Wenqin Cai

Distribution and differences of estrogen receptor beta immunoreactivity in the brain of adult male and female rats.

Studies have shown that estrogen plays important roles in regulating neural structure and function in the brain, but the mechanism remains unclear. The actions of estrogen were thought to be mediated by a single estrogen receptor until the identification of another estrogen receptor, namely estrogen receptor-beta (ER-beta). Here we report a comprehensive study of the localization of ER-beta immunoreactivity and differences in the brains of adult male and female rats on the basis of a nickel ammonium sulfate-enhanced immunocytochemical method using a polyclonal antiserum sc-8974. The results of these studies revealed: (1) ER-beta immunoactive material was mainly localized in the neuronal nucleus, but it was also detectable in the cytoplasm and neuronal processes; (2) in both male and female rats, high levels of ER-beta immunopositive signals were detected in the anterior olfactory nucleus, cerebral cortex, Purkinje cells, vertical limb of the diagonal band, red nucleus, locus ceruleus, and motor trigeminal nucleus. Moderate levels were found in the medial septum, lateral amygdaloid nucleus, substantia nigra, and central gray. Weak signals were localized in other subregions of the hypothalamus and amygdaloid complex; (3) there was an obvious difference of ER-beta immunoreactivity between male and female rats, and its intracellular distribution also showed a sex difference. The above results provide the first detailed evidence that ER-beta protein is widely distributed in both male and female rat brains, but that distinctive sex differences also exist. Estrogen may exert its function in different brain regions in a gender-specific manner.

Downregulation of P2X7 receptor expression in rat oligodendrocyte precursor cells after hypoxia ischemia

Oligodendrocyte precursor cells (OPCs) are the predominant oligodendrocyte‐lineage stage in the cerebral hemispheres of neonatal rat. Prior studies have shown that OPCs are highly vulnerable to hypoxic‐ischemic injury, yet the mechanisms are not well understood. P2X7 receptor (P2X7R) is an ATP‐gated ion channel that has unusual properties and plays very complex roles in a variety of neuropathologic conditions. However, little is known about the involvement of P2X7R in OPCs development and injury. The present study was aimed at examining the presence of P2X7R in OPCs and evaluating the change of the receptor expression after hypoxia ischemia. Using Immunofluorescence, RT‐PCR, and western blot analysis, we demonstrated that OPCs expressed P2X7R in vitro and in vivo. Activation of P2X7R in OPCs in response to 3′‐O‐(4‐benzoyl) benzoyl‐ATP (BzATP) led to an increased mobilization of intracellular calcium [Ca2+]i, formation of large pores and cell death. These functional responses were sensitive to pretreatment of cells with the P2X7R antagonist, Brilliant Blue G (BBG, 100 nM), which was a selective antagonist for P2X7R in nanomole range. A decrease in P2X7R expression was observed in cultured OPCs after exposure to oxygen‐glucose deprivation (OGD) for 2 h in vitro. Using a neonatal hypoxic‐ischemic injury model in postnatal 3 rats, the similar downregulation was also detected in ischemic cerebral cortex, subcortical white matter and hippocampus compared with sham operation controls. In conclusion, the present data demonstrated that OPCs expressed functional P2X7R. The post‐ischemic downregulation of P2X7R suggested a role for this receptor in the pathophysiology of hypoxic‐ischemic brain injury.

Localization and sex-difference of steroid receptor coactivator-1 immunoreactivities in the brain of adult female and male mice

Females and males are different in brain and behaviors. These differences are mediated by steroids and their nuclear receptors which require coactivators to regulate the transcription of target genes. Studies have shown that these coactivators are critical for modulating steroid hormone action in the brain. Steroid receptor coactivator-1 has been implied in the regulation of reproduction, stress, motor learning, and limited studies have reported the sex-specific difference of SRC-1 mRNA or protein expression in specific brain regions, but the expression and differences of SRC-1 immunoreactivities in adult female and male brain remain unclear. In this study we reported that in both sexes, high levels of SRC-1 immunoreactivities were detected in olfactory bulb, cerebral cortex, hippocampus, Purkinje cells, some limited diencephalon and brainstem nuclei. The immunopositive materials were predominantly detected in cell nucleus, but in some regions they were also detected in the processes or fiber-like structures. In most of the brain regions studied, males possessed significantly higher levels of SRC-1 immunoreactivities than that of females. Higher levels of SRC-1 were detected in some nuclei related to learning and memory, motor regulation and reproduction indicated its potential roles in neurodegeneration and sex-dependent behavior and structure; the region- and sex-specific localization of SRC-1 immunoreactivities in agreement with that of some steroid receptors, indicating this coactivator play important roles in these hormone-reactive regions and cell groups related to reproduction, learning and memory, integration of motor and sense.

A developmental study on the expression of PDGFαR immunoreactive cells in the brain of postnatal rats

The platelet-derived growth factor-alpha receptor (PDGFalphaR) has been found specifically expressed in oligodendrocyte precursor cells (OPCs), whereas another membrane protein, NG2, has been widely applied to characterize developing and matured OPCs. In order to investigate whether PDGFalphaR expression is consistent to NG2 in identifying OPCs, we utilized techniques of immunohistochemistry and Western blot to study the PDGFalphaR expression and distribution in rat postnatal brain from a series of ages P0 (postnatal day 0) to P540, and further compared it with NG2. Results showed that PDGFalphaR immunoreactive (PDGFalphaR+) cells existed in both the gray and white matter of the postnatal rat brain, although these cells displayed different features in distinct regions and developmental stages. PDGFalphaR did not express in oligodendrocytes, astrocytes or neurons (indicated by non-co-localization with CC1 and NF200, respectively). Western blot analysis revealed that the expression of PDGFalphaR in the cerebral cortex and hippocampus increased from P0 to P7 and then decreased gradually. PDGFalphaR+ cells displayed similar characteristics as of NG2+ cells in the morphology, distribution and electrophysiology. Like NG2+ cells, the density of PDGFalphaR+ cells had an increase at P7 and a late age-dependent decline, except a lower value from P7 to P540 in cerebral cortex, hippocampus and corpus callosum. PDGFalphaR+ cells exhibited number consistent with NG2+ cells at early developmental stages and were approximately 75% as numerous as NG2+ cells at old age. PDGFalphaR+/NG2- cells were not found. In conclusion, our findings suggest that both PDGFalphaR and NG2 are markers of developing OPCs. PDGFalphaR is specific to the NG2+ OPCs and mainly plays an important role at early developmental stages of OPCs. Aging had an effect on the morphological feature, number and developmental regulation of OPCs in rat CNS. However, further work will be necessary to determine if PDGFalphaR-/NG2+ cells may still maintain the biological characteristics of OPCs or they are other subpopulation of OPCs.

Sex differences and synchronous development of steroid receptor coactivator-1 and synaptic proteins in the hippocampus of postnatal female and male C57BL/6 mice

The structure and function including synaptic plasticity of the hippocampus are deeply affected by steroids in a sex-dependant manner, these processes are believed to be mediated by steroid receptors though their coactivators. Our previous studies have reported the developmental profiles of steroid receptor coactivator-1 (SRC-1) and PSD-95 in the hippocampus of postnatal female rats and the sex-differences of SRC-1 immunoreactivities in the brain of adult mice. However, whether there are any sex differences about postnatal development of SRC-1 and synaptic proteins in the hippocampus remain unclear. In this study, we investigated the postnatal profile of SRC-1 and key synaptic protein synaptophysin (SYN), PSD-95 and GluR1 in the hippocampus of female and male mice using immunohistochemistry and Western blot. The results showed that in the female hippocampus, the highest levels of SRC-1 were detected at P14, SYN and GluR1 at P30 and PSD-95 at P60; while in the males, the highest levels of SRC-1, SYN and GluR1 were detected at P30, and PSD-95 at P60. Female hippocampus tended to have higher levels of SRC-1, SYN and GluR1 before P30 and PSD-95 before P14; while male hippocampus have higher levels of PSD-95 at P14, P60 and GluR1 at P0. Correlation analysis showed the profiles of SRC-1 were highly correlated with each synaptic protein. The above results showed that in the hippocampus, except some minor sex differences detected at some time-point examined, females and males shared similar postnatal developmental profile and SRC-1 may be deeply involved in the regulation of hippocampal synaptogenesis.

Expression of Steroid Receptor Coactivator-1 Was Regulated by Postnatal Development but Not Ovariectomy in the Hippocampus of Rats

Female steroids such as estrogens and progestins, through their nuclear receptors, play important roles in regulation of the structure and function of the hippocampus. Steroid receptor coactivator-1 (SRC-1) has been detected in embryonic and/or adult hippocampus of rodents, and SRC-1 null mice showed significantly longer escape latency in the Morris maze test, indicating a role of this coactivator in the regulation of hippocampus function. Whether this is regulated by development and circulating ovary hormones remains unclear. In this study, postnatal development and ovariectomy for regulation of hippocampal SRC-1 in female rats were investigated by Western blot and immunohistochemistry. The results showed that SRC-1-immunopositive materials were predominantly detected in the CA1 pyramidal cell layer and dentate gyrus granular cell layer. Very low levels of SRC-1 were detected at postnatal day 0, but they increased with development. The highest levels of SRC-1 were detected at postnatal day 14, then they decreased to adult levels from postnatal day 30; significantly lower levels of SRC-1 were detected in the middle-aged (18-month-old) hippocampus when compared with that of the adult. Western blot and immunohistochemistry demonstrated that hippocampal SRC-1 expression was unchanged after ovariectomy, no significant differences were noticed from day 3 to 8 weeks postsurgery when compared with sham animals. The above results showed that hippocampal SRC-1 is regulated by postnatal development but not ovariectomy, and that the exact role of SRC-1 in the estradiol regulation of hippocampus needs further investigation.

Aromatase inhibitor letrozole downregulates steroid receptor coactivator-1 in specific brain regions that primarily related to memory, neuroendocrine and integration

As one of the third generation of aromatase inhibitors, letrozole is a favored drug for the treatment of hormone receptor-positive breast cancer with some adverse effects on the nervous system, but the knowledge is limited and the results are controversial, the mechanism underlying its central action is also unclear. Accumulated evidences have demonstrated that estrogens derived from androgens by aromatase play profound roles in the brain through their receptors, which needs coactivator for the transcription regulation, among which steroid receptor coactivator-1 (SRC-1) has been shown to be multifunctional potentials in the brain, but whether it is regulated by letrozole is currently unknown. In this study, we examined letrozole regulation on SRC-1 expression in adult mice brain using immunohistochemistry. The results showed that letrozole induced dramatic decrease of SRC-1 in the medial septal, hippocampus, medial habenular nucleus, arcuate hypothalamic nucleus and superior colliculus (p<0.01). Significant decrease was detected in the dorsal lateral septal nucleus, bed nucleus of stria terminalis, ventral taenia tecta, dorsomedial and ventromedial hypothalamic nuclei, dorsomedial periaqueductal gray, superior paraolivary nucleus and pontine nucleus (p<0.05). In the hippocampus, levels of estradiol content, androgen receptor, estrogen receptor α and β also decreased significantly after letrozole injection. The above results demonstrated letrozole downregulation of SRC-1 in specific regions that are primarily related to learning and memory, cognition and mood, neuroendocrine as well as information integration, indicating that SRC-1 may be one important downstream central target of letrozole. Furthermore, these potential central adverse effects of letrozole should be taken into serious considerations.

Intriguing roles of hippocampus-synthesized 17β-estradiol in the modulation of hippocampal synaptic plasticity.

Accumulated studies have shown that 17β-estradiol (E2) can be de novo synthesized in the hippocampus, and its role in the regulation of hippocampal synaptic plasticity, which is the basis of learning and memory, has long been exploring. Steroidogenic enzymes (e.g., aromatase) that are essential to the hippocampus-synthesized synthesis of E2 have been detected in the hippocampus. Inhibition of E2 synthesis by aromatase inhibitors significantly reduces the density of hippocampal spine synapses, levels of some synaptic proteins such as spinopholin and synaptophysin. Moreover, the electrophysiological properties of hippocampal neurons are also changed in response to this inhibition. The influences of gonadal and hippocampal E2 on synaptic plasticity may exist some differences, since some reports showed that gonadal (or circulating) estrogens have no obvious effects in the modulation of hippocampal synaptic proteins as evidenced in some ovariectomized animals and postmenopausal women who suffered from Alzheimer’s disease (AD). These evidences leads to a hypothesis that hippocampal E2 may play a more important role in modulation of synaptic plasticity than gonadal E2. The signaling pathways, whereby hippocampal E2 modulates synaptic plasticity, insist of classical chronic genomic pathway and rapid nongenomic pathway, which mediated by nonnuclear estrogen receptor (GPER) and/or nuclear or nonnuclear estrogen receptors, which require coactivators for their transcription activity. Among which steroid receptor coactivator-1 (SRC-1) is the predominant coactivator p160 family members in the brain. Several clues have shown that SRC-1 is expressed in hippocampus and is highly correlated with some key synaptic proteins developmentally or after orchidectomy but not ovariectomy, indicating SRC-1 may be regulated by hippocampus-synthesized E2 and profoundly involved in the mediation of hippocampal E2 regulation of hippocampal synaptic plasticity. Further studies about the exact roles of hippocampus-synthesized E2 and therefore SRC-1 are urgently needed in order to facilitate our understanding of hippocampal E2, which will be very important to the development of novel strategies of estrogen replacement therapy against neurodegenerative deficits such as Alzheimer’s disease (AD).

Immunolocalization of estrogen receptor beta in the hypothalamic paraventricular nucleus of female mice during pregnancy, lactation and postnatal development

Previous studies have shown that estrogen receptor beta (ERbeta) is the predominant estrogen receptor in the hypothalamic paraventricular nucleus (PVN) of mouse, mediating estrogen regulation of the neuroendocrine activities of the PVN, but the exact roles that ERbeta plays in the PVN remain unclear. In this study, we used immunocytochemistry to investigate the expression of ERbeta in the maternal PVN of mice during pregnancy (pregnant days 8, 10, 12, 15 and 18), lactation (postpartum days 1, 4 and 8) as well as in the PVN of the females from postnatal days 1, 3, 5, 7, 9, 15, 30 and 70. We found out that ERbeta was predominantly localized in the magnocellular divisions of PVN. In the pregnant female brain, generally, the ERbeta was lower than that of the postnatal development, the lowest level was found at gestational days 10-12; then from gestational day 18 to postpartum day 1, it increased to higher levels, followed by a decrease from postpartum day 4. During the postnatal development, the highest level of ERbeta was found at early postnatal days (before postnatal day 15), thereafter, it decreased to a lower level. The above results indicate that circulating sex steroids may differentially regulate the expression of ERbeta in the PVN of mice. It also suggests that this receptor may play important roles in the regulation of parturition and in the development, food intake and body weight increases of the newborns by acting on the neuropeptides, which were also detected in the PVN.

A morphological and electrophysiological study on the postnatal development of oligodendrocyte precursor cells in the rat brain

A widespread population of cells in CNS is identified by specific expression of the NG2 chondroitin sulphate proteoglycan and named as oligodendrocyte precursor cell (OPC). OPCs may possess stem cell-like characteristics, including multipotentiality in vitro and in vivo. It was proposed that OPCs in the CNS parenchyma comprise a unique population of glia, distinct from oligodendrocytes and astrocytes. This study confirmed that NG2 immunoreactive OPCs were continuously distributed in cerebral cortex and hippocampus during different postnatal developmental stages. These cells rapidly increased in number over the postnatal 7 days and migrate extensively to populate with abundant processes both in developing cortex and hippocampus. The morphology of OPCs exhibited extremely complex changes with the distribution of long distance primary process gradually increased from neonatal to adult CNS. Immunohistochemical studies showed that OPCs exhibited the morphological properties that can be distinguished from astrocytes. The electrophysiological properties showed that OPCs expressed a small amount of inward Na(+) currents which was distinguished from Na(+) currents in neurons owing to their lower Na-to-K conductance ratio and higher command voltage step depolarized maximum Na(+) current amplitude. These observations suggest that OPCs can be identified as the third type of macroglia because of their distribution in the CNS, the morphological development in process diversity and the electrophysiological difference from astrocyte.