Early-life stress modulates the development of cortico-limbic circuits and increases vulnerability to adult psychopathology. Given the important stress-buffering role of endocannabinoid (eCB) signaling, we performed a comprehensive investigation of the developmental trajectory of the eCB system and the impact of exposure to early life stress induced by repeated maternal separation (MS; 3 h/day) from postnatal day 2 (PND2) to PND12. Tissue levels of the eCB molecules anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured after MS exposures, as well under basal conditions at juvenile (PND14), adolescent (PND40) and adult (PND70) timepoints in the prefrontal cortex (PFC), amygdala and hippocampus. We also examined the effects of MS on CB1 receptor binding in these three brain regions at PND40 and PND70. AEA content was found to increase from PND2 into adulthood in a linear manner across all brain regions, while 2-AG was found to exhibit a transient spike during the juvenile period (PND12-14) within the amygdala and PFC, but increased in a linear manner across development in the hippocampus. Exposure to MS resulted in bidirectional changes in AEA and 2-AG tissue levels within the amygdala and hippocampus and produced a sustained reduction in eCB function in the hippocampus at adulthood. CB1 receptor densities across all brain regions were generally found to be downregulated later in life following exposure to MS. Collectively, these data demonstrate that early life stress can alter the normative ontogeny of the eCB system, resulting in a sustained deficit in eCB function, particularly within the hippocampus, in adulthood.

Drug addiction requires associative learning processes that critically involve hippocampal circuits, including the opioid system. We recently found that acute and chronic stress, important regulators of addictive processes, affect hippocampal opioid levels and mu opioid receptor trafficking in a sexually dimorphic manner. Here, we examined whether acute and chronic stress similarly alters the levels and trafficking of hippocampal delta opioid receptors (DORs). Immediately after acute immobilization stress (AIS) or one-day after chronic immobilization stress (CIS), the brains of adult female and male rats were perfusion-fixed with aldehydes. The CA3b region and the dentate hilus of the dorsal hippocampus were quantitatively analyzed by light microscopy using DOR immunoperoxidase or dual label electron microscopy for DOR using silver intensified immunogold particles (SIG) and GABA using immunoperoxidase. At baseline, females compared to males had more DORs near the plasmalemma of pyramidal cell dendrites and about 3 times more DOR-labeled CA3 dendritic spines contacted by mossy fibers. In AIS females, near-plasmalemmal DOR-SIGs decreased in GABAergic hilar dendrites. However, in AIS males, near-plasmalemmal DOR-SIGs increased in CA3 pyramidal cell and hilar GABAergic dendrites and the percentage of CA3 dendritic spines contacted by mossy fibers increased to about half that seen in unstressed females. Conversely, after CIS, near-plasmalemmal DOR-SIGs increased in hilar GABA-labeled dendrites of females whereas in males plasmalemmal DOR-SIGs decreased in CA3 pyramidal cell dendrites and near-plasmalemmal DOR-SIGs decreased hilar GABA-labeled dendrites. As CIS in females, but not males, redistributed DOR-SIGs near the plasmalemmal of hilar GABAergic dendrites, a subsequent experiment examined the acute affect of oxycodone on the redistribution of DOR-SIGs in a separate cohort of CIS females. Plasmalemmal DOR-SIGs were significantly elevated on hilar interneuron dendrites one-hour after oxycodone (3 mg/kg, I.P.) administration compared to saline administration in CIS females. These data indicate that DORs redistribute within CA3 pyramidal cells and dentate hilar GABAergic interneurons in a sexually dimorphic manner that would promote activation and drug related learning in males after AIS and in females after CIS.

The development of medial temporal lobe circuits is critical for subsequent learning and memory functions later in life. The present study reports the expression of progesterone receptor (PR), a powerful transcription factor of the nuclear steroid receptor superfamily, in Cajal-Retzius cells of the molecular layer of the dentate gyrus of rats. PR was transiently expressed from the day of birth through postnatal day 21, but was absent thereafter. Although PR immunoreactive (PR-ir) cells did not clearly express typical markers of mature neurons, they possessed an ultrastructural morphology consistent with neurons. PRir cells did not express markers for GABAergic neurons, neuronal precursor cells, nor radial glia. However, virtually all PR cells co-expressed the calcium binding protein, calretinin, and the glycoprotein, reelin, both reliable markers for Cajal-Retzius neurons, a transient population of developmentally critical pioneer neurons that guide synaptogenesis of perforant path afferents and histogenesis of the dentate gyrus. Indeed, inhibition of PR activity during the first two weeks of life impaired adult performance on both the novel object recognition and object placement memory tasks, two behavioral tasks hypothesized to describe facets of episodic-like memory in rodents. These findings suggest that PR plays an unexplored and important role in the development of hippocampal circuitry and adult memory function.

Basal differences in the brain may account for why some individuals are more vulnerable to stress than others. Although trait anxiety behavior varies greatly in human populations, most animal models of anxiety disorders tend to focus on the development of anxiety after a stressful experience. In this study, adult male Sprague-Dawley and Lewis rats were grouped according to baseline anxiety-like behavior in the open field, measured by time spent and distance traveled in the center. Individuals that fell one standard deviation above and below the mean, approximately the top and bottom 15%, were selected for the Low and High Anxiety groups. Pyramidal neurons from layer II/III of the prelimbic region of the medial prefrontal cortex were iontophoretically loaded with Lucifer yellow dye and reconstructed. In both strains, animals in the High Anxiety group had smaller apical dendrites than those in the Low Anxiety group. No difference was found in basal dendrites. Sholl analysis revealed a strain difference in the distribution of dendritic material between anxiety groups. These results illustrate significant variability in dendritic morphology in the prefrontal cortex of healthy adult male rats prior to experimental manipulation that correlates with baseline levels of anxiety-like behavior.

The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity.

The ovarian hormone estrogen increases the axospinous synapse density in the hippocampal CA1 region of young female rats but fails to do so in aged rats. This estrogen-mediated alteration of spine synapse structures suggests the coincident requirement for the structural reorganization of the underlying actin cytoskeleton network. Actin reorganization is known to require the deactivation of Cofilin, an actin depolymerization factor. Cofilin is deactivated by LIM kinase (LIMK), and LIMK activity is modulated by the phosphorylation of specific residues. We have previously demonstrated that estrogen is able to increase phosphorylated LIMK (pLIMK) immunoreactivity (IR) in the hippocampus in vivo and that this estrogen-stimulated pLIMK-IR is decreased in the aged brain. Because Cofilin phosphorylation allows for actin filament elongation and spine synapse growth, we sought to determine if estrogen acts through Cofilin and if such estrogen action requires the observed LIMK activity. Using both hippocampal neurons and the NG108-15 neuroblastoma cell line, we demonstrate here that estrogen stimulates the phosphorylation of Cofilin in vitro. Furthermore, this estrogen action on Cofilin requires LIMK. Lastly, while initiating the phosphorylation of LIMK and Cofilin, estrogen can also stimulate the formation of filopodial extensions, an early step in the formation of nascent spines, demonstrating that estrogen can alter the actin-dependent neuronal morphology. This linkage of estrogen communication to Cofilin via LIMK provides the functionality to the age-sensitive pLIMK-IR that we have observed in vivo.

Circulating estrogen levels and hippocampal-dependent cognitive functions decline with aging. Moreover, the responses of hippocampal synaptic structure to estrogens differ between aged and young rats. We recently reported that estrogens increase levels of post-synaptic proteins, including PSD-95, and opioid peptides leu-enkephalin and dynorphin in the hippocampus of young animals. However, the influence of ovarian hormones on synaptic protein and opioid peptide levels in the aging hippocampus is understudied. Here, young (3- to 5-month-old), middle-aged (9- to 12-month-old), and aged (about 22-month-old) female rats were ovariectomized and then, 4 weeks later, subcutaneously implanted with a silastic capsule containing vehicle or 17β-estradiol. After 48 h, rats were subcutaneously injected with progesterone or vehicle and sacrificed 1 day later. Coronal sections through the dorsal hippocampus were processed for quantitative peroxidase immunohistochemistry of leu-enkephalin, dynorphin, synaptophysin, and PSD-95. With age, females showed opposing changes in leu-enkephalin and dynorphin levels in the mossy fiber pathway, particularly within the hilus, and regionally specific changes in synaptic protein levels. 17β-estradiol, with or without progesterone, altered leu-enkephalin levels in the dentate gyrus and synaptophysin levels in the CA1 of young but not middle-aged or aged females. Additionally, 17β-estradiol decreased synaptophysin levels in the CA3 of middle-aged females. Our results support and extend previous findings indicating 17β-estradiol modulation of hippocampal opioid peptides and synaptic proteins while demonstrating regional and age-specific effects. Moreover, they lend credence to the "window of opportunity" hypothesis during which hormone replacement can modulate hippocampal structure and circuitry to improve cognitive outcomes.

In the hippocampal formation (HF), the enkephalin opioids and estrogen are each known to modulate learning and cognitive performance relevant to drug abuse. Within the HF, leu-enkephalin (LENK) is most prominent in the mossy fiber (MF) pathway formed by the axons of dentate gyrus (DG) granule cells. To examine the influence of ovarian steroids on MF pathway LENK levels, we used quantitative light microscopic immunocytochemistry to evaluate LENK levels in normal cycling rats and in estrogen-treated ovariectomized rats. Rats in estrus had increased levels of LENK-immunoreactivity (ir) in the DG hilus compared to rats in diestrus or proestrus. Rats in estrus and proestrus had higher levels of LENK-ir in CA3a-c compared to rats in diestrus. Ovariectomized (OVX) rats 24 h (but not 6 or 72 h) after estradiol benzoate (EB; 10 microg) administration had increased LENK-ir in the DG hilus and CA3c. Electron microscopy showed a larger proportion of LENK-labeled small terminals and axons in the DG hilus compared to CA3 which may have contributed to region-specific changes in LENK-ir densities. Next we evaluated the subcellular relationships of estrogen receptor (ER) alpha, ERbeta and progestin receptor (PR) with LENK-labeled MF pathway profiles using dual-labeling electron microscopy. ERbeta-ir colocalized in some LENK-labeled MF terminals and smaller terminals while PR-ir was mostly in CA3 axons, some of which also showed colocalization with LENK. ERalpha-ir was in dendritic spines, but no colocalization with LENK-labeled profiles was observed. The present studies indicate that estrogen can modulate LENK in subregions of the MF pathway in a dose-and time-dependent manner. These effects might be triggered by direct activation of ERbeta or PR in LENK-containing terminals.

There is growing evidence that stress leads to contrasting patterns of structural plasticity in the hippocampus and amygdala, two brain areas implicated in the cognitive and affective symptoms of stress-related psychiatric disorders. Acute stress has been shown to trigger a delayed increase in the density of dendritic spines in the basolateral amygdala (BLA) of rodents. However, the physiological correlates of this delayed spinogenesis in the BLA remain unexplored. Furthermore, NMDA receptors (NMDARs) have been known to underlie chronic stress-induced structural plasticity in the hippocampus, but nothing is known about the role of these receptors in the delayed spinogenesis, and its physiological consequences, in the BLA following acute stress. Here, using whole-cell recordings in rat brain slices, we find that a single exposure to 2-h immobilization stress enhances the frequency, but not amplitude, of miniature excitatory postsynaptic currents (mEPSCs) recorded from principal neurons in the BLA 10 days later. This was also accompanied by faster use-dependent block of NMDA receptor currents during repeated stimulation of thalamic inputs to the BLA, which is indicative of higher presynaptic release probability at these inputs 10 days later. Furthermore, targeted in vivo infusion of the NMDAR-antagonist APV into the BLA during the acute stress prevents the increase in mEPSC frequency and spine density 10 days later. Together, these results identify a role for NMDARs during acute stress in both the physiological and morphological strengthening of synaptic connectivity in the BLA in a delayed fashion. These findings also raise the possibility that activation of NMDA receptors during stress may serve as a common molecular mechanism despite the divergent patterns of plasticity that eventually emerge after stress in the amygdala and hippocampus.

This study reveals the presence of dendritic cells (DCs) in the pituitary gland, which play a role in communicating immune activation to the hypothalamic pituitary adrenal (HPA) axis. Using enhanced yellow fluorescent protein (eyfp) expression as a reporter for CD11c, a marker of DCs, we demonstrate anatomically the presence of CD11c/eyfp+ cells throughout the pituitary. Flow cytometric analysis shows that the predominant cellular phenotype of pituitary CD11c/eyfp+ cells resembles that of non-lymphoid DCs. In vivo and in vitro immune challenge with lipopolysaccharide (LPS) stimulates these pituitary CD11c/eyfp+ DCs, but not eyfp(neg) cells, to increase levels of pro-inflammatory cytokines, IL-6, IL-1β, and TNF-α. In vivo analysis of plasma glucocorticoid (GC) and adrenocorticotropic hormone (ACTH) levels at this early phase of the immune response to LPS suggest that pro-inflammatory cytokine production by DCs within the pituitary may activate the release of GCs from the adrenals via ACTH. Pituitary CD11c/eyfp+ cells also express annexin A1 (ANXA1), indicating a role in GC signal attenuation. In summary, our data demonstrate that a resident DC population of the pituitary gland coordinates GC release in the early phase of systemic immune activation, thereby providing an essential immune signaling sentinel for the initial shaping of the systemic immune response to LPS.

Age and estrogens may impact the mobility of N-methyl-D-aspartate receptors (NMDARs) in hippocampal synapses. Here, we used serial section immunogold electron microscopy to examine whether phosphorylated tyrosine 1472 NR2B (pY1472), which is involved in the surface expression of NMDARs, is altered in the dorsal hippocampus of young (3-4 months old) and aged (∼24 months old) ovariectomized rats treated with 17β-estradiol or vehicle for 2 days. The number of gold particles labeling pY1472 was higher in presynaptic and postsynaptic compartments of aged rats with low estradiol (vehicle-treated) compared to other groups. In terminals, pY1472 levels were elevated in aged rats but reduced by estradiol treatment to levels seen in young rats. Conversely, the mitochondria number was lower in aged females but was restored to young levels by estradiol. In the postsynaptic density and dendritic spines, estradiol reduced pY1472 in young and aged rats. As phosphorylation at Y1472 blocks NR2B endocytosis, reduction of pY1472 by estradiol suggests another mechanism through which estrogen enhances synaptic plasticity by altering localization of NMDAR subunits within synapses.

Obstructive sleep apnea (OSA), a chronic sleep disorder characterized by repetitive reduction or cessation of airflow during sleep, is widely prevalent and is associated with adverse neurocognitive sequelae including increased risk of Alzheimer's disease (AD). In humans, OSA is more common in elderly males. OSA is characterized by sleep fragmentation and chronic intermittent hypoxia (CIH), and recent epidemiological studies point to CIH as the best predictor of neurocognitive sequelae associated with OSA. The sex- and age- specific effects of OSA-associated CIH on specific cell populations such as γ-aminobutyric acid (GABA)-ergic neurons in the hippocampus and the medial prefrontal cortex (mPFC), regions important for cognitive function, remain largely unknown. The present study examined the effect of 35 days of either moderate (10% oxygen) or severe (5% oxygen) CIH on GABAergic neurons in the mPFC and hippocampus of young and aged male and female mice as well as post-accelerated ovarian failure (AOF) female mice. In the mPFC and hippocampus, the number of GABA-labeled neurons increased in aged and young severe CIH males compared to controls but not in young moderate CIH males. This change was not representative of the individual GABAergic cell subpopulations, as the number of parvalbumin-labeled neurons decreased while the number of somatostatin-labeled neurons increased in the hippocampus of severe CIH young males only. In all female groups, the number of GABA-labeled cells was not different between CIH and controls. However, in the mPFC, CIH increased the number of parvalbumin-labeled neurons in young females and the number of somatostatin-labeled cells in AOF females but decreased the number of somatostatin-labeled cells in aged females. In the hippocampus, CIH decreased the number of somatostatin-labeled neurons in young females. CIH decreased the density of vesicular GABA transporter in the mPFC of AOF females only. These findings suggest sex-specific changes in GABAergic neurons in the hippocampus and mPFC with males showing an increase of this cell population as compared to their female counterparts following CIH. Age at exposure and severity of CIH also differentially affect the GABAergic cell population in mice.

Prescription opioid abuse is a serious public health issue. Recently, we showed that female and male Sprague-Dawley rats acquire conditioned place preference (CPP) to the mu opioid receptor agonist oxycodone. Anatomical analysis of the hippocampus from these rats unveiled sex differences in the opioid system in a way that would support excitation and opiate associative learning processes especially in females. In this study, we examined the expression and protein densities of opioid, plasticity, stress and related kinase and signaling molecules in the hippocampus of female and male rats following oxycodone CPP. Oxycodone CPP females have: a) increases in ARC (activity regulated cytoskeletal-associated protein)-immunoreactivity (ir) in CA3 pyramidal cells; b) decreases in Npy (neuropeptide Y) gene expression in the medial hippocampus but higher numbers of NPY-containing hilar interneurons compared to males; c) increases in Crhr2 (corticotropin releasing factor receptor 2) expression in CA2/3; d) increases in Akt1 (AKT serine/threonine kinase 1) expression in medial hippocampus; and e) decreases in phosphorylated MAPK (mitogen activated protein kinase)-ir in CA1 and dentate gyrus. Oxycodone CPP males have: a) increases in Bdnf (brain derived-neurotrophic factor) expression, which is known to be produced in granule cells, relative to females; b) elevated Mapk1 expression and pMAPK-ir in the dentate hilus which harbors newly generated granule cells; and c) increases in CRHR1-ir in CA3 pyramidal cell soma. These sex-specific changes in plasticity, stress and kinase markers in hippocampal circuitry parallel previously observed sex differences in the opioid system after oxycodone CPP.

The multifactorial etiology of stress-related disorders necessitates a constant interrogation of the molecular convergences in preclinical models of stress that use disparate paradigms as stressors spanning from environmental challenges to genetic predisposition to hormonal signaling. Using RNA-sequencing, we investigated the genomic signatures in the ventral hippocampus common to mouse models of stress. Chronic oral corticosterone (CORT) induced increased anxiety- and depression-like behavior in wild-type male mice and male mice heterozygous for the gene coding for brain-derived neurotrophic factor Val66Met, a variant associated with genetic susceptibility to stress. In a separate set of male mice, chronic social defeat stress (CSDS) led to a susceptible or a resilient population, whose proportion was dependent on housing conditions, namely standard housing or enriched environment. Rank-rank-hypergeometric overlap (RRHO), a threshold-free approach that ranks genes by their p value and effect size direction, was used to identify genes from a continuous gradient of significancy that were concordant across groups. In mice treated with CORT and in standard-housed susceptible mice, differentially expressed genes (DEGs) were concordant for gene networks involved in neurotransmission, cytoskeleton function, and vascularization. Weighted gene co-expression analysis generated 54 gene hub modules and revealed two modules in which both CORT and CSDS-induced enrichment in DEGs, whose function was concordant with the RRHO predictions, and correlated with behavioral resilience or susceptibility. These data showed transcriptional concordance across models in which the stress coping depends upon hormonal, environmental, or genetic factors revealing common genomic drivers that embody the multifaceted nature of stress-related disorders.

Hypertension susceptibility in women increases at the transition to menopause, termed perimenopause, a state characterized by erratic estrogen fluctuation and extended hormone cycles. Elucidating the role of estrogen signaling in the emergence of hypertension during perimenopause has been hindered by animal models that are confounded by abrupt estrogen cessation or effects of aging. In the present study, accelerated ovarian failure (AOF) in estrogen receptor β (ERβ) reporter mice was induced by 4-vinylcyclohexene diepoxide in young mice to model early-stage ovarian failure (peri-AOF) characteristic of peri-menopause. It was found that administering ERβ agonists suppressed elevated blood pressure in a model of neurogenic hypertension induced by angiotensin II (AngII) in peri-AOF, but not in age-matched male mice. It was also found that ERβ agonist administration in peri-AOF females, but not males, suppressed the heightened NMDAR signaling and reactive oxygen production in ERβ neurons in the hypothalamic paraventricular nucleus (PVN), a critical neural regulator of blood pressure. It was further shown that deleting ERβ in the PVN of gonadally intact females produced a phenotype marked by a sensitivity to AngII hypertension. These results suggest that ERβ signaling in the PVN plays an important role in blood pressure regulation in female mice and contributes to hypertension susceptibility in females at an early stage of ovarian failure comparable to human perimenopause.

The genomic effects of circulating glucocorticoids are particularly relevant in cortico-limbic structures, which express a high concentration of steroid hormone receptors. To date, no studies have investigated genomic differences in hippocampal subregions, namely the dorsal (dHPC) and ventral (vHPC) hippocampus, in preclinical models treated with exogenous glucocorticoids. Chronic oral corticosterone (CORT) in mouse is a pharmacological approach that disrupts the activity of the hypothalamic-pituitary-adrenal axis, increases affective behavior, and induces genomic changes after stress in the HPC of wildtype (WT) mice and mice heterozygous for the gene coding for brain-derived neurotrophic factor Val66Met (hMet), a variant associated with genetic susceptibility to stress. Using RNA-sequencing, we investigated the genomic signatures of oral CORT in the dHPC and vHPC of WT and hMet male and female mice, and examined sex and genotype differences in response to oral CORT. Males under CORT showed lower glycemia and increased anxiety- and depression-like behavior compared to females that showed instead opposite affective behavior in response to CORT. Rank-rank-hypergeometric overlap (RRHO) was used to identify genes from a continuous gradient of significancy that were concordant across groups. RRHO showed that CORT-induced differentially expressed genes (DEGs) in WT mice and hMet mice converged in the dHPC of males and females, while in the vHPC, DEGs converged in males and diverged in females. The vHPC showed a higher number of DEGs compared to the dHPC and exhibited sex differences related to glucocorticoid receptor (GR)-binding genes and epigenetic modifiers. Methyl-DNA-immunoprecipitation in the vHPC revealed differential methylation of the exons 1C and 1F of the GR gene (Nr3c1) in hMet females. Together, we report behavioral and endocrinological sex differences in response to CORT, as well as epigenetic signatures that i) differ in the dHPC and vHPC,ii) are distinct in males and females, and iii) implicate differential methylation of Nr3c1 selectively in hMet females.

In the rat, repeated brief exposures to novelty early in life can induce long-lasting enhancements in adult cognitive, social, emotional, and neuroendocrine function. Family-to-family variations in these intervention effects on adult offspring are predicted by the mother's ability to mount a rapid corticosterone (CORT) response to the onset of an acute stressor. Here, in Long-Evans rats, we investigated whether neonatal and adulthood novelty exposure, each individually and in combination, can enhance offspring hypothalamic-pituitary-adrenal (HPA) regulation. Using a 2 × 2 within-litter design, one half of each litter were exposed to a relatively novel non-home environment for 3-min (Neo_Novel) daily during infancy (PND 1-21) and the other half of the litter remained in the home cage (Neo_Home); we further exposed half of these two groups to early adulthood (PND 54-63) novelty exposure in an open field and the remaining siblings stayed in their home cages. Two aspects of HPA regulation were assessed: the ability to maintain a low level of resting CORT (CORTB) and the ability to mount a large rapid CORT response (CORTE) to the onset of an acute stressor. Assessment of adult offspring's ability to regulate HPA regulation began at 370 days of age. We further investigated whether the novelty exposure effects on offspring HPA regulation are sensitive to the context of maternal HPA regulation by assessing maternal HPA regulation similarly beginning 7 days after her pups were weaned. We found that at the population level, rats receiving neonatal, but not early adulthood exposure or both, showed a greater rapid CORTE than their home-staying siblings. At the individual family level, these novelty effects are positively associated with maternal CORTE. These results suggest that early experience of novelty can enhance the offspring's ability to mount a rapid response to environmental challenge and the success of such early life intervention is critically dependent upon the context of maternal HPA regulation.

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 an approximate 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 approximately 50%. Furthermore, the age-related increase in pLIMK occurred selectively within the largest subset of prelimbic PFC synapses. Because 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.

The immune response to stroke is comprised of inflammatory and regulatory processes. One cell type involved in both innate and adaptive immunity is the dendritic cell (DC). A DC population residing in the healthy brain (bDC) was identified using a transgenic mouse expressing enhanced yellow fluorescent protein (EYFP) under the promoter for the DC marker, CD11c (CD11c/EYFP Tg). To determine if bDC are involved in the immune response to cerebral ischemia, transient (40 min) middle cerebral artery occlusion (MCAO) followed by 6, 24, or 72 h reperfusion was conducted in CD11c/EYFP Tg mice. Our results demonstrated that DC accumulated in the ischemic hemisphere at 24 h post-MCAO-reperfusion, particularly in the border region of the infarct where T lymphocytes accrued. To distinguish resident bDC from the infiltrating peripheral DC, radiation chimeras [1. wild type (WT) hosts restored with CD11c/EYFP Tg bone marrow (BM) or 2. CD11c/EYFP Tg hosts restored with WT BM] were generated and examined by immunocytochemistry. These data confirmed that DC populating the core of the infarct at 72 h were of peripheral origin, whereas those in the border region were comprised primarily of resident bDC. The brain resident (CD45 intermediate) cells of CD11c/EYFP Tg mice were analyzed by flow cytometry. Compared to microglia, bDC displayed increased major histocompatibility class II (MHC II) and co-stimulatory molecules following MCAO-reperfusion. High levels of MHC II and the co-stimulatory molecule CD80 on bDC at 72 h corresponded to peak lymphocyte infiltration, and suggested a functional interaction between these two immune cell populations.

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 female 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 post-synaptically than pre-synaptically 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.