively associated impairment [52], but similar data from PFC are scarce. To with working memory and recognition memory (and their address this shortcoming, Luebke and Amatrudo [53••]per- affiliated neural circuits), this distinction is not complete be- formed in vitro recording experiments using tissue from cause MTL lesions may impair DR performance [44]andthe young and aged monkeys to determine whether the AHP dlPFC is apparently necessary for DNMS acquisition [45, 46••, and other electrophysiological parameters of neurons in the 47••]. However, data obtained from these tasks consistently dlPFC are changed by age and associated with working mem- demonstrate working memory decline in monkeys starting after ory. Importantly, parameters of layer III neurons, which con- 20 years of age [48, 49]. By using tissue from monkeys trained nect to other cortical regions, were contrasted with layer V on these tasks, two recent publications have shed light on neurons, which connect primarily to subcortical regions. Us- changes to the structure and physiology of dlPFC neurons in ing this strategy, Luebke and Amatrudo [53••] found that the aged monkeys that may explain age-related impairments in AHP was significantly increased in layer III neurons, and working memory. greater increases in the AHP current were associated with worse working memory performance. While an increase in Prefrontal Neurons and Working Memory Impairment AHP was initially hypothesized to decrease cell firing, the firing rate of layer III neurons was actually increased in aged While we have previously discussed evidence demonstrating monkeys and not related to AHP. These divergent results that long projection pathways (perforant path and fornix) suggest that some other factor, such as intracellular calcium deteriorate with age, the effects of lost input on postsynaptic (which can regulate both cell excitability as well as AHP targets also warrant investigation. Dumitriu and colleagues amplitude), may influence these parameters. Interestingly, [46••] offer evidence that working memory deficits are strong- these robust changes apparent in layer III neurons of aged ly associated with layer-specific changes to a particular sub- monkeys were not present in layer V; this layer-specific effect class of dendritic spines. Spines are specialized postsynaptic may indicate that circuit-level changes in the aged PFC play structures that contain a high concentration of ionotropic some role in the manifestation of enhanced AHP. glutamate receptors, and their morphology and receptor con- The morphological data of Dumitriu and colleagues [46••] tent are modulated by presynpatic activity. Specifically, demonstrate that aged dlPFC is less amenable to modification smaller, thinner spines are more plastic and motile and express by “thin” spine loss, and Luebke and Amatrudo [53••]present greater levels of NMDA receptors, while larger spines are congruent physiologic evidence that dlPFC neurons exhibit more stable and express a greater proportion of AMPA (α- enhanced AHP, rendering these neurons less adaptive to sub- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) recep- sequent activity-dependent modulation. The significance of tors [50]. The existence of the more plastic “thin” spines is these alterations to normal cognition was supported in each theorized to render the dlPFC more amenable to behavioral study by reliable associations between age-related changes to modification; conversely, larger spines have been stabilized “thin” spine density or AHP and severity of working memory by prior experience and resist alteration. Using a combination deficits. of traditional postmortem histological techniques, Dumitriu and colleagues [46••] observed that there was a significant Assessing Brain Systems and Working Memory Impairment age-related reduction in spine density within the dlPFC, but this reduction was specific to “thin” spines; the larger “mush- The anatomical similarities between the human and monkey room” spines were not affected. Additionally, “thin” spines brain not only facilitate translational research through ex
233 Curr Tran Geriatr Gerontol Rep vivo morphological and physiological assessments, but also PFC across hemispheres and to other cortical regions, it is permit the use of brain imaging technology identical to that notable that both studies found that indices of myelin damage, employed in clinical settings for human patients. Human either DT-MRI or histologically-derived, were associated with studies of aging generally require large numbers of partic- impairments in working memory [55, 56••]. ipants to achieve reliable results separating true effects of aging from confounds, including health factors, lifestyle, and other experiences that can affect clinical outcomes. It Conclusions is possible to control for many of these factors in aged monkeys and, therefore, conduct well-powered imaging Even in the absence of any overt neurological illness, aging studies using fewer subjects while yielding results that can is associated with a decline in cognitive capacities. The directly inform human aging studies and clinical practice. severity of this decline is highly variable, with some indi- Perhaps of greatest interest, the use of noninvasive in vivo viduals showing little evidence of loss and others presenting brain imaging techniques opens the door to future longitu- with more obvious deficits. Despite species differences, dinal studies of brain structure and behavior that could not many of the key neural circuits are present in traditional be accomplished using the ex vivo approaches that have laboratory animals, including rodents and monkeys, and traditionally defined neurological assessments of the aged species-appropriate tasks have revealed age-related declines brain. in cognitive function similar to humans. Using these cogni- Using structural magnetic resonance imaging (MRI), tively defined models, we now appreciate that normal aging Shamy and colleagues [47••] compared regional volumes is free from widespread neurodegeneration. However, we do between young and aged monkeys and determined the observe a selective loss of synapses and connections and dlPFC and striatum both are smaller in aged monkeys. changes to neuron physiology that compromise the func- Furthermore, smaller volumes in both of these regions were tions of brain networks that support normal cognition. These significantly associated with poorer working memory. This subtle changes may, in turn, predispose the aged brain to the study elegantly demonstrates that normal aging is not asso- subsequent manifestation of pathological conditions such as ciated with widespread cerebral atrophy; rather, specific AD. However, in the absence of pathology, the more modest brain regions are more sensitive to the aging process. Inter- changes observed in nonpathological aging also may pro- estingly, the brain regions that were identified in these aged vide a window of opportunity to intervene and prevent monkeys, PFC and striatum, comprise the frontostriatal cognitive decline. This latter consideration underscores the network that is also compromised in aged rodents [39••, need for future work to develop novel therapies that better 42]. However, disruptions to the integrity of neural networks apply this knowledge regarding the mechanistic basis of are related to changes not only in grey matter, but also the cognitive aging. white matter (WM) fiber tracts that contain axons that con- vey information between brain regions. Shamy and col- leagues [47••] revealed that a tendency toward decreased frontal WM volume in older monkeys was associated with Disclosures No potential conflicts of interest relevant to this article poorer DNMS performance. Similarly, Wisco and col- were reported. leagues [54•] reported decreased frontal WM volume in aged monkeys, although this reduction was not related to cognitive impairment. Despite general agreement, core dif- References ferences in the conclusions of these studies warrant a more refined analysis of WM integrity in the aged PFC. Accord- ingly, Makris and colleagues [55] utilized diffusion tensor Papers of particular interest, published recently, have been MRI (DT-MRI), which measures the directional diffusion of highlighted as: • water along myelinated axons, to evaluate the integrity of Of importance, •• discrete fiber pathways that connect with the PFC. It was Of major importance found that the integrity of the superior longitudinal fascicu- lus, the cingulum bundle, and anterior corpus callosum were 1. United States Census Bureau: 2009 U.S. Population Projections. Available at: http://www.census.gov/population/www/projections/ compromised in older monkeys. This DT-MRI evidence was 2009projections.html. Accessed November 2011. complimented by a subsequent histological analysis that 2. Salthouse TA. Are individual differences in rates of aging greater reported both a decrease in myelinated axon density and at older ages? Neurobiol Aging. 2011; In press. increased frequency of myelin degeneration in the cingulum 3. Christensen H, Mackinnon AJ, Korten AE, et al. An analysis of •• diversity in the cognitive performance of elderly community and anterior corpus callosum of aged monkeys [56 ]. Be- dwellers: individual differences in change scores as a function of cause each of these fiber pathways serves to interconnect the age. Psychol Aging. 1999;14:365–79.
234 Curr Tran Geriatr Gerontol Rep
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A re-examination of the trix molecules and synaptic plasticity: immunomapping of intra- role of basal forebrain cholinergic neurons in spatial working cellular and secreted reelin in the adult rat brain. Eur J Neurosci. memory. Neuropharmacology. 1998;37:481–7. 2006;23:401–22. 32. Fletcher BR, Baxter MG, Guzowski JF, et al. Selective cholinergic 18. •• Stranahan AM, Haberman RP, Gallagher M. Cognitive decline is depletion of the hippocampus spares both behaviorally induced associated with reduced reelin expression in the entorhinal cortex Arc transcription and spatial learning and memory. Hippocampus. of aged rats. Cereb Cortex. 2011; 21:392–4. This article reports a 2007;17:227–34. loss of reelinergic entorhinal neurons specific to aged rats with 33. Foster TC. Calcium homeostasis and modulation of synaptic plasticity cognitive impairment. Given prior work describing lost perforant in the aged brain. Aging Cell. 2007;6:319–25. path connections arising from the entorhinal cortex in aged- 34. •• Murchison D, McDermott AN, Lasarge CL, et al. Enhanced impaired rats, the authors argue that reelin may be a central calcium buffering in F344 rat cholinergic basal forebrain neurons molecule in mediating this effect. is associated with age-related cognitive impairment. J Neurophy- 19. •• Stranahan AM, Salas-Vega S, Jiam NT, et al. Interference with siol. 2009; 102:2194–207. This paper used electrophysiological reelin signaling in the lateral entorhinal cortex impairs spatial techniques to determine that calcium buffering is enhanced in memory. Neurobiol Learn Mem. 2011; 96:150–5. Having previ- basal forebrain cholinergic neurons of aged-impaired rats, while ously observed decreased reelin in the entorhinal cortex of aged aged-unimpaired rats had buffering capacity similar to young. rats with spatial learning impairment, the authors of this paper Furthermore, the calcium-buffering enhancement was observed demonstrate that experimentally blocking reelin activity in the ento- in middle-aged rats that do not exhibit spatial learning deficits, rhinal cortex of young rats will induce age-like spatial learning suggesting that alterations to calcium buffering precede overt deficits. Similarly, decreased reelin activity also triggers synapse loss cognitive impairment. within the entorhinal cortex. 35. Salthouse TA. What cognitive abilities are involved in trail-making 20. Amaral DG, Kurz J. An analysis of the origins of the cholinergic performance? Intelligence. 2011;39:222–32. and noncholinergic septal projections to the hippocampal forma- 36. Chudasama Y. 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38. Barense MD, Fox MT, Baxter MG. Aged rats are impaired on an aged rhesus monkeys. Cereb Cortex. 2011; 21:1559–73. This attentional set-shifting task sensitive to medial frontal cortex damage paper used MRI to measure regional volumes throughout the entire in young rats. Learn Mem. 2002;9:191–201. primate brain. They found selective reductions in prefrontal and 39. •• Nieves-Martinez E, Haynes K, Childers SR, et al. Muscarinic striatal volumes that correlated with working memory receptor/G-protein coupling is reduced in the dorsomedial striatum performance. of cognitively impaired aged rats. Behav Brain Res. 2011. This 48. Herndon JG, Moss MB, Rosene DL, et al. Patterns of cognitive paper examined cognitive flexibility, a core component of executive decline in aged rhesus monkeys. Behav Brain Res. 1997;87:25–34. function, in aged rats and observed age-related impairments in 49. Moore TL, Killiany RJ, Herndon JG, et al. Executive system reversal-learning and set shifting. 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This zation in hippocampal CA1 neurons covaries with spatial learning paper investigated age-related changes in decision making, another ability in aged Fisher 344 rats. J Neurosci. 2005;25:2609–16. important aspect of executive function. The observations strongly 53. •• Luebke JI, Amatrudo JM. Age-related increase of sI(AHP) in suggest that aging, separate from subjective life experiences, modu- prefrontal pyramidal cells of monkeys: relationship to cognition. lates decision making, possibly in a circuit-dependent fashion. Neurobiol Aging. 2011. This paper reported increased AHP am- 42. Nicolle MM, Baxter MG. Glutamate receptor binding in the frontal plitude in layer III neurons of aged monkeys is associated with cortex and dorsal striatum of aged rats with impaired attentional worse working memory performance. Increased AHP was not set-shifting. Eur J Neurosci. 2003;18:3335–42. related to cell firing, which also was increased in aged monkeys. 43. Green L, Myerson J, Lichtman D, et al. Temporal discounting in These data reveal layer-specific changes in prefrontal neuron choice between delayed rewards: the role of age and income. activity, although the basis for the changes warrants additional Psychol Aging. 1996;11:79–84. investigation. 44. Zola-Morgan S, Squire LR, Amaral DG. Human amnesia and the 54. • Wisco JJ, Killiany RJ, Guttmann CR, et al. An MRI study of age- medial temporal region: enduring memory impairment following a related white and gray matter volume changes in the rhesus monkey. bilateral lesion limited to field CA1 of the hippocampus. J Neurosci. Neurobiol Aging. 2008; 29:1563–75. This paper used structural MRI 1986;6:2950–67. to reveal that prefrontal grey and white matter volumes are reduced 45. Peters A, Sethares C, Luebke JI. Synapses are lost during aging in in aged monkeys. However, these reductions were not specifically the primate prefrontal cortex. Neuroscience. 2008;152:970–81. associated with working memory impairment. 46. •• Dumitriu D, Hao J, Hara Y, et al. Selective changes in thin spine 55. Makris N, Papadimitriou GM, van der Kouwe A, et al. Frontal density and morphology in monkey prefrontal cortex correlate with connections and cognitive changes in normal aging rhesus aging-related cognitive impairment. J Neurosci. 2011; 30:7507– monkeys: a DTI study. Neurobiol Aging. 2007;28:1556–67. 15. This paper reported a selective loss of thin spines in the 56. •• Bowley MP, Cabral H, Rosene DL, et al. Age changes in prefrontal cortex of aged monkeys with working memory impair- myelinated nerve fibers of the cingulate bundle and corpus cal- ment. In contrast, mushroom spines, which resist alteration, are losum in the rhesus monkey. J Comp Neurol. 2010; 518:3046–64. not lost with age. The authors argue that the loss of thin spines is This paper used electron microscopy to directly evaluate changes critical for executive function because these spines are highly to myelinated fibers in monkey frontal white matter pathways. In plastic and likely support novel rule-learning. aged monkeys, the density of myelinated fibers was decreased 47. •• Shamy JL, Habeck C, Hof PR, et al. Volumetric correlates of while signs of myelin degeneration were increased. This paper spatiotemporal working and recognition memory impairment in provides useful histological evidence to support DTI findings.
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Supplier Elsevier Limited The Boulevard,Langford Lane Kidlington,Oxford,OX5 1GB,UK Registered Company 1982084 Number Customer name Joseph McQuail Customer address Wake Forest University Winston-Salem, NC 27157 License number 3096060353223 License date Feb 25, 2013 Licensed content publisher Elsevier Licensed content publication Neurobiology of Aging Licensed content title GABABreceptor GTP-binding is decreased in the prefrontal cortex but not the hippocampus of aged rats Licensed content author Joseph A. McQuail,Cristina Bañuelos,Candi L. LaSarge,Michelle M. Nicolle,Jennifer L. Bizon Licensed content date June 2012 Licensed content volume 33 number Licensed content issue 6 number Number of pages 12 Start Page 1124.e1 End Page 1124.e12 Type of Use reuse in a thesis/dissertation Intended publisher of new other work Portion full article Format electronic Are you the author of this Yes Elsevier article? Will you be translating? No Order reference number
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Supplier Elsevier Limited The Boulevard,Langford Lane Kidlington,Oxford,OX5 1GB,UK Registered Company 1982084 Number Customer name Joseph McQuail Customer address Wake Forest University Winston-Salem, NC 27157 License number 3096060434948 License date Feb 25, 2013 Licensed content publisher Elsevier Licensed content publication Neuropharmacology Licensed content title Hippocampal Gαq/11but not Gαo-coupled receptors are altered in aging Licensed content author Joseph A. McQuail,Kathleen N. Davis,Frances Miller,Robert E. Hampson,Samuel A. Deadwyler,Allyn C. Howlett,Michelle M. Nicolle Licensed content date July 2013 Licensed content volume 70 number Licensed content issue number Number of pages 11 Start Page 63 End Page 73 Type of Use reuse in a thesis/dissertation Intended publisher of new other work Portion full article Format electronic Are you the author of this Yes Elsevier article? Will you be translating? No Order reference number
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Title of your Normal Aging and Cognition: System-Specific Changes to G-Protein thesis/dissertation Coupled Receptor-Mediated Signal Transduction within the Hippocampus Expected completion date May 2013 Estimated size (number of 250 pages) Elsevier VAT number GB 494 6272 12 Permissions price 0.00 USD VAT/Local Sales Tax 0.0 USD / 0.0 GBP Total 0.00 USD Terms and Conditions
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License Number 3096480355827 License date Feb 26, 2013 Licensed content publisher Springer Licensed content publication Current Translational Geriatrics & Experimental Gerontology Reports Licensed content title Animal Models of Aging and Cognition Licensed content author Joseph A. McQuail Licensed content date Jan 1, 2012 Type of Use Thesis/Dissertation Portion Full text Number of copies 5 Author of this Springer Yes and you are the sole author of the new work article Order reference number Title of your thesis / Normal Aging and Cognition: System-Specific Changes to G-Protein dissertation Coupled Receptor-Mediated Signal Transduction within the Hippocampus Expected completion date May 2013 Estimated size(pages) 250 Total 0.00 USD Terms and Conditions
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Curriculum Vitae
256
JOSEPH ALOYSIUS MCQUAIL
Program in Neuroscience • Wake Forest University Medical Center Boulevard • Winston-Salem, NC 27157 PHONE: (336) 713-1515 • FAX: (336) 716-8603 • EMAIL: [email protected]
EDUCATION
2013 Ph.D. Neuroscience, Wake Forest University (Expected) Dissertation: Normal aging and cognition: system-specific changes to G-protein coupled receptor-mediated signal transduction within the hippocampus (Mentor: Michelle M. Nicolle, Ph.D.) 2004 B.S. Neuroscience with Highest Honors, College of William & Mary Honors Thesis: Effects of cholinergic receptor antagonists on sustained attention performance in the rat (Mentor: Joshua A. Burk, Ph.D.)
PROFESSIONAL AND RESEARCH EXPERIENCE
2007-2013 Graduate Research Student in the Laboratory of Michelle M. Nicolle, Ph.D., Department of Internal Medicine – Section on Gerontology, Department of Physiology & Pharmacology and Program in Neuroscience, Wake Forest University 2006-2007 Research Assistant in the Laboratory of Jessica A. Mong, Ph.D., Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine 2004-2006 Senior Laboratory Technician in the Laboratory of Michela Gallagher, Ph.D., Department of Psychological and Brain Sciences, Johns Hopkins University 2003-2004 Honors Research Student in the Laboratory of Joshua A. Burk, Ph.D., Department of Psychology, College of William & Mary
PROFESSIONAL MEMBERSHIPS
2004-Present Society for Neuroscience 2007-Present Western North Carolina Society for Neuroscience
ACADEMIC AND PROFESSIONAL AWARDS
2008 Alumni Travel Award, Wake Forest University Graduate School of Arts & Sciences 2007-2008 Trainee, Wake Forest University NIH Predoctoral Training Program in Neuroscience (T32-NS007422) 2004 Highest Honors in Neuroscience, College of William & Mary
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RESEARCH GRANTS, FELLOWSHIPS AND OTHER SUPPORT
Oxidative damage to receptor:G-protein coupling in the aged hippocampus 8/1/2011-7/31/2013 F31-AG038266 – Ruth L. Kirschstein Pre-Doctoral National Research Service Award National Institute on Aging, National Institutes of Health This fellowship supports pre-doctoral training to evaluate the relationship between deficits in cholinergic, glutamatergic and GABAergic G-protein coupled receptor-mediated signal transduction, oxidative stress and behavioral impairments in aged rats. Role: P.I.
RESEARCH ARTICLES
McQuail JA, Davis KN, Miller F, Hampson RE, Deadwyler SA, Howlett AC, Nicolle MM (2013) Hippocampal Gαq/11 but not Gαo-coupled receptors are altered in aging. Neuropharmacology. 70:63-73. Hua K, Schindler MK, McQuail JA, Forbes ME, Riddle DR (2012) Regionally distinct responses of microglia and glial progenitor cells to whole brain irradiation in adult and aging rats. PLoS ONE 7(12): e52728. Blutstein T, Castello MA, Viechweg SS, Hadjimarkou MM, McQuail JA, Holder M, Thompson LP, Mong JA (2012 Nov 29 [Epub ahead of print]) Differential responses of hippocampal neurons and astrocytes to nicotine and hypoxia in the fetal guinea pig. Neurotoxicity Research. DOI: 10.1007/s12640-012-9363-2. Bañuelos C, LaSarge CL, McQuail JA, Hartman JJ, Gilbert RJ, Ormerod BK, Bizon JL (2013) Age-related changes in rostral basal forebrain cholinergic and GABAergic projection neurons: Relationship with spatial impairment. Neurobiology of Aging. 34(3):845-862. McQuail JA, Bañuelos C, LaSarge CL, Nicolle MM, Bizon JL (2012) GABAB receptor GTP- binding is decreased in the prefrontal cortex but not the hippocampus of aged rats. Neurobiology of Aging. 33(6):1124.e1–1124.e12. Sergeant S, McQuail JA, Riddle DR, Chilton FH, Ortmeier S, Jessup JA, Groban L, Nicolle MM (2011) Dietary fish oil modestly attenuates the effect of age on diastolic dysfunction but has no effect on memory or brain inflammation in aged rats. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences. 66(5):521-33. McQuail JA, Riddle DR, Nicolle MM (2011) Neuroinflammation not associated with cholinergic degeneration in aged-impaired brain. Neurobiology of Aging. 32(12):2322.e1–2322.e 4. McQuail JA, Burk JA (2006) Evaluation of muscarinic and nicotinic receptor antagonists on attention and working memory. Pharmacology, Biochemistry and Behavior. 85(4):796- 803.
INVITED REVIEW
McQuail JA, Nicolle MM (2012) Animal Models of Aging and Cognition. Current Translational Geriatrics and Experimental Gerontology Reports. 1:21-28.
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NATIONAL AND INTERNATIONAL CONFERENCE PRESENTATIONS
McQuail JA, Davis KN, Horman BM, Bass CE, Nicolle MM (2012) Hippocampal phospholipase Cβ1 is necessary for rapid acquisition of spatial information. 42nd Annual Meeting of the Society for Neuroscience. New Orleans, LA, USA. Bañuelos C, Beas BS, McQuail JA, Gilbert RJ, Setlow B, Bizon JL (2012) GABAB receptor blockade enhances delayed match-to-sample working memory performance in aged but not young rats. 42nd Annual Meeting of the Society for Neuroscience. New Orleans, LA, USA. McQuail JA, Sink KM, Nicolle MM, Peiffer AM, Alzheimer’s Disease Neuroimaging Initiative (2012) Correcting for volumetric changes reveals novel patterns of glucose metabolism in Alzheimer’s disease brain. Alzheimer’s Association International Conference 2012. Vancouver, BC, Canada. McQuail JA, Davis KN, Miller F, Hampson RE, Deadwyler SA, Howlett AC, Nicolle MM (2011) Functional coupling of muscarinic receptors to Gαq/11 in the aged hippocampus. 41st Annual Meeting of the Society for Neuroscience. Washington, DC, USA. McQuail JA, Bañuelos C, LaSarge CL, Gilbert RJ, Bizon JL, Nicolle MM (2010) Baclofen- mediated GTP-binding is decreased in the prefrontal cortex, but not hippocampus, of aged F344 rats. 40th Annual Meeting of the Society for Neuroscience. San Diego, CA, USA. Nicolle MM, Watson ML, McQuail JA, Sambamurti K, Brady AE, Jones C, Conn PJ, Lindsey CW (2009) Allosteric potentiation of the muscarinic M1 receptor restores reversal learning impairment in a transgenic mouse model of Alzheimer’s disease. 39th Annual Meeting of the Society for Neuroscience. Chicago, IL, USA. McQuail JA, Riddle DR, Nicolle MM (2009) Microglial activation but no loss of cholinergic neurons in basal forebrain of aged-impaired F344 × Brown Norway rats. 39th Annual Meeting of the Society for Neuroscience. Chicago, IL, USA. Nicolle MM, Watson ML, McQuail JA, Sambamurti K, Brady AE, Jones C, Conn PJ, Lindsey, CW (2009) Acute administration of a novel M1 receptor allosteric potentiator (BQCA) restores reversal learning in a transgenic mouse model of Alzheimer’s disease (Tg2576). Alzheimer’s Association 2009 International Conference on Alzheimer’s Disease. Vienna, Austria. Comberiate Jr MA, McQuail JA, Anant P, Hadjimarkou MM, Blutstein T, Holder MK, Thompson L, Mong, JA (2008) Neural inflammation induced by prenatal nicotine exposure. 38th Annual Meeting of the Society for Neuroscience. Washington, DC, USA. McQuail JA, Schindler MK, Riddle DR (2008) The neuroinflammatory response of the adult rat hippocampus to whole brain irradiation is age-dependent and sub-region specific. 38th Annual Meeting of the Society for Neuroscience. Washington, DC, USA. Koh MT, Haberman RP, McQuail JA, Hoyt EC, Lund PK, Gallagher M (2006) Benefit in aged rats with cognitive impairment by treatment strategies targeting hippocampal hyperfunction. 36th Annual Meeting of the Society for Neuroscience. Atlanta, GA, USA. McQuail JA, Burk JA (2004) Effects of muscarinic and nicotinic receptor antagonists on sustained attention in the rat. 34th Meeting of the Society for Neuroscience. San Diego, CA, USA.
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EDITORIAL AND REVIEW ACTIVITIES
Ad hoc reviewer for Cerebral Cortex (with David R. Riddle, Ph.D.)
TEACHING EXPERIENCE
Wake Forest University Graduate School NEUR 701: Introduction to Neuroscience I, Neuroanatomy (Graduate Teaching Assistant under the direction of Mary Lou Voytko, Ph.D.)
MENTORSHIP OF UNDERGRADUATE RESEARCH STUDENTS
James P. Hibble (B.S., Biology/Neuroscience, 2013, Wake Forest Univ.) 1/2012-5/2013 Bradley D. Shugoll (B.S., Biophysics/Neuroscience, 2013, Wake Forest Univ.) 1/2012-5/2013
INSTITUTIONAL SERVICE AND COMMUNITY OUTREACH
2009-2012 Graduate School Honor Panel, Neuroscience Program Representative, Wake Forest University 2009-2010 Orientation Aid, Neuroscience Program, Wake Forest University 2008-2009 Neuroscience Teaching Award, Class Representative, Wake Forest University 2008-2010 Brain Awareness Council, Student Volunteer, Wake Forest University
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