Thymelaea lythroides extract attenuates microglial activation and depressive-like behavior in LPS-induced inflammation in adult male rats.

Thymelaea lythroides extract is widely used as a traditional folk medicine in Morocco, especially for the treatment of diabetes, rheumatism and Inflammatory disease. The aim of the study is to evaluate the possible effect of methanolic extract of Thymelaea lythroides in repressing the inflammatory responses and long-lasting depression-like behavior associated with neuroinflammation in adult rats after neonatal LPS exposure. Male rat pups were treated systemically with either LPS (250??g/kg) or vehicle (phosphate buffer saline) on postnatal day 14. Six hours later, the LPS groups were assigned to intraperitoneal (ip) injection of Minocycline (50?mg/kg) or Thymelaea lythroides (200?mg/kg). Thereafter, in adulthood (postnatal days 90-97), the spontaneous locomotor activity and depression-like behavior were assessed successively in open field and forced swim tests. The levels of proinflammatory cytokines, oxidative damage, and activation of microglia were determined in the hippocampus (HP) of male rats on (PND90-97). Our results showed that open field hypoactivity and increased immobility period in LPS-induced adult rats were normalized on treatment with Thymelaea lythroides and minocycline. Both treatments attenuate the overactivated microglial cells in the CA1 and CA3 of hippocampus (HP) and significantly reduced the oxidative-nitrosative stress markers and cytokine (TNF ?) production in the HP. Thymelaea lythroides seems to have similar neuroprotective effects to Minocycline, and such protection may be due to: reduction of oxidative stress, upregulation of inflammatory mediators production, antidepressant behavior which all are associated with neuroinflammation.

Estrogen, synaptic plasticity and hypothalamic reproductive aging.

Unlike primates who undergo ovarian failure and loss of sex steroids at the end of reproduction, aging rodents undergo constant vaginal estrus followed by constant diestrus and finally anestrus, which indicates the absence of responsive ovarian follicles. The latter state is analogous to menopause in women. The timing of the appearance of constant estrus is determined by many factors including estrogen exposure in the brain during development and the number of times that the animal gets pregnant. The chief site of this reproductive aging in rat brains is the arcuate nucleus of the hypothalamus. The transition from normal cycles to constant estrus parallels the females' gradually decreased ability to respond to administered estradiol with a cycle of inhibition followed by disinhibition of gonadotrophin-releasing hormone. Evidence has accumulated indicating this to be due to a loss of the rat's ability to respond to markedly elevated estradiol with the usual arcuate nucleus neuro-glial plasticity that supports the estrogen-induced gonadotrophin surge (EIGS). Just as male rats are not capable of an EIGS, aged females loose this ability through repeated EIGS. Experiments indicate that in male rats the hypothalamic synaptology that develops as a result of exposure to testicular androgens in the perinatal period (brain sexual differentiation) is a result of conversion of testosterone from the testes to estrogen in the brain and is therefore due to early estrogen exposure. Aging females appear to reach a synaptology similar to males and constant estrus as a result of repeated exposure to ovarian estrogens during their reproductive careers. The relative role of aging and hormonal factors remains unclear. Morphological evidence is presented that indicates the above effects of estrogen involve changes in hypothalamic arcuate nucleus neurons and glia, including changes in the organization of perikaryal membranes as well as arcuate nucleus synaptology and the load of peroxidase in the astroglia. A possible role for free radicals (reactive oxygen species) in hypothalamic reproductive aging has been proposed. Such a mechanism is supported by evidence that the anti-oxidant vitamin E delays the onset of constant estrus and the accumulation of glial peroxidase in aging female rats. However, since the synaptology and peroxidase load in constant estrus females is independent of the age at which the constant estrus occurs, it appears that the role of (repeated) estradiol exposure is more deterministic of hypothalamic failure than is aging, per se.

Effects of early undernutrition on the brain insulin-like growth factor-I system.

Undernutrition reduces circulating concentrations of insulin-like growth factor (IGF)-I, but how it affects the brain IGF system, especially during development, is largely unknown. We have studied IGF-I, IGF-II, IGF receptor and IGF binding protein (BP)-2 mRNA expression in the hypothalamus, cerebellum and cerebral cortex of neonatal rats that were food restricted beginning on gestational day 16. One group was refed starting on postnatal day 14. Rats were killed on postnatal day 8 or 22. Undernutrition did not produce an overall reduction in brain weight at either age but, at 22 days, both the cerebellum and hypothalamus weighed significantly less. At 8 days, no change was detected in the central IGF axis in response to undernutrition. However, in 22-day-old undernourished rats, IGF-I and IGF receptor mRNA expression were increased in both the hypothalamus and cerebellum, while IGFBP-2 was decreased, but only in the hypothalamus. Refeeding had no effect on any of these parameters. These results suggest that the hypothalamus and cerebellum respond to malnutrition and the decrease in circulating IGF-I, a peptide fundamental for growth and development, by increasing the local production of both the growth factor and its receptor in attempt to maintain normal development.

Insulin-like growth factor I receptors and estrogen receptors colocalize in female rat brain.

Several findings indicate that there is a close interaction between estrogen and insulin-like growth factor I in different brain regions. In adult brain, both estrogen and insulin-like growth factor I have co-ordinated effects in the regulation of neuroendocrine events, synaptic plasticity and neural response to injury. In this study we have qualitatively assessed whether estrogen receptors and insulin-like growth factor I receptor are colocalized in the same cells in the preoptic area, hypothalamus, hippocampus, cerebral cortex and cerebellum of female rat brain using confocal microscopy. Immunoreactivity for estrogen receptors alpha and beta was colocalized with immunoreactivity for insulin-like growth factor I receptor in many neurons from the preoptic area, hypothalamus, hippocampus and cerebral cortex. Furthermore, estrogen receptor beta and insulin-like growth factor I receptor immunoreactivities were colocalized in the Purkinje cells of the cerebellum. Colocalization of estrogen receptor beta and insulin-like growth factor I receptor was also detected in cells with the morphology of astrocytes in all regions assessed. The co-expression of estrogen receptors and insulin-like growth factor I receptor in the same neurons may allow a cross-coupling of their signaling pathways. Furthermore, the colocalization of immunoreactivity for estrogen receptor beta and insulin-like growth factor I receptor in glial cells suggests that glia may also play a role in the interactions of insulin-like growth factor I and estrogen in the rat brain. In conclusion, the co-expression of estrogen receptors and insulin-like growth factor I receptors in the same neural cells suggests that the co-ordinated actions of estrogen and insulin-like growth factor I in the brain may be integrated at the cellular level.

Estradiol and progesterone regulate the expression of insulin-like growth factor-I receptor and insulin-like growth factor binding protein-2 in the hypothalamus of adult female rats.

Gonadal hormones interact with insulin-like growthfactor-I (IGF-I) to regulate synaptic plasticity during the estrous cycle in the rat mediobasal hypothalamus. It has been proposed that tanycytes, specialized glial cells lining the ventral region of the third ventricle, may regulate the availability of IGF-I to hypothalamic neurons. IGF-I levels in tanycytes fluctuate during the estrous cycle. Furthermore, estrogen administration to ovariectomized rats increases IGF-I levels in tanycytes, while progesterone, injected simultaneously with estrogen, blocks the estrogen-induced increase of IGF-I levels in tanycytes. To test whether hormonal regulation of IGF-I receptor (IGF-IR) and IGF binding protein-2 (IGFBP-2) may be involved in the accumulation of IGF-I in tanycytes, we assessed the effect of ovarian hormones on the levels of these molecules in the mediobasal hypothalamus of adult female rats. Ovariectomized animals were treated with either oil, estrogen, progesterone, or estrogen and progesterone simultaneously and then killed 6 or 24 h later. Some neurons, some astrocytes, and many tanycytes in the mediobasal hypothalamus were found by confocal microscopy to be immunoreactive for IGF-IR. IGFBP-2 immunoreactivity was restricted almost exclusively to tanycytes and ependymal cells and was colocalized with IGF-IR immunoreactivity in tanycytes. By electron microscope immunocytochemistry using colloidal gold labeling, IGF-IR and IGFBP-2 immunoreactivities were observed in the microvilli of tanycytes in the lumen of the third ventricle. IGF-IR and IGFBP-2 immunoreactive levels on the apical surface of tanycytes were significantly decreased by the administration of progesterone, either alone or in the presence of estradiol. IGF-IR levels in the mediobasal hypothalamus, measured by Western blotting, were not significantly affected by the separate administration of estradiol or progesterone to ovariectomized rats. However, the simultaneous administration of both hormones resulted in a marked decrease in IGF-IR protein levels. Estradiol administration to ovariectomized rats increased IGFBP-2 immunoreactive levels in the hypothalamus. While progesterone did not significantly affect IGFBP-2 expression, the simultaneous injection of estradiol and progesterone resulted in a marked decrease in IGFBP-2 protein levels. The effect of estradiol on IGFBP-2 was observed both in protein and mRNA levels, suggesting a transcriptional regulation. However, the simultaneous administration of progesterone and estradiol had different effects on IGF-IR protein and IGF-IR mRNA levels, as well as on IGFBP-2 protein and IGFBP-2 mRNA levels, suggesting a postranscriptional action. These findings indicate that estradiol and progesterone regulate the expression of IGF-IR and IGFBP-2 in the mediobasal hypothalamus of adult female rats. Regulation of the hypothalamic IGF-I system by ovarian hormones may be physiologically relevant for neuroendocrine regulation and for synaptic plasticity during the estrous cycle. These results do not support the hypothesis that estrogen-induced accumulation of IGF-I by tanycytes is mediated by the hormonal regulation of IGF-IR. However, estrogen-induced up-regulation of IGFBP-2 and progesterone-induced down-regulation of IGF-IR and IGFBP-2 levels in the apical plasma membrane of tanycytes may be involved in the fluctuation of IGF-I levels in the mediobasal hypothalamus during the estrous cycle.

Anatomically specific changes in the expression of somatostatin, growth hormone-releasing hormone and growth hormone receptor mRNA in diabetic rats.

Growth hormone (GH) secretion is altered in poorly controlled diabetic animals. However, modifications in the hypothalamic neuropeptides that control GH secretion, somatostatin and GH-releasing hormone (GHRH), as well as changes in the sensitivity of the hypothalamus and pituitary to the feedback effects of GH, are less clear. We have used RNase protection assays and in-situ hybridization to address whether the mRNA expression of GH, somatostatin and GHRH, as well as of the GH receptor (GHR) in the hypothalamus and anterior pituitary, are altered in streptozotocin-induced diabetic rats. After induction of diabetes, rats were treated with insulin twice daily for 3 weeks to obtain either poorly controlled (mean plasma glucose >300 mg/dl) or well-controlled diabetic rats. Although no significant change in pituitary GH mRNA expression was found, the hypothalamic expression of GHRH and somatostatin mRNA was reduced in poorly-controlled diabetic rats and returned to control values with normalisation of plasma glucose concentrations (P<0.0001 and P<0.002, respectively). Somatostatin mRNA expression was reduced only in the central portion of the periventricular nucleus, with no change being seen in the other areas of the periventricular nucleus or in the arcuate, suprachiasmatic or paraventricular nuclei. A significant decline in GHRH mRNA expression was observed in both the arcuate nucleus and ventromedial hypothalamus. Anterior pituitary GHR mRNA expression was significantly reduced in both well and poorly-controlled diabetic rats, while there was no change in the hypothalamus. To examine whether the evolution time of the diabetes influences these parameters, in a subsequent experiment, diabetic rats received no insulin for 2 months. A significant decline in GHRH and somatostatin mRNA expression was also observed in these rats. In addition, pituitary GH mRNA expression declined significantly in long-term diabetic rats. These results demonstrate that: (1) the expression of both GHRH and somatostatin declines specifically in anatomical areas involved in anterior pituitary hormone control; (2) GHR mRNA expression is decreased in the pituitary of diabetic rats, but not in the hypothalamus, and does not return to control values with normalisation of mean blood glucose concentrations; and (3) the evolution time of the diabetes is important for detecting some changes, including the decrease in pituitary GH mRNA expression.

Cellular composition of the adult rat anterior pituitary is influenced by the neonatal sex steroid environment.

Growth hormone (GH) and prolactin (PRL) secretion differ significantly between adult males and females and this is due, at least in part, to the postpubertal hormone environment which affects GH and PRL gene expression, as well as somatotrope and lactotrope proliferation. However, the role of the neonatal steroid environment in this phenomenon is less well understood. We have used in situ hybridization to determine the number of GH and PRL mRNA containing cells, as well as the level of expression of these two hormones and of the pituitary transcription factor 1 (Pit-1). Neonatally castrated male rats that had been exposed to testosterone during the neonatal period, adulthood or during both periods, males castrated as adults, normal adult males and normal proestrous females were used. Orchidectomy of adult rats had no effect on the number of somatotropes or lactotropes, but significantly reduced GH and PRL mRNA levels. Neonatal castration significantly reduced the percentage of somatotropes and increased that of lactotropes in the adult male. In addition, GH and Pit-1 mRNA levels were reduced significantly, but PRL mRNA levels were not modified. Treatment of neonatally castrated males with testosterone during the neonatal period significantly increased the percentage of somatotropes and decreased the percentage of lactotropes compared to vehicle-treated animals. It also increased GH and Pit-1 mRNA levels, but did not affect PRL mRNA levels. Adult testosterone treatment significantly increased the percentage of both somatotropes and lactotropes, as well as GH, PRL and Pit-1 mRNA levels. Treatment of neonatally castrated males with testosterone during both the neonatal and adult periods returned the percentage of somatotropes and lactotropes, as well as GH, PRL and Pit-1 mRNA levels, to that of the intact male. These results suggest that, although the postpubertal steroid environment is important in determining anterior pituitary hormone synthesis and cellular composition, the neonatal steroid environment also plays an important role in this phenomenon.

Role of astroglia and insulin-like growth factor-I in gonadal hormone-dependent synaptic plasticity.

Gonadal hormones exert a critical influence over the architecture of specific brain areas affecting the formation of neuronal contacts. Cellular mechanisms mediating gonadal hormone actions on synapses have been studied extensively in the rat arcuate nucleus, a hypothalamic center involved in the feed-back regulation of gonadotropins. Gonadal steroids exert organizational and activational effects on arcuate nucleus synaptic connectivity. Perinatal testosterone induces a sexual dimorphic pattern of synaptic contacts. Furthermore, during the preovulatory and ovulatory phases of the estrous cycle there is a transient disconnection of inhibitory synaptic inputs to the somas of arcuate neurons. This synaptic remodeling is induced by estradiol, blocked by progesterone, and begins with the onset of puberty in females. Astroglia appear to play a significant role in the organizational and the activational hormone effects on neuronal connectivity by regulating the amount of neuronal membrane available for the formation of synaptic contacts and by releasing soluble factors, such as insulin-like growth factor I (IGF-I), which promote the differentiation of neural processes. Recent evidence indicates that gonadal steroids and IGF-I may interact in their trophic effects on the neuroendocrine hypothalamus. Estradiol and IGF-I promote the survival and morphological differentiation of rat hypothalamic neurons in primary cultures. The effect of estradiol depends on IGF-I, while the effects of both estradiol and IGF-I depend on estrogen receptors. Furthermore, estrogen activation of astroglia in hypothalamic tissue fragments depends on IGF-I receptors. These findings indicate that IGF-I may mediate some of the developmental and activational effects of gonadal steroids on the brain and suggest that IGF-I may activate the estrogen receptor to induce its neurotrophic effects on hypothalamic cells. In addition, IGF-I levels in the neuroendocrine hypothalamus are regulated by gonadal steroids. IGF-I levels in tanycytes, a specific astroglia cell type present in the arcuate nucleus and median eminence, increase at puberty, are affected by neonatal androgen levels, show sex differences, and fluctuate in accordance to the natural variations in plasma levels of ovarian steroids that are associated with the estrous cycle. These changes appear to be mediated by hormonal regulation of IGF-I uptake from blood or cerebrospinal fluid by tanycytes. These results suggest that tanycytes may be involved in the regulation of neuroendocrine events in adult rats by regulating the availability of IGF-I to hypothalamic neurons. In summary, IGF-I and different forms of neuron-astroglia communication are involved in the effects of estradiol on synaptic plasticity in the hypothalamic arcuate nucleus.

Endocrine-dependent accumulation of IGF-I by hypothalamic glia.

Tanycytes are specialized glial cells of the hypothalamus and median eminence. Immunoreactive insulin-like growth factor I (IGF-I) levels fluctuate in tanycytes with the natural variations in sex steroids associated with the ovarian cycle. To determine whether these changes are as a result of differences in IGF-I accumulation, the peptide was labelled with digoxigenin and injected into the lateral cerebral ventricle. Tanycyte-like cells specifically accumulated digoxigenin-labelled IGF-I. This accumulation was mediated by IGF-I receptors and showed marked differences during the oestrous cycle, being low in the afternoon of pro-oestrus and high in the afternoon of oestrus. These results indicate that the accumulation by tanycytes of IGF-I or IGF-I fragments capable of receptor-mediated internalization is under endocrine control, suggesting that hypothalamic glia may be involved in neuroendocrine regulation.

Interaction of insulin-like growth factor-I and estradiol signaling pathways on hypothalamic neuronal differentiation.

Neurotrophic effects of estradiol and insulin-like growth factor-I were assessed in primary cultures from fetal rat hypothalamus. Cultured neurons were immunostained with an antibody for the microtubule-associated protein-2. While both estradiol and insulin-like growth factor-I increased the number of microtubule-associated protein-2-immunoreactive neurons and the extension of immunoreactive processes, the effect of these two factors was not additive. The estradiol-induced increases in neuronal numbers and extension of neuronal processes were blocked by either the estrogen receptor antagonist ICI 182,780 or by an anti-sense oligonucleotide to the estrogen receptor. Furthermore, incubation of the cultures with an anti-sense oligonucleotide directed against the insulin-like growth factor-I messenger RNA also blocked the effect of estradiol. In turn, the effects of insulin-like growth factor-I were blocked by the estrogen receptor antagonist ICI 182,780 and by the anti-sense oligonucleotide to the estrogen receptor. These findings suggest that estradiol-induced activation of the estrogen receptor in developing hypothalamic cells requires the presence of insulin-like growth factor-I, and that both estradiol and insulin-like growth factor-I use the estrogen receptor as a mediator of their trophic effects on hypothalamic neurons.

A new organotypic culture method to study the actions of steroid hormones on the nervous system.

A new organotypic culture method for growing slices of nervous system tissue, based on the use of hyaluronic acid as a growth supporting milieu, is described. This method allows cultures derived from either fetuses or newborns to grow and develop with markedly reduced amounts of added serum. Organotypic cultures from fetal rat hypothalamus were exposed to 17 beta estradiol and compared to control cultures exposed to the ethanol vehicle. When exposed to estradiol, cultures showed an outgrowth of thick nerve fibers that was accompanied by an elevation in the number of microtubules present in the neuronal processes, an increment in the number of synapses, and an increased morphological differentiation of synaptic terminals. Freeze-fracture analysis of neuronal membranes from estradiol-treated cultures revealed a significant increase in the number of exoendocytotic images and a decrease in the number of intramembranous particles. Estradiol's effects parallel those found in in vivo studies, indicating that hyaluronic acid-based organotypic cultures represent an appropriate model to study hormonal influences on the developing nervous system.

The role of glia in the neuroendocrine hypothalamus: possible implications in hormone secretion.

Recent evidence indicates that glia may play a significant role in the link between the endocrine and nervous systems. Gonadal steroids modulate astroglia morphology, differentiation and gene expression in different brain areas. Hormonal effects on glia may have important consequences for neuronal development, metabolism and activity, for the formation and plasticity of synaptic connections, and for the modulation of hypothalamic hormone release. Perinatal and adult testosterone levels modulate the expression of the specific astroglia cytoskeletal marker glial fibrillary acidic protein in the arcuate nucleus of the rat hypothalamus. These changes parallel hormonal effects on the expression of growth hormone-releasing hormone (GHRH) and the number of GHRH neurones in the arcuate nucleus. The effects of testosterone and its metabolite oestradiol on hypothalamic neurones may be dependent on the release by hypothalamic astroglia of insulin-like growth factor I, a molecule involved in the control of growth hormone secretion.

Interaction of the signalling pathways of insulin-like growth factor-I and sex steroids in the neuroendocrine hypothalamus.

Among the numerous endocrine signals that affect the central nervous system, sex steroids play an important role. It has been recently postulated that part of the effects of these hormones on the brain may be mediated by trophic factors, such as insulin-like growth factor I (IGF-I). Both estradiol and IGF-I increase the survival and differentiation of developing fetal rat hypothalamic neurons in culture. The effect of estradiol is blocked by the pure estrogen receptor antagonist ICI 182,780, by an antisense oligonucleotide to the estrogen receptor, and by an antisense oligonucleotide to IGF-I. In turn, the effect of IGF-I is blocked by ICI 182,780 and by the antisense oligonucleotide to the estrogen receptor. These findings indicate that estrogen-induced activation of the estrogen receptor in developing hypothalamic neurons requires the presence of IGF-I and that both estradiol and IGF-I use the estrogen receptor to mediate their trophic effects on hypothalamic cells. In vivo, sex steroids affect IGF-I levels in the endocrine hypothalamus. IGF-I levels in tanycytes, a specific subtype of glial cells present in the arcuate nucleus and median eminence, are sexually dimorphic in the rat, increase with the onset of puberty, and are regulated by perinatal and adult levels of sex steroids. These changes may be due to hormonal modifications of IGF-I uptake by tanycytes from blood or cerebrospinal fluid. Therefore, this type of glial cell appears to play a central role in the interaction of sex steroids and IGF-I in the hypothalamus.

Sexual dimorphism and sex steroid modulation of glial fibrillary acidic protein messenger RNA and immunoreactivity levels in the rat hypothalamus.

By using the techniques of in situ hybridization histochemistry and immunocytochemistry, we have found that both glial fibrillary acidic protein messenger RNA levels and glial fibrillary acidic protein immunoreactive surface density in the arcuate nucleus and median eminence are modulated by both the neonatal and adult sex steroid environments. No effect was seen on the number of immunoreactive glia. Intact adult males had significantly higher glial fibrillary acidic protein messenger RNA levels and glial fibrillary acidic protein immunoreactive surface density than females. Both adult and neonatal castration of male animals significantly reduced glial fibrillary acidic protein messenger RNA levels and glial fibrillary acidic protein immunoreactive surface density. Neonatal and adult testosterone treatment increased both of these parameters in both sexes; however, there was no additive effect of the steroid treatments. Glial cells are involved in the proliferation, survival, migration and maturation of neurons, as well as in the modulation of synaptic connectivity, and therefore it follows that hormonal modulation of glia may mediate some of the known steroid effects on neurons. The data reported here show that astroglia are significantly influenced by both the neonatal and adult sex steroid environments and suggest that some of the steroid effects on neurons during both of these developmental periods may be mediated, at least in part, through modulation of glial cells.

Gonadal hormone regulation of neuronal-glial interactions in the developing neuroendocrine hypothalamus.

Recent evidence indicates that, in addition to their well known effects on neurons, gonadal steroids may exert part of their neural effects through astroglia. In adult female rats astroglia participate in the phasic remodelling of synapses that takes place during the estrous cycle in the arcuate nucleus of the hypothalamus under the influence of estradiol. Astroglia also appear to be involved in the genesis of sex differences in synaptic connectivity. Gonadal steroids influence hypothalamic astroglia differentiation in vitro and in vivo. In monolayer mixed neuronal-glial cultures from fetal rat hypothalami, estradiol induces a progressive differentiation of astrocytes from a flattened epithelioid morphology to bipolar, radial and stellate shapes. This effect of estradiol on astroglia is dependent on the expression of specific molecules on the neuronal surface, such as the polysialic acid-rich form of the neural cell adhesion molecule. In the rat arcuate nucleus in situ, perinatal androgen influences astroglia gene expression and differentiation, resulting in a sex difference in astroglia organization by postnatal day 20. By this day, the amount of neuronal surface covered by astroglial processes is higher in males than in females. This difference in the coverage of neuronal surface by astroglia may be directly related to the reduced number of synaptic contacts that is established on the soma of male neurons compared to females.

Estradiol promotion of changes in the morphology of astroglia growing in culture depends on the expression of polysialic acid of neural membranes.

Gonadal steroids are known to affect astroglial morphology in developing and adult animals. Earlier studies of mixed neuronal-glial cultures from fetal rat hypothalamus showed that glial fibrillary acidic protein (GFAP)-immunoreactive cells with a polygonal shape were transformed into process-bearing cells upon exposure to the ovarian hormone estradiol. This effect was dependent on a direct contact of astroglia with living hypothalamic neurons. The present study shows that somata and processes of neurons in such cultures were immunoreactive for polysialic acid (PSA); astroglia were immunonegative. PSA appears to participate in the estradiol-induced shape changes since treatment with endoneuraminidase, an enzyme that specifically removes PSA from the cell surface, abolished PSA immunostaining and prevented the 17 beta-estradiol-induced morphological changes of astroglia. In contrast, treatment with endoneuraminidase did not affect astroglial shape changes induced by basic fibroblast growth factor (bFGF), nor those induced by the addition of neurons to glial cultures. These results suggest that PSA on neuronal membranes, probably linked to the highly sialylated isoform of the neural cell adhesion molecule, is necessary for the expression of certain hormonally-regulated neuro-glial interactions.

Regulation of arcuate nucleus synaptology by estrogen.

Estrogen modulates the synaptology of the hypothalamic arcuate nucleus during sexual differentiation of the rat brain in both males and females. In males, testosterone of gonadal origin is converted to estrogen in the brain by an enzyme, aromatase, which is also present in females. The exposure of the male's hypothalamus to relatively high levels of estrogen (following a perinatal testosterone surge) leads to the development of a pattern of synaptogenesis which does not support an estrogen-induced gonadotrophin surge in the adult. In female rats, hypothalamic development occurs with permissively low levels of estrogen, enabling a midcycle estrogen-induced gonadotrophin surge and ovulation in adulthood. During adult reproductive life in female rats, circulating estrogen modulates the synaptology of the arcuate nucleus. The most physiological example of this is the 30-50% loss of axosomatic synapses following the preovulatory estrogen surge on diestrus-proestrus. Studies on post-synaptic membranes of the arcuate nucleus reveal sex differences in membrane organization and protein content which are estrogen-dependent. Estrogen apparently stimulates endocytosis of areas of post-synaptic membrane that are dense with small intramembranous protein particles, resulting in a reduction in the number of small intramembranous particles. This also appears to be the physiologic mechanism of neuronal changes in females during the estrus cycle. Repeated exposure to preovulatory levels of estrogen may lead to an age-related decline in reproductive capacity in female rats. Aging females lose the estrogen-induced gonadotrophin surge responsible for ovulation. This loss of function may result from a cumulative estrogen effect during the repeated ovarian cycles which results in a reorganization of the synaptology on which regulates the estrogen-induced gonadotrophin surge. The membrane organization of the senescent constant estrus aged female appears indistinguishable from the males. The hypothalamic circuits modulated by estrogen have yet to be delineated. However, recent research has shown that GABA, the monoamines, and several neuropeptides are participants in the estrogen-sensitive network which regulates GNRH secretion. In this regard, present work shows estrogen-induced changes in GABA and dopamine synapses in the arcuate nucleus.

Gonadal hormone regulation of insulin-like growth factor-I-like immunoreactivity in hypothalamic astroglia of developing and adult rats.

The influence of gonadal steroids on insulin-like growth factor I (IGF-I)-like immunoreactivity was assessed in the rat arcuate nucleus, an area of the hypothalamus that regulates pituitary secretion. IGF-I-like immunoreactivity was observed in hypothalamic cells with the morphological aspects of tanycytes and astrocytes. The surface density of IGF-I-like immunoreactive glia increased with puberty in the arcuate nucleus of male and female rats, while decreasing with age in other brain areas. Gender differences in the surface density of IGF-I-like immunoreactive glia were detected in adult animals, with males and androgenized females having significantly higher values than normal females. In the latter, the surface density of IGF-I-like immunoreactive glia was increased in the afternoon of proestrus and in the morning of estrus compared to the morning of proestrus, diestrus and metestrus. In addition, IGF-I-like immunoreactivity showed a dose-dependent increase in ovariectomized rats injected with 17 beta-estradiol, but not in those receiving 17 alpha-estradiol. The effect of 17 beta-estradiol was blocked by simultaneous administration of progesterone, while this hormone alone had no effect. These results indicate that IGF-I-like immunoreactivity in arcuate glial cells is affected by the hormonal environment and suggest that IGF-I-like immunoreactive glia may be involved in neuroendocrine events within the hypothalamus.

Astroglial induction of in vivo angiogenesis.

A purified population of astrocytes was prepared from embryonic rat hypothalamus. These cells were transplanted into the cerebral cortex of adult rats with survival time of 5 days and studied by glial fibrillary acidic protein (GFAP) immunohistochemistry and electron microscopy. A progressive gradation from the edges of the implant to the intact host tissue was observed in relation to the vascularization process. The regenerating tissue showed cells with cavities resembling capillary central lumens and their cytoplasms revealed gold particles when they were immunostained for electron microscopy. Dark processes were seen in capillary-like lumens and, on other occasions, evaginations of endothelial cells were in contact with astroglial processes. These findings lead us to suggest that capillary-like structures might develop from cavitated astroglial cells, which would permit the migration of endothelial cells into their lumens. Astroglial-endothelial interactions persist until endothelial cells are morphologically differentiated. One possible interpretation of the present data is that astrocytes might participate directly and actively in the regulation of capillary formation.