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1.  Modulation of Higher Primate Adrenal Androgen Secretion with Estradiol or Estradiol and Progesterone Intervention 
Menopause (New York, N.Y.)  2013;20(3):10.1097/GME.0b013e318273a070.
Background
Circulating adrenal steroids rise during the menopausal transition (MT) in most mid-aged women and may contribute to differences in between-woman symptoms as well as ultimate health outcomes. However, the mechanism(s) for this shift in adrenal steroid production in mid-aged women is not known.
Objective
To determine if hormone replacement therapy (HT) for one year can modulate adrenal androgen production.
Method
Younger (9.8 +/− 0.4 y/o, n=20) and older (22.7+/−0.4 y/o, n=37) female laboratory macaques were ovariectomized (OVX), and then each group was treated with different regimens of HT for up to one year. Changes in adrenal histology and circulating adrenal androgens were monitored following estradiol treatment alone (E) or estrogen plus progesterone (E+P), and these changes were compare to the same measures in similar aged animals given vehicle (V).
Results
Zona reticularis (ZR) area and serum dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) were higher in younger compared to older V-treated animals (P< 0.02). Both E and E+P treatments decreased circulating DHEAS in the younger group (P<0.05). While E also decreased DHEAS in the older group, this was not statistically significant. In contrast, E+P treatment in the older group resulted in a rise in DHEAS over V, which was significantly higher than the results of E alone (p< 0.01). Circulating concentrations of DHEA exhibited similar trends but these changes did not reach statistical significance.
Conclusion
These data demonstrate that intervention with ovarian steroids can modulate adrenal androgen production in female higher primates and that both animal age and type of HT regimen determines the adrenal response.
doi:10.1097/GME.0b013e318273a070
PMCID: PMC3610787  PMID: 23435030
Adrenal; Steroids; Hormone Therapy; Replacement
2.  NMDA Receptors Subserve Persistent Neuronal Firing During Working Memory In Dorsolateral Prefrontal Cortex 
Neuron  2013;77(4):736-749.
Summary
Neurons in the primate dorsolateral prefrontal cortex (dlPFC) generate persistent firing in the absence of sensory stimulation, the foundation of mental representation. Persistent firing arises from recurrent excitation within a network of pyramidal Delay cells. Here, we examined glutamate receptor influences underlying persistent firing in primate dlPFC during a spatial working memory task. Computational models predicted dependence on NMDA receptor (NMDAR) NR2B stimulation, and Delay cell persistent firing was abolished by local NR2B NMDAR blockade or by systemic ketamine administration. AMPA receptors (AMPAR) contributed background depolarization to sustain network firing. In contrast, many Response cells -which likely predominate in rodent PFC- were sensitive to AMPAR blockade and increased firing following systemic ketamine, indicating that models of ketamine actions should be refined to reflect neuronal heterogeneity. The reliance of Delay cells on NMDAR may explain why insults to NMDARs in schizophrenia or Alzheimer’s Disease profoundly impair cognition.
doi:10.1016/j.neuron.2012.12.032
PMCID: PMC3584418  PMID: 23439125
3.  Morphological and molecular changes in aging rat prelimbic prefrontal cortical synapses 
Neurobiology of aging  2012;34(1):200-210.
Age-related impairments of executive functions appear to be related to reductions of the number and plasticity of dendritic spine synapses in the prefrontal cortex (PFC). Experimental evidence suggests that synaptic plasticity is mediated by the spine actin cytoskeleton, and a major pathway regulating actin-based plasticity is controlled by phosphorylated LIM kinase (pLIMK). We asked whether aging resulted in altered synaptic density, morphology, and pLIMK expression in the rat prelimbic region of the PFC. Using unbiased electron microscopy, we found a ~50% decrease in the density of small synapses with aging, while the density of large synapses remained unchanged. Postembedding immunogold revealed that pLIMK localized predominantly to the postsynaptic density where it was increased in aging synapses by ~50%. Furthermore, the age-related increase in pLIMK occurred selectively within the largest subset of prelimbic PFC synapses. Since pLIMK is known to inhibit actin filament plasticity, these data support the hypothesis that age-related increases in pLIMK may explain the stability of large synapses at the expense of their plasticity.
doi:10.1016/j.neurobiolaging.2012.05.014
PMCID: PMC3483440  PMID: 22727942
Aging; synapse; prefrontal cortex; actin; electron microscopy; postembedding immunogold; perforated synapse; postsynaptic density; structural plasticity
4.  Neuronal and morphological bases of cognitive decline in aged rhesus monkeys 
Age  2011;34(5):1051-1073.
Rhesus monkeys provide a valuable model for studying the basis of cognitive aging because they are vulnerable to age-related decline in executive function and memory in a manner similar to humans. Some of the behavioral tasks sensitive to the effects of aging are the delayed response working memory test, recognition memory tests including the delayed nonmatching-to-sample and the delayed recognition span task, and tests of executive function including reversal learning and conceptual set-shifting task. Much effort has been directed toward discovering the neurobiological parameters that are coupled to individual differences in age-related cognitive decline. Area 46 of the dorsolateral prefrontal cortex (dlPFC) has been extensively studied for its critical role in executive function while the hippocampus and related cortical regions have been a major target of research for memory function. Some of the key age-related changes in area 46 include decreases in volume, microcolumn strength, synapse density, and α1- and α2-adrenergic receptor binding densities. All of these measures significantly correlate with cognitive scores. Interestingly, the critical synaptic subtypes associated with cognitive function appear to be different between the dlPFC and the hippocampus. For example, the dendritic spine subtype most critical to task acquisition and vulnerable to aging in area 46 is the thin spine, whereas in the dentate gyrus, the density of large mushroom spines with perforated synapses correlates with memory performance. This review summarizes age-related changes in anatomical, neuronal, and synaptic parameters within brain areas implicated in cognition and whether these changes are associated with cognitive decline.
doi:10.1007/s11357-011-9278-5
PMCID: PMC3448991  PMID: 21710198
Aging; Area 46; Dentate gyrus; Executive function; Recognition memory; Perforated synapse
5.  Session II: Mechanisms of Age-Related Cognitive Change and Targets for Intervention: Neural Circuits, Networks, and Plasticity 
Age-related changes in neural circuits, neural networks, and their plasticity are central to our understanding of age changes in cognition and brain structure and function. This paper summarizes selected findings on these topics presented at the Cognitive Aging Summit II. Specific areas discussed were synaptic vulnerability and plasticity, including the role of different types of synaptic spines, and hormonal effects in the dorsolateral prefrontal cortex of nonhuman primates, the impact of both compensatory processes and dedifferentiation on demand-dependent differences in prefrontal activation in relation to age and performance, the role of vascular disease, indexed by white matter signal abnormalities, on prefrontal activation during a functional magnetic resonance imaging-based cognitive control paradigm, and the influence of amyloid-β neuropathology on memory performance in older adults and the networks of brain activity underlying variability in performance. A greater understanding of age-related changes in brain plasticity and neural networks in healthy aging and in the presence of underlying vascular disease or amyloid pathology will be essential to identify new targets for intervention. Moreover, this understanding will assist in promoting the utilization of existing interventions, such as lifestyle and therapeutic modifiers of vascular disease.
doi:10.1093/gerona/gls111
PMCID: PMC3732094  PMID: 22570135
cognitive changes; aging; Neural networks
6.  Clinically relevant hormone treatments fail to induce spinogenesis in prefrontal cortex of aged female rhesus monkeys 
Preclinical animal models have provided strong evidence that estrogen therapy (ET) enhances cognition and induces spinogenesis in neuronal circuits. However, clinical studies have been inconsistent, with some studies revealing adverse effects of ET, including an increased risk of dementia. In an effort to bridge this disconnect between the preclinical and clinical data, we have developed a non-human primate (NHP) model of ET combined with high-resolution dendritic spine analysis of dorsolateral prefrontal cortical (dlPFC) neurons. Previously, we reported cyclic ET in aged, ovariectomized NHPs increased spine density on dlPFC neurons. Here, we report that monkeys treated with cyclic E treatment paired with cyclic progesterone (P), continuous E combined with P (either cyclic or continuous), or unopposed continuous E failed to increase spines on dlPFC neurons. Given that the most prevalent form of ET prescribed to women is a combined and continuous E and P, these data bring into convergence the human neuropsychological findings and preclinical neurobiological evidence that standard hormone therapy in women is unlikely to yield the synaptic benefit presumed to underlie the cognitive enhancement reported in animal models.
doi:10.1523/JNEUROSCI.1881-12.2012
PMCID: PMC3657730  PMID: 22915112
7.  EFFECTS OF ESTROGEN AND AGING ON THE SYNAPTIC DISTRIBUTION OF PHOSPHORYLATED AKT-IMMUNOREACTIVITY IN THE CA1 REGION OF THE FEMALE RAT HIPPOCAMPUS 
Brain research  2010;1379:98-108.
The estrogen 17β-estradiol (E) increases the axospinous synaptic density and plasticity in the hippocampal CA1 region of young female rats but fails to do so in aged female rats. This E stimulus on synaptic plasticity is associated with the phosphorylation-dependent activation of Akt kinase. Our previous findings demonstrated that increased estrogen levels subsequently increase phosphorylated Akt (pAkt)-immunoreactivity (-IR) within the dendritic shafts and spines of pyramidal neurons in young female rats. Therefore, because Akt can promote cell survival and growth, we tested the hypothesis that the less plastic synapses of aged female rats would contain less E-stimulated pAkt-IR. Here, young (3-4 months) and aged (22-23 months) female rats were ovariectomized seven days prior to a 48-hour administration of either vehicle or E. The pAkt-IR synaptic distribution was then analyzed using post-embedding electron microscopy. In both young and aged rats, pAkt-IR was found in dendritic spines and terminals, and pAkt-IR was particularly abundant at the post-synaptic density. Quantitative analyses revealed that the percentage of pAkt-labeled synapses was significantly greater in young rats compared to aged rats. Nonetheless, E treatment significantly increased pAkt-IR in pre- and post-synaptic profiles of both young and aged rats, although the stimulus in young rats was notably more widespread. These data support the evidence that hormone-activated signaling associated with cell growth and survival is diminished in the aged brain. However, the observation that E can still increase pAkt-IR in aged synapses presents this signaling component as a candidate target for hormone replacement therapies.
doi:10.1016/j.brainres.2010.07.053
PMCID: PMC3657736  PMID: 20709039
8.  The Aging Cortical Synapse: Hallmarks and Implications for Cognitive Decline 
Nature reviews. Neuroscience  2012;13(4):240-250.
Normal aging is associated with impairments in cognitive function, including memory, and with specific and relatively subtle synaptic alterations in the hippocampus and prefrontal cortex. The authors describe these structural changes reported in monkeys and rodents, how they might affect age-associated cognitive decline and potential strategies to limit their impact.
doi:10.1038/nrn3200
PMCID: PMC3592200  PMID: 22395804
9.  Clinically Relevant Hormone Treatments Fail to Induce Spinogenesis in Prefrontal Cortex of Aged Female Rhesus Monkeys 
The Journal of Neuroscience  2012;32(34):11700-11705.
Preclinical animal models have provided strong evidence that estrogen (E) therapy (ET) enhances cognition and induces spinogenesis in neuronal circuits. However, clinical studies have been inconsistent, with some studies revealing adverse effects of ET, including an increased risk of dementia. In an effort to bridge this disconnect between the preclinical and clinical data, we have developed a nonhuman primate (NHP) model of ET combined with high-resolution dendritic spine analysis of dorsolateral prefrontal cortical (dlPFC) neurons. Previously, we reported cyclic ET in aged, ovariectomized NHPs increased spine density on dlPFC neurons. Here, we report that monkeys treated with cyclic E treatment paired with cyclic progesterone (P), continuous E combined with P (either cyclic or continuous), or unopposed continuous E failed to increase spines on dlPFC neurons. Given that the most prevalent form of ET prescribed to women is a combined and continuous E and P, these data bring into convergence the human neuropsychological findings and preclinical neurobiological evidence that standard hormone therapy in women is unlikely to yield the synaptic benefit presumed to underlie the cognitive enhancement reported in animal models.
doi:10.1523/JNEUROSCI.1881-12.2012
PMCID: PMC3657730  PMID: 22915112
10.  High throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy 
Nature protocols  2011;6(9):1391-1411.
Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high resolution confocal microscopy, deconvolution and image analysis using NeuronStudio. Recent technical advancements include better preservation of tissue resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual Z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically for the identification of both optimal resolution as well as highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing 3 experimental groups with 8 subjects in each can take as little as one month if optimized for speed, or approximately 4 to 5 months if the highest resolution and morphometric detail is sought.
doi:10.1038/nprot.2011.389
PMCID: PMC3566769  PMID: 21886104
microinjection; dendritic spines; confocal microscopy; morphometrics; image analysis
11.  Synaptic correlates of memory and menopause in the hippocampal dentate gyrus in rhesus monkeys 
Neurobiology of aging  2010;33(2):421.e17-421.e28.
Aged rhesus monkeys exhibit deficits in hippocampus-dependent memory, similar to aging humans. Here we explored the basis of cognitive decline by first testing young adult and aged monkeys on a standard recognition memory test (delayed nonmatching-to-sample test; DNMS). Next we quantified synaptic density and morphology in the hippocampal dentate gyrus (DG) outer (OML) and inner molecular layer (IML). Consistent with previous findings, aged monkeys were slow to learn DNMS initially, and they performed significantly worse than young subjects when challenged with longer retention intervals. Although OML and IML synaptic parameters failed to differ across the young and aged groups, the density of perforated synapses in the OML was coupled with recognition memory accuracy. Independent of chronological age, monkeys classified on the basis of menses data as peri/post-menopausal scored worse on DNMS, and displayed lower OML perforated synapse density, than pre-menopausal monkeys. These results suggest that naturally occurring reproductive senescence potently influences synaptic connectivity in the DG OML, contributing to individual differences in the course of normal cognitive aging.
doi:10.1016/j.neurobiolaging.2010.09.014
PMCID: PMC3031995  PMID: 21030115
delayed nonmatching-to-sample; disector method; estrogen; hippocampus; menopause; outer molecular layer; perforated synapse; post-synaptic density; recognition memory
12.  Synaptic distributions of GluA2 and PKM ζ in the monkey dentate gyrus and their relationships with aging and memory 
The Journal of Neuroscience  2012;32(21):7336-7344.
Rhesus monkeys provide a valuable model for studying the neurobiological basis of cognitive aging, because they are vulnerable to age-related memory decline in a manner similar to humans. In this study, young and aged monkeys were first tested on a well-characterized recognition memory test (delayed nonmatching-to-sample; DNMS). Then, electron microscopic immunocytochemistry was performed to determine the subcellular localization of two proteins in the hippocampal dentate gyrus (DG): the GluA2 subunit of the glutamate alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate (AMPA) receptor and the atypical protein kinase C ζ isoform (PKMζ). PKMζ promotes memory storage by regulating GluA2-containing AMPA receptor trafficking. Thus, we examined whether the distribution of GluA2 and PKMζ is altered with aging in DG axospinous synapses and whether it is coupled with memory deficits. Monkeys with faster DNMS task acquisition and more accurate recognition memory exhibited higher proportions of dendritic spines coexpressing GluA2 and PKMζ. These double-labeled spines had larger synapses, as measured by postsynaptic density area, than single- and unlabeled spines. Within this population of double-labeled spines, aged monkeys compared to young expressed a lower density of synaptic GluA2 immunogold labeling, which correlated with lower recognition accuracy. Additionally, higher density of synaptic PKMζ labeling in double-labeled spines correlated with both faster task acquisition and better retention. Together, these findings suggest that age-related impairment in maintenance of GluA2 at the synapse in the primate hippocampus is coupled with memory deficits.
doi:10.1523/JNEUROSCI.0605-12.2012
PMCID: PMC3391702  PMID: 22623679
AMPA receptor; delayed nonmatching-to-sample test; GluR2; immunogold; PKMζ; recognition memory
13.  Subregional, dendritic compartment, and spine subtype specificity in cocaine-regulation of dendritic spines in the nucleus accumbens 
The Journal of Neuroscience  2012;32(20):6957-6966.
Numerous studies have found that chronic cocaine increases dendritic spine density of medium spiny neurons in the nucleus accumbens (NAc). Here, we employed single cell microinjections and advanced 3D imaging and analysis techniques to extend these findings in several important ways: by assessing cocaine regulation of dendritic spines in the core versus shell subregions of NAc in the mouse, over a broad time course (4 hours, 24 hours, or 28 days) of withdrawal from chronic cocaine, and with a particular focus on proximal versus distal dendrites. Our data demonstrate subregion-specific, and in some cases opposite, regulation of spines by cocaine on proximal but not distal dendrites. Notably, all observed density changes were attributable to selective regulation of thin spines. At 4 hours post-injection, the proximal spine density is unchanged in the core but significantly increased in the shell. At 24 hours, the density of proximal dendritic spines is reduced in the core but increased in the shell. Such down-regulation of thin spines in the core persists through 28 days of withdrawal, while the spine density in the shell returns to baseline levels. Consistent with previous results, dendritic tips exhibited up-regulation of dendritic spines after 24 hours of withdrawal, an effect localized to the shell. The divergence in regulation of proximal spine density in NAc core versus shell by cocaine correlates with recently reported electrophysiological data from a similar drug administration regimen and might represent a key mediator of changes in the reward circuit that drive aspects of addiction.
doi:10.1523/JNEUROSCI.5718-11.2012
PMCID: PMC3360066  PMID: 22593064
14.  Interactive Effects of Age and Estrogen on Cortical Neurons: Implications for Cognitive Aging 
Neuroscience  2011;191:148-158.
In the past few decades it has become clear that estrogen signaling plays a much larger role in modulating the cognitive centers of the brain than previously thought possible. We have developed a nonhuman primate (NHP) model to investigate the relationships between estradiol (E) and cognitive aging. Our studies of cyclical E treatment in ovariectomized (OVX) young and aged rhesus monkeys have revealed compelling cognitive and synaptic effects of E in the context of aging. Delayed response (DR), a task that is particularly dependent on integrity of dorsolateral prefrontal cortex (dlPFC) area 46 revealed the following: 1) that young OVX rhesus monkeys perform equally well whether treated with E or vehicle (V), and 2) that aged OVX animals given E perform as well as young adults with or without E, whereas OVX V-treated aged animals display significant DR impairment. We have analyzed the structure of layer III pyramidal cells in area 46 in these same monkeys. We found both age and treatment effects on these neurons that are consistent with behavioral data. Briefly, reconstructions of pyramidal neurons in area 46 from these monkeys showed that cyclical E increased the density of small, thin spines in both young and aged monkeys. However, this effect of E was against a background of age-related loss of small, thin spines, leaving aged V-treated monkeys with a particularly low density of these highly plastic spines and vulnerable to cognitive decline. Our current interpretation is that E not only plays a critically important role in maintaining spine number, but also enables synaptic plasticity through a cyclical increase in small highly plastic spines that may be stabilized in the context of learning. Interestingly, recent studies demonstrate that chronic E is less effective at inducing spinogenesis than cyclical E. We have begun to link certain molecular attributes of excitatory synapses in area 46 to E effects and cognitive performance in these monkeys. Given the importance of synaptic estrogen receptor α (ER-α) in rat hippocampus, we focused our initial studies on synaptic ER-α in area 46. Three key findings have emerged from these studies: 1) synaptic ER-α is present in axospinous synapses in area 46; 2) it is stable across treatment and age groups (which is not the case in rat hippocampus); and 3) the abundance and distribution of synaptic ER-α is a key correlate of individual variation in cognitive performance in certain age and treatment groups. These findings have important implications for the design of hormone treatment strategies for both surgically and naturally menopausal women.
doi:10.1016/j.neuroscience.2011.05.045
PMCID: PMC3166405  PMID: 21664255
Prefrontal cortex; estrogen; aging; primate; cognition; hormone replacement therapy
15.  Evidence for reduced experience-dependent dendritic spine plasticity in the aging prefrontal cortex 
The Journal of Neuroscience  2011;31(21):7831-7839.
Cognitive functions that require the prefrontal cortex are highly sensitive to aging in humans, non-human primates, and rodents, although the neurobiological correlates of this vulnerability remain largely unknown. It has been proposed that dendritic spines represent the primary site of structural plasticity in the adult brain, and recent data have supported the hypothesis that aging is associated with alterations of dendritic spine morphology and plasticity in prefrontal cortex. However, no study to date has directly examined whether aging alters the capacity for experience-dependent spine plasticity in aging prefrontal neurons. To address this possibility we used young, middle-aged, and aged rats in a behavioral stress paradigm known to produce spine remodeling in prefrontal cortical neurons. In young rats, stress resulted in dendritic spine loss and altered patterns of spine morphology; in contrast, spines from middle-aged and aged animals were remarkably stable and did not show evidence of remodeling. The loss of stress-induced spine plasticity observed in aging rats occurred alongside robust age-related reductions in spine density and shifts in remaining spine morphology. Taken together, the data presented here provide the first evidence that experience-dependent spine plasticity is altered by aging in prefrontal cortex, and support a model in which dendritic spines become progressively less plastic in the aging brain.
doi:10.1523/JNEUROSCI.0839-11.2011
PMCID: PMC3398699  PMID: 21613496
stress; resilience; prelimbic cortex; confocal microscopy; synapse
16.  Synaptic characteristics of dentate gyrus axonal boutons and their relationships with aging, menopause, and memory in female rhesus monkeys 
Age-related memory impairment occurs in many mammalian species including humans. Moreover, women undergoing the menopausal transition often complain of problems with memory. We recently reported that rhesus monkeys display age- and menopause-related recognition memory impairment on a hippocampus-reliant test (delayed nonmatching-to-sample; DNMS). In the same monkeys, perforated synapse densities in the dentate gyrus outer molecular layer (OML) correlated with DNMS recognition accuracy, while total axospinous synapse density was similar across age and menses groups. The current study examined whether synaptic characteristics of OML axonal boutons are coupled with age- or menopause-related memory deficits. Using serial section electron microscopy, we measured the frequencies of single-synapse boutons (SSBs), multiple-synapse boutons (MSBs), and boutons with no apparent synaptic contacts (non-synaptic boutons, NSBs) in the OML. Aged females had double the percentage of NSBs as compared to young females and this measure correlated positively and inversely with DNMS acquisition (number of trials to criterion) and delay performance (average accuracy), respectively. Aged compared to young females also had a lower frequency of MSBs and a lower number of synaptic contacts per MSB, and the latter variable inversely correlated with DNMS acquisition. Although proportions of NSBs, SSBs and MSBs were similar across menses groups, compared to pre-menopausal monkeys, peri/post-menopausal monkeys had fewer MSBs contacting one or more segmented perforated synapse and the abundance of this bouton subtype positively correlated with DNMS performance. These results suggest that age- and menopause-related shifts in OML synaptic subtypes may be coupled with deficits in task acquisition and recognition memory.
doi:10.1523/JNEUROSCI.0822-11.2011
PMCID: PMC3103072  PMID: 21613486
delayed nonmatching-to-sample test; menopause; multiple-synapse bouton; serial sections; recognition memory
17.  ESTROGEN AND AGING AFFECT THE SYNAPTIC DISTRIBUTION OF ESTROGEN RECEPTOR BETA-IMMUNOREACTIVITY IN THE CA1 REGION OF FEMALE RAT HIPPOCAMPUS 
Brain research  2010;1379:86-97.
Estradiol (E) mediates increased synaptogenesis in the hippocampal CA1 stratum radiatum (sr) and enhances memory in young and some aged female rats, depending on dose and age. Young females rats express more estrogen receptor α (ERα) immunolabeling in CA1sr spine synapse complexes than aged rats and ERα regulation is E sensitive in young but not aged rats. The current study examined whether estrogen receptor β (ERβ) expression in spine synapse complexes may be altered by age or E treatment. Young (3–4 months) and aged (22–23 months) female rats were ovariectomized 7 days prior to implantation of silastic capsules containing either vehicle (cholesterol) or E (10% in cholesterol) for 2 days. ERβ immunoreactivity (ir) in CA1sr was quantitatively analyzed using post-embedding electron microscopy. ERβ-ir was more prominent postsynaptically than presynaptically and both age and E treatment affected its synaptic distribution. While age decreased the spine synaptic complex localization of ERβ-ir (i.e., within 60 nm of the pre- and post-synaptic membranes), E treatment increased synaptic ERβ in both young and aged rats. In addition, the E treatment, but not age, increased dendritic shaft labeling. This data demonstrates that like ERα the levels of ERβ-ir decrease in CA1 axospinous synapses with age, however, unlike ERα the levels of ERβ-ir increase in these synapses in both young and aged rats in response to E. This suggests that synaptic ERβ may be a more responsive target to E, particularly in aged females.
doi:10.1016/j.brainres.2010.09.069
PMCID: PMC3046233  PMID: 20875808
18.  IκB Kinase Regulates Social Defeat Stress-Induced Synaptic and Behavioral Plasticity 
The neurobiological underpinnings of mood and anxiety disorders have been linked to the nucleus accumbens (NAc), a region important in processing the rewarding and emotional salience of stimuli. Using chronic social defeat stress, an animal model of mood and anxiety disorders, we investigated whether alterations in synaptic plasticity are responsible for the long-lasting behavioral symptoms induced by this form of stress. We hypothesized that chronic social defeat stress alters synaptic strength or connectivity of medium spiny neurons (MSNs) in the NAc to induce social avoidance. To test this, we analyzed the synaptic profile of MSNs via confocal imaging of Lucifer-yellow-filled cells, ultrastructural analysis of the postsynaptic density, and electrophysiological recordings of miniature EPSCs (mEPSCs) in mice after social defeat. We found that NAc MSNs have more stubby spine structures with smaller postsynaptic densities and an increase in the frequency of mEPSCs after social defeat. In parallel to these structural changes, we observed significant increases in IκB kinase (IKK) in the NAc after social defeat, a molecular pathway that has been shown to regulate neuronal morphology. Indeed, we find using viral-mediated gene transfer of dominant-negative and constitutively active IKK mutants that activation of IKK signaling pathways during social defeat is both necessary and sufficient to induce synaptic alterations and behavioral effects of the stress. These studies establish a causal role for IKK in regulating stress-induced adaptive plasticity and may present a novel target for drug development in the treatment of mood and anxiety disorders in humans.
doi:10.1523/JNEUROSCI.4763-10.2011
PMCID: PMC3219041  PMID: 21209217
19.  Estrogen Promotes Stress Sensitivity in a Prefrontal Cortex–Amygdala Pathway 
Cerebral Cortex (New York, NY)  2010;20(11):2560-2567.
We have recently reported in male rats that medial prefrontal cortex (mPFC) neurons that project to the basolateral nucleus of the amygdala (BLA) are resilient to stress-induced dendritic remodeling. The present study investigated whether this also occurs in female rats. This pathway was identified using the retrograde tracer Fast Blue injected into the BLA of ovariectomized female rats with estrogen replacement (OVX + E) and without (OVX + veh). Animals were exposed for 10 days either to 2-h immobilization stress or to home cage rest, after which layer III mPFC neurons that were either retrogradely labeled by Fast Blue or unlabeled were filled with Lucifer Yellow and analyzed for apical dendritic length and spine density. No dendritic remodeling occurred in unlabeled neurons from OVX + veh or OVX + E animals. In BLA-projecting neurons, however, stress had no effect on length in OVX + veh animals, but stressed OVX + E females showed greater dendritic length than controls at intermediate branches. Stress also caused an increase in spine density in all neurons in OVX + veh animals and a spine density increase in BLA-projecting neurons in OVX + E females. Estrogen also increased spine density on BLA-projecting neurons in unstressed animals. These data demonstrate both independent effects of estrogen on pyramidal cell morphology and effects that are interactive with stress, with the BLA-projecting neurons being sensitive to both kinds of effects.
doi:10.1093/cercor/bhq003
PMCID: PMC2951843  PMID: 20139149
connectivity; dendritic arborization; medial prefrontal cortex; neural plasticity; sex difference
20.  Long-term gonadal hormone treatment and endogenous neurogenesis in the dentate gyrus of the adult female monkey 
Experimental neurology  2010;224(1):252-257.
Neurogenesis occurs continually throughout life in all mammals and the extent of neurogenesis is influenced by many factors including gonadal hormones. Most research regarding hormones and neurogenesis has been performed on non-primate species. To determine whether gonadal hormones can modulate endogenous neurogenesis in the dentate gyrus (DG) of the hippocampus in non-human primates, ovariectomized (OVX) female rhesus monkeys received continuous, unopposed β-estradiol (OVX-E-Con), cyclic unopposed β-estradiol (OVX-E-Cyc), continuous β-estradiol + cyclic progesterone (OVX-E-Con+P-Cyc), or control (OVX-Veh) treatments. At week 29, all monkeys received BrdU injections for four consecutive days, in addition to the ongoing treatment. Twenty days after the last BrdU injection, all animals were sacrificed for tissue collection. In DG of hippocampus, scattered BrdU-ir cells were observed mainly in the subgranular zone (SGZ) and in the granule cell layer and occasionally these BrdU-ir cells in the SGZ formed clusters containing between 2–5 cells. In the granule cell layer and SGZ, virtually none of the BrdU-ir cells were either Dcx, a marker of immature neurons, or GFAP positive. However, an occasional BrdU-ir cell was positive for both neuronal marker NeuN or β III-tubulin. Unbiased stereological analysis of BrdU-ir cells within the SGZ and the granule cell layer of DG revealed that among the experimental groups, there was no significant difference in number of BrdU-ir cells within the SGZ and the granule cell layer of the DG: OVX-E-Con (1801+218.7), OVX-E-Cyc (1783+415.6), OVX-E-Con+P-Cyc (1721+229.6), and OVX-Veh (1263+106.3), but a trend towards increased BrdU-ir cells was observed in all the experimental groups.
doi:10.1016/j.expneurol.2010.03.027
PMCID: PMC2885495  PMID: 20362573
monkey; hippocampal neurogenesis; dentate gyrus; estrogen
21.  POST-PUBERTAL DECREASE IN HIPPOCAMPAL DENDRITIC SPINES OF FEMALE RATS 
Experimental neurology  2007;210(2):339-348.
Hippocampal dendritic spine and synapse numbers in female rats vary across the estrous cycle and following experimental manipulation of hormone levels in adulthood. Based on behavioral studies demonstrating that learning patterns are altered following puberty, we hypothesized that dendritic spine number in rat hippocampal CA1 region would change post-pubertally. Female Sprague-Dawley rats were divided into prepubertal (postnatal day (P) 22), peripubertal (P35) and post-pubertal (P49) groups, with the progression of puberty evaluated by vaginal opening, and estrous cyclicity subsequently assessed by daily vaginal smears. Spinophilin immunoreactivity in dendritic spines was used as an index of spinogenesis in area CA1 stratum radiatum (CA1sr) of hippocampus. First, electron microscopy analyses confirmed the presence of spinophilin specifically in dendritic spines of CA1sr, supporting spinophilin as a reliable marker of hippocampal spines in young female rats. Second, stereologic analysis was performed to assess the total number of spinophilin-immunoreactive puncta (i.e. spines) and CA1sr volume in developing rats. Our results indicated that the number of spinophilin-immunoreactive spines in CA1sr was decreased 46% in the post-pubertal group compared to the two younger groups, whereas the volume of the hippocampus underwent an overall increase during this same developmental time frame. Third, to determine a potential role of estradiol in this process, an additional group of rats was ovariectomized (OVX) prepubertally at P22, then treated with estradiol or vehicle at P35, and spinophilin quantified as above in rats perfused on P49. No difference in spinophilin puncta number was found in OVX rats between the two hormone groups, suggesting that this developmental decrease is independent of peripheral estradiol. These changes in spine density coincident with puberty may be related to altered hippocampal plasticity and synaptic consolidation at this phase of maturity.
doi:10.1016/j.expneurol.2007.11.003
PMCID: PMC3075007  PMID: 18096161
Puberty; dendritic spine; hippocampus; CA1; spinophilin; estrogen; synaptic plasticity; spinogenesis
22.  Structural and functional alterations to rat medial prefrontal cortex following chronic restraint stress and recovery 
Neuroscience  2009;164(2):798-808.
Chronic stress has been shown in animal models to result in altered dendritic morphology of pyramidal neurons of the medial prefrontal cortex (mPFC). It has been hypothesized that the stress-induced dendritic retractions and spine loss lead to disrupted connectivity that results in stress-induced functional impairment of mPFC. While these alterations were initially viewed as a neurodegenerative event, it has recently been established that stress induced dendritic alterations are reversible if animals are given time to recover from chronic stress. However, whether spine growth accompanies dendritic extension remains to be demonstrated. It is also not known if recovery-phase dendritic extension allows for re-establishment of functional capacity. The goal of this study, therefore, was to characterize the structural and functional effects of chronic stress and recovery on the infralimbic (IL) region of the rat mPFC. We compared neuronal morphology of layer V IL pyramidal neurons from animals subjected to 21 days of chronic restraint stress (CRS) to those that experienced CRS followed by a 21 day recovery period. Layer V pyramidal cell functional capacity was assessed by intra-IL long-term potentiation (LTP) both in the absence and presence of SKF38393, a dopamine receptor partial agonist and a known PFC LTP modulator. We found that stress-induced IL apical dendritic retraction and spine loss co-occur with receptor-mediated impairments to catecholaminergic facilitation of synaptic plasticity. We also found that while post-stress recovery did not reverse distal dendritic retraction, it did result in over-extension of proximal dendritic neuroarchitecture and spine growth as well as a full reversal of CRS-induced impairments to catecholaminergic-mediated synaptic plasticity. Our results support the hypothesis that disease-related PFC dysfunction is a consequence of network disruption secondary to altered structural and functional plasticity and that circuitry reestablishment may underlie elements of recovery. Accordingly, we believe that pharmacological treatments targeted at preventing dendritic retraction and spine loss or encouraging circuitry reestablishment and stabilization may be advantageous in the prevention and treatment of mood and anxiety disorders.
doi:10.1016/j.neuroscience.2009.08.053
PMCID: PMC2762025  PMID: 19723561
infralimbic; dendritic morphology; dendritic spines; dopamine; long-term potentiation
23.  Interactive effects of stress and aging on structural plasticity in the prefrontal cortex 
Neuronal networks in the prefrontal cortex mediate the highest levels of cognitive processing and decision making, and the capacity to perform these functions is among the cognitive features most vulnerable to aging. Despite much research, the neurobiological basis of age-related compromised prefrontal function remains elusive. Many investigators have hypothesized that exposure to stress may accelerate cognitive aging, though few studies have directly tested this hypothesis and even fewer have investigated a neuronal basis for such effects. It is known that in young animals, stress causes morphological remodeling of prefrontal pyramidal neurons that is reversible. The present studies sought to determine whether age influences the reversibility of stress-induced morphological plasticity in rat prefrontal neurons. We hypothesized that neocortical structural resilience is compromised in normal aging. To directly test this hypothesis we utilized a well-characterized chronic restraint stress paradigm, with an additional group allowed to recover from the stress paradigm, in 3, 12 and 20-month old male rats. In young animals, stress induced reductions of apical dendritic length and branch number, which were reversed with recovery; in contrast, middle-aged and aged rats failed to show reversible morphological remodeling when subjected to the same stress and recovery paradigm. The data presented here provide evidence that aging is accompanied by selective impairments in long-term neocortical morphological plasticity.
doi:10.1523/JNEUROSCI.0759-10.2010
PMCID: PMC2888496  PMID: 20463234
aging; stress; prefrontal cortex; neuron; dendrite; plasticity
24.  Stress-induced dendritic remodeling in the medial prefrontal cortex: effects of circuit, hormones and rest 
Brain research  2009;1293:108-113.
The medial prefrontal cortex (mPFC) has been implicated as a site of dysfunction and abnormal morphology in major depressive disorder and post-traumatic stress disorder, two illnesses that can be brought on by exposure to stress. In animal models, stress has long been shown to induce impairments in tasks known to be mediated by the mPFC, and recent work has demonstrated that chronic stress can lead to morphological changes in mPFC pyramidal cells. This review explores the current literature on stress-induced dendritic remodeling in the mPFC, with particular focus on new findings that illuminate modulators of these effects.
doi:10.1016/j.brainres.2009.03.062
PMCID: PMC2748148  PMID: 19361488
25.  Stress-Induced Dendritic Remodeling in the Prefrontal Cortex is Circuit Specific 
Cerebral Cortex (New York, NY)  2009;19(10):2479-2484.
Chronic stress exposure has been reported to induce dendritic remodeling in several brain regions, but it is not known whether individual neural circuits show distinct patterns of remodeling. The current study tested the hypothesis that the projections from the infralimbic (IL) area of the medial prefrontal cortex (mPFC) to the basolateral nucleus of the amygdala (BLA), a pathway relevant to stress-related mental illnesses like depression and post-traumatic stress disorder, would have a unique pattern of remodeling in response to chronic stress. The retrograde tracer FastBlue was injected into male rats’ BLA or entorhinal cortex (EC) 1 week prior to 10 days of immobilization stress. After cessation of stress, FastBlue-labeled and unlabeled IL pyaramidal neurons were loaded with fluorescent dye Lucifer Yellow to visualize dendritic arborization and spine density. As has been previously reported, randomly selected (non-FastBlue-labeled) neurons showed stress-induced dendritic retraction in apical dendrites, an effect also seen in EC-projecting neurons. In contrast, BLA-projecting neurons showed no remodeling with stress, suggesting that this pathway may be particularly resilient against the effects of stress. No neurons showed stress-related changes in spine density, contrasting with reports that more dorsal areas of the mPFC show stress-induced decreases in spine density. Such region- and circuit-specificity in response to stress could contribute to the development of stress-related mental illnesses.
doi:10.1093/cercor/bhp003
PMCID: PMC2742599  PMID: 19193712
amygdala; chronic stress; connectivity; infralimbic cortex; neural plasticity; spine density

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