Automated rodent in situ muscle contraction assay and myofiber organization analysis in sarcopenia animal models.

Age-related sarcopenia results in frailty and decreased mobility, which are associated with increased falls and long-term disability in the elderly. Given the global increase in lifespan, sarcopenia is a growing, unmet medical need. This report aims to systematically characterize muscle aging in preclinical models, which may facilitate the development of sarcopenia therapies. Naïve rats and mice were subjected to noninvasive micro X-ray computed tomography (micro-CT) imaging, terminal in situ muscle function characterizations, and ATPase-based myofiber analysis. We developed a Definiens (Parsippany, NJ)-based algorithm to automate micro-CT image analysis, which facilitates longitudinal in vivo muscle mass analysis. We report development and characterization of translational in situ skeletal muscle performance assay systems in rat and mouse. The systems incorporate a custom-designed animal assay stage, resulting in enhanced force measurement precision, and LabVIEW (National Instruments, Austin, TX)-based algorithms to support automated data acquisition and data analysis. We used ATPase-staining techniques for myofibers to characterize fiber subtypes and distribution. Major parameters contributing to muscle performance were identified using data mining and integration, enabled by Labmatrix (BioFortis, Columbia, MD). These technologies enabled the systemic and accurate monitoring of muscle aging from a large number of animals. The data indicated that longitudinal muscle cross-sectional area measurement effectively monitors change of muscle mass and function during aging. Furthermore, the data showed that muscle performance during aging is also modulated by myofiber remodeling factors, such as changes in myofiber distribution patterns and changes in fiber shape, which affect myofiber interaction. This in vivo muscle assay platform has been applied to support identification and validation of novel targets for the treatment of sarcopenia.

Differential distribution of estrogen receptor (ER)-alpha and ER-beta in the midbrain raphe nuclei and periaqueductal gray in male mouse: Predominant role of ER-beta in midbrain serotonergic systems.

We examined the distribution of estrogen receptor (ER)-alpha and ER-beta immunoreactive (ir) cells in the dorsal (DRN) and median/paramedian (MPRN) raphe nuclei in male mice. ER-alpha ir neurons were scattered across the three subdivisions (ventral, dorsal, and lateral) of the DRN and the MPRN. Robust ER-beta ir cells were observed throughout the raphe nuclei, and were particularly abundant in the ventral and dorsal subdivisions of the DRN. Using dual-label immunocytochemistry for ER-alpha or ER-beta with tryptophan hydroxylase (TPH), the rate-limiting enzyme for 5-hydroxytryptamine (5-HT) synthesis, over 90% of ER-beta ir cells exhibited TPH-ir in all DRN subdivisions, whereas only 23% of ER-alpha ir cells contained TPH. Comparisons of ER-alpha knockout (alphaERKO) as well as ER-beta knockout (betaERKO) mice with their respective wild-type (WT) littermates revealed that gene disruption of either ER-alpha or ER-beta did not affect the other ER subtype expression in the raphe nuclei. In situ hybridization histochemistry revealed that there was a small but statistically significant decrease in TPH mRNA expression in the ventral DRN subdivision in betaERKO mice compared with betaWT mice, whereas TPH mRNA levels were not affected in alphaERKO mice. These findings support a hypothesis that ER-beta activation may contribute to the estrogenic regulation of neuroendocrine and behavioral functions, in part, by acting directly on 5-HT neurons in the raphe nuclei in male mice.

TrkA immunoreactive astrocytes in dendritic fields of the hippocampal formation across estrous.

Neurotrophins are important modulators of structural synaptic plasticity. (Through trophic action (Jordan. J Neurobiol 40:434-445, 1999), astrocytes serve as permissive substrates to support axonal regrowth (Ridet et al. Trends Neurosci 20:570-571, 1997), and are involved in estrogen-induced synaptic structural plasticity (Garcia-Segura et al. Cell Mol Neurobiol 16:225-237, 1996). Previously, we reported that tyrosine kinase A receptor (TrkA) immunoreactivity was present both in presynaptic neuronal processes (axons and terminals) and in select astrocytes of the male rat hippocampal formation (Barker-Gibb et al. J Comp Neurol 430:182-199, 2001). We show that the number of TrkA-immunoreactive astrocytes in female rats fluctuates 16-fold across the estrous cycle in dendritic fields of the hippocampal formation, with the greatest number at estrus after the peak plasma estradiol concentration of proestrus. Few TrkA-labeled astrocytes were found in ovariectomized animals; after estrogen replacement, this number increased by 12-fold in the hippocampal formation, indicating estrogen-mediated induction. Dual-labeling studies showed that TrkA-labeled astrocytes were also immunoreactive for vimentin, a protein expressed by reactive astrocytes. Ultrastructural analysis of the dentate gyrus molecular layer demonstrated that TrkA immunoreactive astrocytes are positioned primarily next to dendrites and unmyelinated axons. Because nerve growth factor (NGF) has been reported to stimulate astrocytes to function as substrates for axon growth (Kawaja and Gage. Neuron 7:1019-1030, 1991), these findings are consistent with the theory that TrkA immunoreactive astrocytes serve a role in structural plasticity, axon guidance, and synaptic regeneration across the estrous cycle in the hippocampal formation.

Novel target sites for estrogen action in the dorsal hippocampus: an examination of synaptic proteins.

Structural studies have shown that estrogens increase dendritic spine number in the dorsal CA1 field of rat hippocampus using Golgi impregnation as well as the number of dorsal CA1 synapses visualized via electron microscopy. The present study was carried out to further these findings by examining changes in the levels of pre- and postsynaptic proteins using radioimmunocytochemistry (RICC). In this study, 2 days of estradiol-benzoate treatment produced significant and comparable increases in synaptophysin, syntaxin, and spinophilin immunoreactivity (IR) in the CA1 region of the dorsal hippocampus of ovariectomized female rats. For spinophilin, IR was also increased in the hilar region of the dentate gyrus as well as CA3. In all cases, the nonsteroidal estrogen antagonist CI628, which has been previously shown to block spine formation, inhibited the effects of estrogen. However, these protein differences were not detected in whole hippocampus using Western blots. These findings add to a growing body of evidence that estrogens increase synapses in the CA1 region of hippocampus along with changes in previously unidentified sites. These results also suggest that RICC is a rapid and sensitive method for examining molecular changes in synaptic profiles in anatomically distinct brain regions.

Ultrastructural evidence that hippocampal alpha estrogen receptors are located at extranuclear sites.

Estrogen may mediate some of its effects on hippocampal function through the alpha isoform of the estrogen receptor (ERalpha). By light microscopy, ERalpha-immunoreactivity (-I) is found in the nuclei of scattered inhibitory gamma-aminobutyric acid (GABA)ergic interneurons. However, several lines of evidence indicate that estrogen also may exert some of its effects through rapid nongenomic mechanisms, possibly by binding to plasma membranes. Thus, to determine whether ERalpha is found in extranuclear sites in the hippocampal formation (HF), four different antibodies to ERalpha were localized by immunoelectron microscopy in proestrous rats. Ultrastructural analysis revealed that in addition to interneuronal nuclei, ERalpha-I was affiliated with the cytoplasmic plasmalemma of select interneurons and with endosomes of a subset of principal (pyramidal and granule) cells. Moreover, ERalpha labeling was found in profiles dispersed throughout the HF, but slightly more numerous in CA1 stratum radiatum. Approximately 50% of the ERalpha-labeled profiles were unmyelinated axons and axon terminals that contained numerous small, synaptic vesicles. ERalpha-labeled terminals formed both asymmetric and symmetric synapses on dendritic shafts and spines, suggesting that ERalphas arise from sources in addition to inhibitory interneurons. About 25% of the ERalpha-I was found in dendritic spines, many originating from principal cells. Within spines, ERalpha-I often was associated with spine apparati and/or polyribosomes, suggesting that estrogen might act locally through the ERalpha to influence calcium availability, protein translation, or synaptic growth. The remaining 25% of ERalpha-labeled profiles were astrocytes, often located near the spines of principal cells. Collectively, these results suggest that ERalpha may serve as both a genomic and nongenomic transducer of estrogen action in the HF.

Estrogen-regulated progestin receptors are found in the midbrain raphe but not hippocampus of estrogen receptor alpha (ER alpha) gene-disrupted mice.

Estrogen and progesterone may modulate serotonergic function through intracellular receptors, alpha (ER alpha) and/or beta (ER beta), and the progestin receptor (PR). Studies in macaque and rat suggest species differences in steroid action. Presently, we examined the mouse. To identify whether ER alpha is involved in estrogen induction of PR in midbrain raphe, we studied the ER alpha gene-disrupted (alpha ERKO) mouse. The hippocampus was examined as another estrogen/progestin-sensitive brain area reported to express ER alpha, ER beta, and PR. Female and male homozygous alpha ERKO and wildtype mice were gonadectomized and given estradiol benzoate or vehicle. Dual-label immunocytochemistry was performed for PR or ER alpha and the serotonin-synthesizing enzyme, tryptophan hydroxylase (TPH). Cells exhibiting PR immunoreactivity (PR-ir) or ER alpha-ir were observed in dorsal and median raphe and hippocampus in both sexes. No ER alpha-ir cells were observed in alpha ERKO brains. In raphe, PR-ir or ER alpha-ir often colocalized with TPH-ir. Thus, estrogen and progesterone may directly modulate gene expression in select serotonergic neurons via ER alpha and PR in female and male mice. Estrogen significantly increased the number of PR-ir cells, and the percentage of PR-ir cells colocalizing TPH-ir in both raphe nuclei, regardless of sex and genotype. Although less among alpha ERKO mice, the significant estrogen induction of PRs implicates the involvement of another ER, perhaps ER beta. In hippocampus, distinct estrogen-induced PR-ir cells were observed only in wildtype animals, demonstrating an ER alpha-mediated event in this forebrain region. Collectively, these findings suggest that estrogen can regulate the expression of one gene (the PR) via multiple mechanisms, based upon brain region.

Differential colocalization of estrogen receptor beta (ERbeta) with oxytocin and vasopressin in the paraventricular and supraoptic nuclei of the female rat brain: an immunocytochemical study.

Evidence exists for the localization of the newly identified estrogen receptor beta (ERbeta) within the rat paraventricular nucleus (PVN) and supraoptic nucleus (SON), regions which lack ERalpha. Presently, we investigate whether ERbeta-like-immunoreactivity (-ir) is found within cells of several major neuropeptide systems of these regions. Young adult Sprague-Dawley rats were ovariectomized (OVX), and 1 week later half of the animals received estradiol-17beta (E). Dual-label immunocytochemistry was performed on adjacent sections by using an ERbeta antibody, followed by an antibody to either oxytocin (OT), arginine-vasopressin (AVP), or corticotropin releasing hormone. Nuclear ERbeta-ir was identified within SON and retrochiasmatic SON, and in specific PVN subnuclei: medial parvicellular part, ventral and dorsal zones, dorsal and lateral parvicellular parts, and in the posterior magnocellular part, medial and lateral zones. However, the ERbeta-ir within magnocellular areas was noticeably less intense. OT-/ERbeta-ir colocalization was confirmed in neurons of the parvicellular subnuclei, in both OVX and OVX+E brains ( approximately 50% of OT and 25% of ERbeta-labeled cells between bregma -1.78 and -2.00). In contrast, few PVN parvicellular neurons contained both AVP- and ERbeta-ir. As well, very little overlap was observed in the distribution of cells containing corticotropin releasing hormone- or ERbeta-ir. In the SON, most nuclear ERbeta-ir colocalized with AVP-ir, whereas few OT-/ERbeta-ir dual-labeled cells were observed. These findings suggest that estrogen can directly modulate specific OT and AVP systems through an ERbeta-mediated mechanism, in a tissue-specific manner.

Immunocytochemical localization of nuclear estrogen receptors and progestin receptors within the rat dorsal raphe nucleus.

Estradiol and progesterone modulate central serotonergic activity; however, the mechanism(s) of action remain unclear. Recently, estradiol-induced progestin receptors (PRs) have been localized within the majority of serotonin (5-HT) neurons in the female macaque dorsal raphe nucleus (DRN; Bethea [1994] Neuroendocrinology 60:50-61). In the present study, we investigated whether estrogen receptors (ERs) and/or PRs exist within 5-HT and/or non-5-HT cells in the female and male rat DRN and whether estradiol treatment alters the expression of these receptors. Young adult female and male Sprague-Dawley rats were gonadectomized, and 1 week later, half of the animals received a subcutaneous Silastic implant of estradiol-17beta. Animals were transcardially perfused 2 days later with acrolein and paraformaldehyde, and sequential dual-label immunocytochemistry was performed on adjacent sections by using either a PR antibody or an ERalpha antibody. This was followed by an antibody to either the 5-HT-synthesizing enzyme, tryptophan hydroxylase (TPH), or to the astrocytic marker, glial fibrillary acidic protein (GFAP). Cells containing immunoreactivity (ir) for nuclear ERs or PRs were identified within the rat DRN in a region-specific distribution in both sexes. No colocalization of nuclear ER-ir or PR-ir with cytoplasmic TPH-ir or GFAP-ir was observed in either sex or treatment, indicating that the steroid target cells are neither 5-HT neurons nor astrocytes. Females were found to have approximately 30% more PR-labeled cells compared with males throughout the DRN (P < 0.05), but no sex difference was detected in the number of neurons demonstrating ER-ir. In both sexes, 2 days of estradiol exposure decreased the number of cells with ER-ir, whereas it greatly increased the number of cells containing PR-ir in several DRN regions (P < 0.005). Collectively, these findings demonstrate the existence of nonserotonergic cells that contain nuclear ERs or PRs within the female and male rat DRN, including estradiol-inducible PRs. These findings point to a species difference in ovarian steroid regulation of 5-HT activity between the macaque and the rat, perhaps transsynaptically via local neurons in the rat brain.

Ovarian steroids and the brain: implications for cognition and aging.

Ovarian steroids have many effects on the brain throughout the lifespan, beginning during gestation and continuing into senescence. These hormones affect areas of the brain that are not primarily involved in reproduction, such as the basal forebrain, hippocampus, caudate putamen, midbrain raphe, and brainstem locus coeruleus. Here we discuss three effects of estrogens and progestins that are especially relevant to memory processes and identify hormonal alterations associated with aging and neurodegenerative diseases. First, estrogens and progestins regulate synaptogenesis in the CA1 region of the hippocampus during the 4- to 5-day estrous cycle of the female rat. Formation of new excitatory synapses is induced by estradiol and involves N-methyl-D-aspartate (NMDA) receptors, whereas synaptic downregulation involves intracellular progestin receptors. Second, there are developmentally programmed sex differences in the hippocampal structure that mat help explain why male and female rats use different strategies to solve spatial navigation problems. During the period of development when testosterone is elevated in the male, aromatase and estrogen receptors are transiently expressed in the hippocampus. Recent data on behavior and synapse induction strongly suggest that this pathway is involved in the masculinization or defeminization of hippocampal structure and function. Third, ovarian steroids have effects throughout the brain, including effects on brainstem and midbrain catecholaminergic neurons, midbrain serotonergic pathways, and the basal forebrain cholinergic system. Regulation of the serotonergic system appears to be linked to the presence of estrogen- and progestin-sensitive neurons in the midbrain raphe, whereas the ovarian steroid influence on cholinergic function involves induction of choline acetyltransferase and acetylcholinesterase according to a sexually dimorphic pattern. Because of these widespread influences on these various neuronal systems, it is not surprising that ovarian steroids produce measurable cognitive effects after ovariectomy and during aging.