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1.  Selective changes in thin spine density and morphology in monkey prefrontal cortex correlate with aging-related cognitive impairment 
Age-associated memory impairment (AAMI) occurs in many mammalian species including humans. In contrast to Alzheimer’s disease (AD) where circuit disruption occurs through neuron death, AAMI is due to circuit and synapse disruption in the absence of significant neuron loss and thus may be more amenable to prevention or treatment. We have investigated the effects of aging on pyramidal neurons and synapse density in Layer III of area 46 in dorsolateral prefrontal cortex of young and aged, male and female Rhesus monkeys (Macaca mulatta) that were tested for cognitive status through the delayed non-matching-to-sample (DNMS) and delayed response (DR) tasks. Cognitive tests revealed an age-related decrement in both acquisition and performance on DNMS. Our morphometric analyses revealed both an age-related loss of spines (33%, p< 0.05) on pyramidal cells and decreased density of axo-spinous synapses (32%, p<0.01) in layer III of area 46. In addition, there was an age-related shift in the distribution of spine types reflecting a selective vulnerability of small, thin spines, thought to be particularly plastic and linked to learning. While both synapse density and the overall spine size average of an animal were predictive of number of trials required for acquisition of DNMS (i.e., learning the task), the strongest correlate of behavior was found to be the head volume of thin spines, with no correlation between behavior and mushroom spine size or density. No synaptic index correlated with memory performance once the task was learned.
doi:10.1523/JNEUROSCI.6410-09.2010
PMCID: PMC2892969  PMID: 20519525
aging; dendritic spines; primate; prefrontal cortex; cognition; plasticity
2.  Continuously delivered ovarian steroids do not alter dendritic spine density or morphology in macaque dorsolateral prefrontal cortical neurons 
Neuroscience  2013;255:219-225.
Aged ovariectomized female monkeys, a model for menopause in humans, show declines in spine density in the dlPFC and diminished performance in cognitive tasks requiring this brain region. Previous studies in our laboratory have shown that long-term cyclic treatment with 17β-estradiol (E) produces an increase in spine density and in the proportion of thinner spines in layer III pyramidal neurons in the dorsolateral prefrontal cortex (dlPFC) of both young and aged ovariectomized rhesus monkeys. Here we used 3D reconstruction of Lucifer yellow-loaded neurons to investigate whether clinically relevant schedules of hormone therapy would produce similar changes in prefrontal cortical neuronal morphology as long-term cyclic E treatment in young female monkeys. We found that continuously delivered E, with or without a cyclic progesterone treatment, did not alter spine density or morphology in the dlPFC of young adult OVX rhesus monkeys. We also found that the increased density of thinner spines evident in the dlPFC 24 hours after E administration in the context of long-term cyclic E therapy is no longer detectable 20 days after E treatment. When compared with the results of our previously published investigations, our results suggest that cyclic fluctuations in serum E levels may cause corresponding fluctuations in the density of thin spines in the dlPFC. By contrast, continuous administration of E does not support sustained increases in thin spine density. Physiological fluctuations in E concentration may be necessary to maintain the morphological sensitivity of the dlPFC to E.
doi:10.1016/j.neuroscience.2013.09.062
PMCID: PMC3957098  PMID: 24120552
Hormone replacement therapy; aging; primate; menopause; dendritic spine; dorsolateral prefrontal cortex; Prefrontal cortex; estrogen; progesterone
3.  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
4.  Synaptic estrogen receptor-alpha levels in prefrontal cortex in female rhesus monkeys and their correlation with cognitive performance 
In rat hippocampus, estrogen receptor-alpha (ER-α) can initiate non-genomic signaling mechanisms that modulate synaptic plasticity in response to either circulating or locally synthesized estradiol (E). Here we report quantitative electron microscopic data demonstrating that ER-α is present within excitatory synapses in dorsolateral prefrontal cortex (dlPFC) of young and aged ovariectomized female rhesus monkeys with and without E treatment. There were no treatment or age effects on the percentage of excitatory synapses containing ER-α, nor were there any group differences in distribution of ER-α within the synapse. However, the mean size of synapses containing ER-α was larger than unlabeled excitatory synapses. All monkeys were tested on delayed response (DR), a cognitive test of working memory that requires dlPFC. In young ovariectomized monkeys without E treatment, presynaptic ER-α correlated with DR accuracy across memory delays. In aged monkeys that received E treatment, ER-α within the postsynaptic density (30–60 nm from the synaptic membrane) positively correlated with DR performance. Thus, while the lack of group effects suggests that ER-α is primarily in synapses that are stable across age and treatment, synaptic abundance of ER-α is correlated with individual performance in two key age/treatment groups. These data have important implications for individual differences in the cognitive outcome among menopausal women and promote a focus on cortical estrogen receptors for therapeutic efficacy with respect to cognition.
doi:10.1523/JNEUROSCI.3192-10.2010
PMCID: PMC2951003  PMID: 20861381
estradiol; menopause; aging; cognition; dendritic spines; electron microscopy
5.  Aromatase Distribution in the Monkey Temporal Neocortex and Hippocampus 
Brain research  2008;1209:115-127.
Numerous studies have shown that neuronal plasticity in the hippocampus and neocortex is regulated by estrogen and that aromatase, the key enzyme for estrogen biosynthesis, is present in cerebral cortex. Although the expression pattern of aromatase mRNA has been described in the monkey brain, its precise cellular distribution has not been determined. In addition, the degree to which neuronal aromatase is affected by gonadal estrogen has not been investigated. In this study, we examined the immunohistochemical distribution of aromatase in young ovariectomized female rhesus monkeys with or without long-term cyclic estradiol treatment. Both experimental groups showed that aromatase is localized in a large population of CA1-3 pyramidal cells, in granule cells of the dentate gyrus and in some interneurons in which it was co-expressed with the calcium binding proteins calbindin, calretinin, and parvalbumin. Moreover, numerous pyramidal cells were immunoreactive for aromatase in the neocortex, whereas only small subpopulations of neocortical interneurons were immunoreactive for aromatase. The widespread expression of the protein in a large neuronal population suggests that local intraneuroral estrogen synthesis may contribute to estrogen-induced synaptic plasticity in monkey hippocampus and neocortex of female rhesus monkeys. In addition, the apparent absence of obvious differences in aromatase distribution between the two experimental groups suggests that these localization patterns are not dependent on plasma estradiol levels.
doi:10.1016/j.brainres.2008.02.061
PMCID: PMC2423000  PMID: 18402929
aromatase; estrogen; hippocampus; interneuron; neocortex; pyramidal cells
6.  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
7.  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
8.  Loss of Cdc42 leads to defects in synaptic plasticity and remote memory recall 
eLife  2014;3:e02839.
Cdc42 is a signaling protein important for reorganization of actin cytoskeleton and morphogenesis of cells. However, the functional role of Cdc42 in synaptic plasticity and in behaviors such as learning and memory are not well understood. Here we report that postnatal forebrain deletion of Cdc42 leads to deficits in synaptic plasticity and in remote memory recall using conditional knockout of Cdc42. We found that deletion of Cdc42 impaired LTP in the Schaffer collateral synapses and postsynaptic structural plasticity of dendritic spines in CA1 pyramidal neurons in the hippocampus. Additionally, loss of Cdc42 did not affect memory acquisition, but instead significantly impaired remote memory recall. Together these results indicate that the postnatal functions of Cdc42 may be crucial for the synaptic plasticity in hippocampal neurons, which contribute to the capacity for remote memory recall.
DOI: http://dx.doi.org/10.7554/eLife.02839.001
eLife digest
Neurons communicate with one another at junctions called synapses, which are typically formed between the dendrite of one neuron and the axon terminus of another. The dendrites are protrusions coming out of the cell body that receive inputs from other cells; the axon is a cable-like structure that enables neurons to contact other cells. In excitatory neurons in part of the brain called the hippocampus, the dendrites are themselves covered in structures called spines, so most synapses are formed between an axon terminus (belonging to the presynaptic cell) and a dendritic spine (on the postsynaptic cell). The hippocampus is necessary for the formation of long-term memories.
The strength of a synapse can increase or decrease over time—a property that is called synaptic plasticity. Changes in the strength of synapses are thought to underlie learning and memory, and long-lasting changes in synaptic strength involve increases or decreases in the number and size of dendritic spines. Such changes are possible because spines have an internal skeleton that can be assembled and disassembled in a matter of minutes. This ‘remodeling’ process is regulated by a family of enzymes called small GTPases. One of these, known as Cdc42, has been shown to promote the formation and maintenance of spines in cell culture, but its role in synaptic plasticity, learning and memory remains unknown.
Now, Kim, Wang et al. have used genetically modified mice who have had Cdc42 deleted from excitatory neurons in their forebrain to examine the functions of this enzyme in living animals. These ‘knockout’ mice showed a small but statistically significant reduction in the number of dendritic spines in the hippocampus. They also showed smaller changes in spine volume and impaired long-term synaptic plasticity in the hippocampus.
When the mice performed long-term memory tests where they learnt to associate a specific set of visual cues with an impending electric shock, the knockout mice performed well for up to a few days. However, when tested again on the same task 45 days later, the knockout mice did not perform as well as normal mice. This is surprising, given the presumed role of long-term synaptic plasticity in learning and memory, and indicates that Cdc42 is required for ‘remote memory’, the form of memory lasting for many days. Similar results were obtained with another memory test using a water maze, where the animals have to remember the location of a hidden platform. Normal mice remember the location for more than 30 days. In contrast, the knockout mice could only remember the location for a few days.
As well as providing the first demonstration of the role of Cdc42 in synaptic plasticity in live animals, the work of Kim, Wang et al. has provided new insights into the functions of this enzyme in memory. Further work is required to determine how Cdc42 interacts with other proteins present at synapses.
DOI: http://dx.doi.org/10.7554/eLife.02839.002
doi:10.7554/eLife.02839
PMCID: PMC4115656  PMID: 25006034
small GTPase; LTP; hippocampus; structural plasticity; dendritic spine; mouse
9.  Ovarian Steroids Increase Spinogenetic Proteins in the Macaque Dorsal Raphe 
Neuroscience  2012;208C:27-40.
Dendritic spines are the basic structural units of neuronal plasticity. Intracellular signaling cascades that promote spinogenesis have centered on RhoGTPases. We found that ovarian steroids increase gene expression of RhoGTPases (RhoA, Cdc42 and Rac) in laser-captured serotonin neurons. We sought to confirm that the increases observed in gene expression translate to the protein level. In addition, a preliminary study was conducted to determine whether an increase in spines occurs via detection of the spine marker protein, PSD-95. Adult ovariectomized (Ovx) monkeys were treated with estradiol (E), progesterone (P) or E+P for 1 month. Sections through the dorsal raphe nucleus were immunostained for RhoA and Cdc42 (n = 3-4/group). The number and positive pixel area of RhoA-positive cells, and the positive pixel area of Cdc42-positive fibers were determined. Upon combining E and E+P treated groups, there was a significant increase in the average and total cell number and positive pixel area of RhoA-positive cells. E, P and E+P treatments individually or combined, also increased the average and total positive pixel area of Cdc42-positive fibers. With remaining sections from 2 animals in each group, we conducted a preliminary examination of the regulation of PSD-95 protein expression. PSD-95, a postsynaptic scaffold protein, was examined with immunogold silver staining (n = 2/group) and the total number of PSD-95-positive puncta was determined with stereology across 4 levels of the dorsal raphe. E, P and E+P treatment significantly increased the total number of PSD-95-positive puncta. Together, these findings indicate that ovarian steroids act to increase gene and protein expression of two pivotal RhoGTPases involved in spinogenesis and preliminarily indicate that an increased number of spines and/or synapses result from this action. Increased spinogenesis on serotonin dendrites would facilitate excitatory glutamatergic input and in turn, increase serotonin neuronal activity throughout the brain.
doi:10.1016/j.neuroscience.2012.02.002
PMCID: PMC3312750  PMID: 22342969
estradiol; immunocytochemistry; PSD-95; progesterone; RhoGTPase; serotonin
10.  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
11.  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
12.  Ovarian Steroids Increase PSD-95 Expression and Dendritic Spines in the Dorsal Raphe of Ovariectomized Macaques 
Synapse (New York, N.Y.)  2013;67(12):897-908.
Estradiol (E) and progesterone (P) promote spinogenesis in several brain areas. Intracellular signaling cascades that promote spinogenesis involve RhoGTPases, glutamate signaling and synapse assembly. We found that in serotonin neurons, E±P administration increases (a) gene and protein expression of RhoGTPases, (b) gene expression of glutamate receptors (c) gene expression of pivotal synapse assembly proteins. Therefore, in this study we determined whether structural changes in dendritic spines in the dorsal raphe follow the observed changes in gene and protein expression. Dendritic spines were examined with immunogold silver staining of a spine marker protein, postsynaptic density-95 (PSD-95) and with Golgi staining. In the PSD-95 study, adult Ovx monkeys received placebo, E, P, or E+P for 1 month (n=3/group). Sections were immunostained for PSD-95 and the number of PSD-95-positive puncta was determined with stereology. E, P and E+P treatment significantly increased the total number of PSD-95-positive puncta (ANOVA, P=0.04). In the Golgi study, adult Ovx monkeys received placebo, E or E+P for 1 month (n=3–4) and the midbrain was Golgi-stained. A total of 80 neurons were analyzed with Neurolucida software. There was a significant difference in spine density that depended on branch order (two-way ANOVA). E+P treatment significantly increased spine density in higher-order (3–5°) dendritic branches relative to Ovx group (Bonferroni, P<0.05). In summary, E+P leads to the elaboration of dendritic spines on dorsal raphe neurons. The ability of E to induce PSD-95, but not actual spines, suggests either a sampling or time lag issue. Increased spinogenesis on serotonin dendrites would facilitate excitatory glutamatergic input and, in turn, increase serotonin neurotransmission throughout the brain.
doi:10.1002/syn.21702
PMCID: PMC3975919  PMID: 23959764
dendritic spines; estradiol; golgi; progesterone; serotonin
13.  Regional-Specific Effects of Ovarian Hormone Loss on Synaptic Plasticity in Adult Human APOE Targeted Replacement Mice 
PLoS ONE  2014;9(4):e94071.
The human apolipoprotein ε4 allele (APOE4) has been implicated as one of the strongest genetic risk factors associated with Alzheimer’s disease (AD) and in influencing normal cognitive functioning. Previous studies have demonstrated that mice expressing human apoE4 display deficits in behavioral and neurophysiological outcomes compared to those with apoE3. Ovarian hormones have also been shown to be important in modulating synaptic processes underlying cognitive function, yet little is known about how their effects are influenced by apoE. In the current study, female adult human APOE targeted replacement (TR) mice were utilized to examine the effects of human APOE genotype and long-term ovarian hormone loss on synaptic plasticity in limbic regions by measuring dendritic spine density and electrophysiological function. No significant genotype differences were observed on any outcomes within intact mice. However, there was a significant main effect of genotype on total spine density in apical dendrites in the hippocampus, with post-hoc t-tests revealing a significant reduction in spine density in apoE3 ovariectomized (OVX) mice compared to sham operated mice. There was also a significant main effect of OVX on the magnitude of LTP, with post-hoc t-tests revealing a decrease in apoE3 OVX mice relative to sham. In contrast, apoE4 OVX mice showed increased synaptic activity relative to sham. In the lateral amygdala, there was a significant increase in total spine density in apoE4 OVX mice relative to sham. This increase in spine density was consistent with a significant increase in spontaneous excitatory activity in apoE4 OVX mice. These findings suggest that ovarian hormones differentially modulate synaptic integrity in an apoE-dependent manner within brain regions that are susceptible to neurophysiological dysfunction associated with AD.
doi:10.1371/journal.pone.0094071
PMCID: PMC3986067  PMID: 24732142
14.  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
15.  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
16.  SYNAPSES ARE LOST DURING AGING IN THE PRIMATE PREFRONTAL CORTEX 
Neuroscience  2007;152(4):970-981.
An electron microscopic analysis has been carried out on the effects of age on the numerical density of both excitatory (asymmetric) and inhibitory (symmetric) synapses in the neuropil of layers 2/3 and of layer 5 in area 46 from the frontal cortex of behaviorally tested rhesus monkeys. There is no change in the lengths of synaptic junctions with age or in the percentage distribution of synapses relative to the postsynaptic spines and dendritic shafts. However, in layers 2/3 there is an overall loss of about 30% of synapses from 5 to 30 years of age, and both asymmetric and symmetric synapses are lost at the same rate. In layer 5 the situation is different; the overall loss of synapses is only 20% and this is almost entirely due to a loss of asymmetric synapses, since there is no significant loss of symmetric synapses from this layer with age. When the synapse data are correlated with the overall cognitive impairment shown by the monkeys, it is found that there is a strong correlation between the numerical density of asymmetric synapses in layers 2/3 and cognitive impairment, with a weaker correlation between symmetric synapse loss and cognitive impairment. In layer 5 on the other hand there is no correlation between synapse loss and cognitive impairment. However synapse loss is not the only factor causing cognitive impairment, since in previous studies of area 46 we have found that age-related alteration in myelin in this frontal area also significantly contributes to cognitive decline. The synapse loss is also considered in light of earlier studies, which show that the frequency of spontaneous excitatory synaptic responses is reduced with age in layers 2/3 neurons.
doi:10.1016/j.neuroscience.2007.07.014
PMCID: PMC2441531  PMID: 18329176
area 46; neuropil; excitatory and inhibitory synapses; electron microscopy
17.  Cranial Irradiation Alters Dendritic Spine Density and Morphology in the Hippocampus 
PLoS ONE  2012;7(7):e40844.
Therapeutic irradiation of the brain is a common treatment modality for brain tumors, but can lead to impairment of cognitive function. Dendritic spines are sites of excitatory synaptic transmission and changes in spine structure and number are thought to represent a morphological correlate of altered brain functions associated with hippocampal dependent learning and memory. To gain some insight into the temporal and sub region specific cellular changes in the hippocampus following brain irradiation, we investigated the effects of 10 Gy cranial irradiation on dendritic spines in young adult mice. One week or 1 month post irradiation, changes in spine density and morphology in dentate gyrus (DG) granule and CA1 pyramidal neurons were quantified using Golgi staining. Our results showed that in the DG, there were significant reductions in spine density at both 1 week (11.9%) and 1 month (26.9%) after irradiation. In contrast, in the basal dendrites of CA1 pyramidal neurons, irradiation resulted in a significant reduction (18.7%) in spine density only at 1 week post irradiation. Analysis of spine morphology showed that irradiation led to significant decreases in the proportion of mushroom spines at both time points in the DG as well as CA1 basal dendrites. The proportions of stubby spines were significantly increased in both the areas at 1 month post irradiation. Irradiation did not alter spine density in the CA1 apical dendrites, but there were significant changes in the proportion of thin and mushroom spines at both time points post irradiation. Although the mechanisms involved are not clear, these findings are the first to show that brain irradiation of young adult animals leads to alterations in dendritic spine density and morphology in the hippocampus in a time dependent and region specific manner.
doi:10.1371/journal.pone.0040844
PMCID: PMC3397939  PMID: 22815839
18.  Facilitation of AMPA Receptor Synaptic Delivery as a Molecular Mechanism for Cognitive Enhancement 
PLoS Biology  2012;10(2):e1001262.
A small peptide from a neuronal cell adhesion molecule enhances synaptic plasticity in the hippocampus and results in improved cognitive performance in rats.
Cell adhesion molecules and downstream growth factor-dependent signaling are critical for brain development and synaptic plasticity, and they have been linked to cognitive function in adult animals. We have previously developed a mimetic peptide (FGL) from the neural cell adhesion molecule (NCAM) that enhances spatial learning and memory in rats. We have now investigated the cellular and molecular basis of this cognitive enhancement, using biochemical, morphological, electrophysiological, and behavioral analyses. We have found that FGL triggers a long-lasting enhancement of synaptic transmission in hippocampal CA1 neurons. This effect is mediated by a facilitated synaptic delivery of AMPA receptors, which is accompanied by enhanced NMDA receptor-dependent long-term potentiation (LTP). Both LTP and cognitive enhancement are mediated by an initial PKC activation, which is followed by persistent CaMKII activation. These results provide a mechanistic link between facilitation of AMPA receptor synaptic delivery and improved hippocampal-dependent learning, induced by a pharmacological cognitive enhancer.
Author Summary
The human brain contains trillions of neuronal connections, called synapses, whose pattern of activity controls all our cognitive functions. These synaptic connections are dynamic and constantly changing in their strength and properties, and this process of synaptic plasticity is essential for learning and memory. Alterations in synaptic plasticity mechanisms are thought to be responsible for multiple cognitive deficits, such as autism, Alzheimer's disease, and several forms of mental retardation. In this study, we show that synapses can be made more plastic using a small protein fragment (peptide) derived from a neuronal protein involved in cell-to-cell communication. This peptide (FGL) initiates a cascade of events inside the neuron that results in the facilitation of synaptic plasticity. Specifically, we find that FGL triggers delivery of a specific type of glutamate receptor (AMPA receptors) to synapses in a region of the brain called the hippocampus, which is known to be involved in multiple forms of learning and memory. Importantly, when this peptide was administered to rats, their ability to learn and retain spatial information was enhanced. Therefore, this work demonstrates that cognitive function can be improved pharmacologically in adult animals by enhancing the plasticity of synaptic connections in the brain.
doi:10.1371/journal.pbio.1001262
PMCID: PMC3283560  PMID: 22363206
19.  The Environmental Estrogen Bisphenol A Inhibits Estradiol-Induced Hippocampal Synaptogenesis 
Environmental Health Perspectives  2005;113(6):675-679.
Bisphenol A (BPA) is an estrogenic chemical that is widely used in the manufacture of plastics and epoxy resins. Because BPA leaches out of plastic food and drink containers, as well as the BPA-containing plastics used in dental prostheses and sealants, considerable potential exists for human exposure to this compound. In this article we show that treatment of ovariectomized rats with BPA dose-dependently inhibits the estrogen-induced formation of dendritic spine synapses on pyramidal neurons in the CA1 area of the hippocampus. Significant inhibitory effects of BPA were observed at a dose of only 40 μg/kg, below the current U.S. Environmental Protection Agency reference daily limit for human exposure. Because synaptic remodeling has been postulated to contribute to the rapid effects of estrogen on hippocampus-dependent memory, these data suggest that environmental BPA exposure may interfere with the development and expression of normal sex differences in cognitive function, via inhibition of estrogen-dependent hippocampal synapse formation. It may also exacerbate the impairment of hippocampal function observed during normal aging, as endogenous estrogen production declines.
doi:10.1289/ehp.7633
PMCID: PMC1257590  PMID: 15929888
bisphenol A; CA1; estradiol; hippocampus; spine synapse density
20.  Morphologic Evidence for Spatially Clustered Spines in Apical Dendrites of Monkey Neocortical Pyramidal Cells 
The Journal of comparative neurology  2012;520(13):2888-2902.
The general organization of neocortical connectivity in rhesus monkey is relatively well understood. However, mounting evidence points to an organizing principle that involves clustered synapses at the level of individual dendrites. Several synaptic plasticity studies have reported cooperative interaction between neighboring synapses on a given dendritic branch, which may potentially induce synapse clusters. Additionally, theoretical models have predicted that such cooperativity is advantageous, in that it greatly enhances a neuron’s computational repertoire. However, largely because of the lack of sufficient morphologic data, the existence of clustered synapses in neurons on a global scale has never been established. The majority of excitatory synapses are found within dendritic spines. In this study, we demonstrate that spine clusters do exist on pyramidal neurons by analyzing the three-dimensional locations of ~40,000 spines on 280 apical dendritic branches in layer III of the rhesus monkey prefrontal cortex. By using clustering algorithms and Monte Carlo simulations, we quantify the probability that the observed extent of clustering does not occur randomly. This provides a measure that tests for spine clustering on a global scale, whenever high-resolution morphologic data are available. Here we demonstrate that spine clusters occur significantly more frequently than expected by pure chance and that spine clustering is concentrated in apical terminal branches. These findings indicate that spine clustering is driven by systematic biological processes. We also found that mushroom-shaped and stubby spines are predominant in clusters on dendritic segments that display prolific clustering, independently supporting a causal link between spine morphology and synaptic clustering.
doi:10.1002/cne.23070
PMCID: PMC3573331  PMID: 22315181
clustering; dendritic spines; plasticity; morphology; image analysis
21.  Role of Androgens and the Androgen Receptor in Remodeling of Spine Synapses in Limbic Brain Areas 
Hormones and behavior  2007;53(5):638-646.
Accumulating evidence indicates that structural synaptic plasticity in limbic areas plays a vital role not only in normal brain functions, such as cognition and mood, but also in the development of neurological and mental disorders. We have learned from studies investigating neuronal remodeling that estrogens have an exceptional synaptogenic potential that seems to be specific to limbic areas of the adult female brain. On the other hand, structural synaptic plasticity in the adult male brain and the synaptogenic effect of androgens received relatively little attention. During the last five years, the Leranth laboratory provided conclusive evidence that the hippocampus and prefrontal cortex of adult male rodents and non-human primates retain considerable structural synaptic plasticity similar to the female, and that androgens are capable of inducing spine synapse growth in both the hippocampus and prefrontal cortex similar to estrogens. Our recent work also demonstrates that androgen-induced remodeling of spine synapses in the prefrontal cortex of adult male rats is dependent, at least to some extent, on functional androgen receptors, while being entirely independent of the androgen receptor in the hippocampus. Based on these findings and on their many beneficial effects, we believe that androgens hold a great and undeservingly neglected therapeutic potential that could be employed to reverse synaptic pathology in various neurocognitive and neuropsychiatric disorders.
doi:10.1016/j.yhbeh.2007.12.007
PMCID: PMC2408746  PMID: 18262185
Androgen; androgen receptor; testicular feminization mutant; hippocampus; prefrontal cortex; spine synapse; electron microscopic stereology
22.  Estrogen and Aging Affect Synaptic Distribution of Phosphorylated LIM Kinase (LIMK) in CA1 Region of Female Rat Hippocampus 
Neuroscience  2008;152(2):360-370.
17β-Estradiol (E) increases axospinous synapse density in the hippocampal CA1 region of young female rats, but not in aged rats. This may be linked to age-related alterations in signaling pathways activated by synaptic estrogen receptor α (ER-α) that potentially regulate spine formation, such as LIM-Kinase (LIMK), an actin depolymerizing factor/cofilin kinase. We hypothesized that, as with ER-α, phospho-LIMK (pLIMK) may be less abundant or responsive to E in CA1 synapses of aged female rats. To address this, cellular and subcellular distribution of pLIMK-immunoreactivity (pLIMK-IR) in CA1 was analyzed by light and electron microscopy in young and aged female rats that were ovariectomized and treated with either vehicle or E. pLIMK-IR was found primarily in perikarya within the pyramidal cell layer and dendritic shafts and spines in stratum radiatum (SR). While pLIMK-IR was occasionally present in terminals, post-embedding quantitative analysis of SR showed that pLIMK had a predominant post-synaptic localization and was preferentially localized within the postsynaptic density (PSD). The percentage of pLIMK-labeled synapses increased (30%) with E treatment (p<0.02) in young animals, and decreased (43%) with age (p<0.002) regardless of treatment. The pattern of distribution of pLIMK-IR within dendritic spines and synapses was unaffected by age or E treatment, with the exception of an E-induced increase in the non-synaptic core of spines in young females. These data suggest that age-related synaptic alterations similar to those seen with ER-α occur with signaling molecules such as pLIMK, and support the hypothesis that age-related failure of E treatment to increase synapse number in CA1 may be due to changes in the molecular profile of axospinous synapses with respect to signaling pathways linked to formation of additional spines and synapses in response to E.
doi:10.1016/j.neuroscience.2008.01.004
PMCID: PMC2396523  PMID: 18294775
sex steroids; electron microscopy; immunogold; signal transduction; plasticity; synapse
23.  Network Self-Organization Explains the Statistics and Dynamics of Synaptic Connection Strengths in Cortex 
PLoS Computational Biology  2013;9(1):e1002848.
The information processing abilities of neural circuits arise from their synaptic connection patterns. Understanding the laws governing these connectivity patterns is essential for understanding brain function. The overall distribution of synaptic strengths of local excitatory connections in cortex and hippocampus is long-tailed, exhibiting a small number of synaptic connections of very large efficacy. At the same time, new synaptic connections are constantly being created and individual synaptic connection strengths show substantial fluctuations across time. It remains unclear through what mechanisms these properties of neural circuits arise and how they contribute to learning and memory. In this study we show that fundamental characteristics of excitatory synaptic connections in cortex and hippocampus can be explained as a consequence of self-organization in a recurrent network combining spike-timing-dependent plasticity (STDP), structural plasticity and different forms of homeostatic plasticity. In the network, associative synaptic plasticity in the form of STDP induces a rich-get-richer dynamics among synapses, while homeostatic mechanisms induce competition. Under distinctly different initial conditions, the ensuing self-organization produces long-tailed synaptic strength distributions matching experimental findings. We show that this self-organization can take place with a purely additive STDP mechanism and that multiplicative weight dynamics emerge as a consequence of network interactions. The observed patterns of fluctuation of synaptic strengths, including elimination and generation of synaptic connections and long-term persistence of strong connections, are consistent with the dynamics of dendritic spines found in rat hippocampus. Beyond this, the model predicts an approximately power-law scaling of the lifetimes of newly established synaptic connection strengths during development. Our results suggest that the combined action of multiple forms of neuronal plasticity plays an essential role in the formation and maintenance of cortical circuits.
Author Summary
The computations that brain circuits can perform depend on their wiring. While a wiring diagram is still out of reach for major brain structures such as the neocortex and hippocampus, data on the overall distribution of synaptic connection strengths and the temporal fluctuations of individual synapses have recently become available. Specifically, there exists a small population of very strong and stable synaptic connections, which may form the physiological substrate of life-long memories. This population coexists with a big and ever changing population of much smaller and strongly fluctuating synaptic connections. So far it has remained unclear how these properties of networks in neocortex and hippocampus arise. Here we present a computational model that explains these fundamental properties of neural circuits as a consequence of network self-organization resulting from the combined action of different forms of neuronal plasticity. This self-organization is driven by a rich-get-richer effect induced by an associative synaptic learning mechanism which is kept in check by several homeostatic plasticity mechanisms stabilizing the network. The model highlights the role of self-organization in the formation of brain circuits and parsimoniously explains a range of recent findings about their fundamental properties.
doi:10.1371/journal.pcbi.1002848
PMCID: PMC3536614  PMID: 23300431
24.  COORDINATION OF SIZE AND NUMBER OF EXCITATORY AND INHIBITORY SYNAPSES RESULTS IN A BALANCED STRUCTURAL PLASTICITY ALONG MATURE HIPPOCAMPAL CA1 DENDRITES DURING LTP 
Hippocampus  2011;21(4):354-373.
Summary
Enlargement of dendritic spines and synapses correlates with enhanced synaptic strength during long-term potentiation (LTP), especially in immature hippocampal neurons. Less clear is the nature of this structural synaptic plasticity on mature hippocampal neurons, and nothing is known about the structural plasticity of inhibitory synapses during LTP. Here the timing and extent of structural synaptic plasticity and changes in local protein synthesis evidenced by polyribosomes were systematically evaluated at both excitatory and inhibitory synapses on CA1 dendrites from mature rats following induction of LTP with theta-burst stimulation (TBS). Recent work suggests dendritic segments can act as functional units of plasticity. To test whether structural synaptic plasticity is similarly coordinated, we reconstructed from serial section transmission electron microscopy all of the spines and synapses along representative dendritic segments receiving control stimulation or TBS-LTP. At 5 min after TBS, polyribosomes were elevated in large spines suggesting an initial burst of local protein synthesis, and by 2 hr only those spines with further enlarged synapses contained polyribosomes. Rapid induction of synaptogenesis was evidenced by an elevation in asymmetric shaft synapses and stubby spines at 5 min and more nonsynaptic filopodia at 30 min. By 2 hr, the smallest synaptic spines were markedly reduced in number. This synapse loss was perfectly counterbalanced by enlargement of the remaining excitatory synapses such that the summed synaptic surface area per length of dendritic segment was constant across time and conditions. Remarkably, the inhibitory synapses showed a parallel synaptic plasticity, also demonstrating a decrease in number perfectly counterbalanced by an increase in synaptic surface area. Thus, TBS-LTP triggered spinogenesis followed by loss of small excitatory and inhibitory synapses and a subsequent enlargement of the remaining synapses by 2 hours. These data suggest that dendritic segments coordinate structural plasticity across multiple synapses and maintain a homeostatic balance of excitatory and inhibitory inputs through local protein-synthesis and selective capture or redistribution of dendritic resources.
doi:10.1002/hipo.20768
PMCID: PMC2891364  PMID: 20101601
serial section transmission electron microscopy; 3D reconstruction; dendritic spine; polyribosomes; hippocampal slice; adult; ultrastructure
25.  Estrus cyclicity of spinogenesis: underlying mechanisms 
Journal of Neural Transmission  2009;116(11):1417-1425.
Hippocampal spine density varies with the estrus cycle. The cyclic change in estradiol levels in serum was hypothesized to underlie this phenomenon, since treatment of ovariectomized animals with estradiol induced an increase in spine density in hippocampal dendrites of rats, as compared to ovariectomized controls. In contrast, application of estradiol to hippocampal slice cultures did not promote spinogenesis. In addressing this discrepancy, we found that hippocampal neurons themselves are capable of synthesizing estradiol de novo. Estradiol synthesis can be suppressed by aromatase inhibitors and by knock-down of Steroid Acute Regulatory Protein (StAR) and enhanced by substrates of steroidogenesis. Expression of estrogen receptors (ERs) and synaptic proteins, synaptogenesis, and long-term potentiation (LTP) correlated positively with aromatase activity in hippocampal cultures without any difference between genders. All effects due to inhibition of aromatase activity were rescued by application of estradiol to the cultures. Most importantly, gonadotropin-releasing hormone (GnRH) increased estradiol synthesis dose-dependently via an aromatase-mediated mechanism and consistently increased spine synapse density and spinophilin expression. As a consequence, our data suggest that cyclic fluctuations in spine synapse density result from pulsative release of GnRH from the hypothalamus and its effect on hippocampal estradiol synthesis, rather than from varying levels of serum estradiol. This hypothesis is further supported by higher GnRH receptor (GnRH-R) density in the hippocampus than in the cortex and hypothalamus and the specificity of estrus cyclicity of spinogenesis in the hippocampus, as compared to the cortex.
doi:10.1007/s00702-009-0294-x
PMCID: PMC3085745  PMID: 19730783
Estrus cycle; Spinogenesis; GnRH; Estradiol synthesis

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