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The endogenous cannabinoid system is a new signaling system composed by the central (CB1) and the peripheral (CB2) receptors, and several lipid transmitters including anandamide and 2-arachidonylglycerol. This system is the target of natural cannabinoids, the psychoactive constituents of Cannabis sativa preparations (marijuana, hashish). Acute and chronic cannabis exposure has been associated with subjective feelings of pleasure and relaxation, but also to the onset of psychiatric syndromes, a decrease of the efficacy of neuroleptics and alterations in the extrapyramidal system regulation of motor activity. These actions point to a tight association of the cannabinoid system with the brain dopaminergic circuits involved in addiction, the clinical manifestation of positive symptoms of schizophrenia and Parkinson's disease. The present work discusses anatomical, biochemical and pharmacological evidences supporting a role for the endogenous cannabinoid system in the modulation of dopaminergic transmission. Cannabinoid CB1 receptors are present in dopamine projecting brain areas. In primates and certain rat strains it is also located in dopamine cells of the A8, A9 and A10 mesencephalic cell groups, as well as in hypothalamic dopaminergic neurons controlling prolactin secretion. CB1 receptors co-localize with dopamine D1/D2 receptors in dopamine projecting fields. Manipulation of dopaminergic transmission is able to alter the synthesis and release of anandamide as well as the expression of CB1 receptors. Additionally, CB1 receptors can switch its transduction mechanism to oppose to the ongoing dopamine signaling. Acute blockade of CB1 receptor potentiates the facilitatory role of dopamine D2 receptor agonists on movement. CB1 stimulation results in sensitization to the motor effects of indirect dopaminergic agonists. The dynamics of these changes indicate that the cannabinoid system is an activity-dependent modulator of dopaminergic transmission, an hypothesis relevant for the design of new therapeutic strategies for dopamine-related diseases such as the psychosis and Parkinson's disease.

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Pain perception is influenced by sex and aging, with previous studies indicating the involvement of aromatase, the estradiol synthase enzyme, in regulating pain perception. Previous research has established the presence of aromatase in dorsal root ganglia sensory neurons and its role in modulating pain perception. The present study aims to explore the implications of aging and sex on the expression of aromatase and estrogen receptors in the trigeminal ganglion. The study examined mRNA levels of aromatase, ERs, and the androgen receptor (AR) in the trigeminal ganglion of 3-month-old and 27-month-old male and female mice, as well as 3-month-old mice from the four-core genotype (FCG) transgenic model. The latter facilitates the assessment of gonadal hormone and sex chromosome implications for sex-specific traits. Aromatase localization in the ganglion was further assessed through immunohistochemistry. Aromatase immunoreactivity was observed for the first time in sensory neurons within the trigeminal ganglion. Trigeminal ganglion gene expressions were detected for aromatase, ERs, and AR in both sexes. Aromatase, ERβ, and GPER gene expressions were higher in young males versus young females. Analyses of the FCG model indicated that sex differences depended solely on gonadal sex. The aging process induced an enhancement in the expression of aromatase, ERs, and AR genes across both sexes, culminating in a reversal of the previously observed gender-based differences. the potential impact of estrogen synthesis and signaling in the trigeminal ganglion on age and sex differences warrants consideration, particularly in relation to trigeminal sensory functions and pain perception., (© 2024. The Author(s).)

Multiple sclerosis (MS) is an immune-mediated and degenerating disease in which myelin sheaths are damaged as a result of chronic progressive inflammation of the central nervous system. Tibolone [(7α,17α)-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-in-3-one], a synthetic estrogenic compound with tissue-specific actions and used for menopausal hormone therapy, shows neuroprotective and antioxidant properties both in vivo and in vitro. In the present study, we analyzed whether tibolone plays a therapeutic role in experimental autoimmune encephalomyelitis (EAE) mice, a commonly used model of MS. Female C57BL/6 mice were induced with the myelin oligodendrocyte glycoprotein MOG 35-55 and received s.c. tibolone (0.08 mg kg -1 ) injection every other day from the day of induction until death on the acute phase of the disease. Reactive gliosis, Toll like receptor 4 (TLR4), high mobility group box protein 1 (HMGB1), inflammasome parameters, activated Akt levels and myelin were assessed by a real-time polymerase chain reaction, immunohistochemistry, and western blot analysis. Our findings indicated that, in the EAE spinal cord, tibolone reversed the astrocytic and microglial reaction, and reduced the hyperexpression of TLR4 and HMGB1, as well as NLR family pyrin domain containing 3-caspase 1-interleukin-1β inflammasome activation. At the same time, tibolone attenuated the Akt/nuclear factor kappa B pathway and limited the white matter demyelination area. Estrogen receptor expression was unaltered with tibolone treatment. Clinically, tibolone improved neurological symptoms without uterine compromise. Overall, our data suggest that tibolone may serve as a promising agent for the attenuation of MS-related inflammation., (© 2021 British Society for Neuroendocrinology.)

Alzheimer's disease (AD) is the most common form of dementia and has a higher incidence in women. The main component of the senile plaques characteristic of AD is amyloid-beta (Aβ), with surrounding astrocytes contributing to the degenerative process. We hypothesized that the sex difference in the incidence of AD could be partially due to differential astrocytic responses to Aβ. Thus, the effect of Aβ 1-40 on cell viability, the inflammatory response, and oxidative status was studied in cultures of hippocampal astrocytes from male and female rats. Aβ 1-40 increased astrocyte viability in both female and male cultures by activating proliferation and survival pathways. Pro-inflammatory and anti-inflammatory responses were induced in astrocytes from both sexes. Aβ 1-40 did not affect endoplasmic reticulum stress although it induced oxidative stress in male and female astrocytes. Interestingly, male astrocytes had an increase in cell number and significantly lower cell death in response to Aβ 1-40 . Conversely, astrocytes from females displayed a greater inflammatory response after the Aβ 1-40 challenge. These results suggest that the inflammatory and oxidative environment induced by Aβ 1-40 in female astrocytes may contribute to enhance the vulnerability to AD and warrants further studies to unveil the mechanisms underlying sex differences in astrocytic responses., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Including sex is of paramount importance in preclinical and clinical stroke researches, and molecular studies dealing in depth with sex differences in stroke pathophysiology are needed. To gain insight into the molecular sex dimorphism of ischaemic stroke in rat cerebral cortex, male and female adult rats were subjected to transient middle cerebral artery occlusion. The expression of neuroglobin (Ngb) and other functionally related molecules involved in sex steroid signalling (oestrogen and androgen receptors), steroidogenesis (StAR, TSPO and aromatase) and autophagic activity (LC3B-II/LC3B-I ratio, UCP2 and HIF-1α) was assessed in the ipsilateral ischaemic and contralateral non-ischaemic hemispheres. An increased expression of Ngb was detected in the injured female cerebral cortex. In contrast, increased expression of oestrogen receptor α, GPER, StAR, TSPO and UCP2, and decreased androgen receptor expression were detected in the injured male cortex. In both sexes, the ischaemic insult induced an upregulation of LC3B-II/-I ratio, indicative of increased autophagy. Therefore, the cerebral cortex activates both sex-specific and common molecular responses with neuroprotective potential after ischaemia-reperfusion, which globally results in similar stroke outcome in both sexes. Nonetheless, these different potential molecular targets should be taken into account when neuroprotective drugs aiming to reduce brain damage in ischaemic stroke are investigated., (© 2020 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Obesity is one of the most important health problems facing developed countries because being overweight is associated with a higher incidence of type 2 diabetes, cardiovascular disease and cancer, as well as other comorbidities. Although increased weight gain results from a combination of poor dietary habits and decreased energy expenditure, not all individuals have equal propensities to gain weight or to develop secondary complications of obesity. This is partially a result not only of genetics, including sex, but also the time during which an individual is exposed to an obesogenic environment. In the present study, we have compared the response of male and female mice to short-term exposure to a high-fat diet (HFD) or a low-fat diet during the peripubertal period (starting at 42 days of age) because this is a stage of dramatic hormonal and metabolic modifications. After 1 week on a HFD, there was no significant increase in body weight, although females significantly increased their energy intake. Serum leptin levels increased in both sexes, even though no change in fat mass was detected. Glyceamia and homeostasis model assessment increased in males, suggesting a rapid change in glucose metabolism. Hypothalamic pro-opiomelanocortin mRNA levels were significantly higher in females on a HFD compared to all other groups, which may be an attempt to reduce their increased energy intake. Hypothalamic inflammation and gliosis have been implicated in the development of secondary complications of obesity; however, no indication of activation of inflammatory processes or gliosis was found in response to 1 week of HFD in the hypothalamus, hippocampus or cerebellum of these young mice. These results indicate that there are both sex and age effects in the response to poor dietary intake because peripubertal male and female mice respond differently to short-term dietary changes and this response is different from that reported in adult rodents., (© 2019 British Society for Neuroendocrinology.)

Microglial cells become dystrophic with aging; this phenotypic alteration contributes to basal central nervous system (CNS) neuroinflammation being a risk factor for age related neurodegenerative diseases. In previous studies we have observed that insulin like growth factor 1 (IGF1) gene therapy is a feasible approach to target brain cells, and that is effective to modify inflammatory response in vitro and to ameliorate cognitive or motor deficits in vivo . Based on these findings, the main aim of the present study is to investigate the effect of IGF1 gene therapy on microglia distribution and morphology in the senile rat. We found that IGF1 therapy leads to a region-specific modification of aged microglia population.

Parkinson's disease (PD) is the second most frequent age-related neurodegenerative disorder. Sex is an important factor in the development of PD, as reflected by the fact that it is more common in men than in women by an approximate ratio of 2:1. Our hypothesis is that differences in PD among men and women are highly determined by sex-dependent differences in the nigrostriatal dopaminergic system, which arise from environmental, hormonal and genetic influences. Sex hormones, specifically estrogens, influence PD pathogenesis and might play an important role in PD differences between men and women. The objective of this review was to discuss the PD physiopathology and point out sex differences in nigrostriatal degeneration, symptoms, genetics, responsiveness to treatments and biochemical and molecular mechanisms among patients suffering from this disease. Finally, we discuss the role estrogens may have on PD sex differences., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Astrocytes participate in both physiological and pathophysiological responses to metabolic and nutrient signals. Although most studies have focused on the astrocytic response to weight gain due to high-fat/high-carbohydrate intake, surplus intake of a balanced diet also induces excess weight gain. We have accessed the effects of neonatal overnutrition, which has both age- and sex-dependent effects on weight gain, on hypothalamic inflammation/gliosis. Although both male and female Wistar rats accumulate excessive fat mass as early as postnatal day (PND) 10 with neonatal overnutrition, no increase in hypothalamic cytokine levels, markers of astrocytes or microglia, or inflammatory signaling pathways were observed. At PND 50, no effect of neonatal overnutriton was found in either sex, whereas at PND 150, males again weighed significantly more than their controls, and this was coincident with an increase in markers of inflammation and astrogliosis in the hypothalamus. Circulating triglycerides and free fatty acids were also elevated in these males, but not in females or in either sex at PND 10. Thus, the effects of fatty acids and estrogens on astrocytes in vitro were analyzed. Our results indicate that changes in circulating fatty acid levels may be involved in the induction of hypothalamic inflammation/gliosis in excess weight gain, even on a normal diet, and that estrogens could participate in the protection of females from these processes. In conclusion, the interaction of developmental influences, dietary composition, age, and sex determines the central inflammatory response and the associated long-term outcomes of excess weight gain., (Copyright © 2018 Endocrine Society.)

Voltage-gated calcium channels are key regulators of brain function, and their dysfunction has been associated with multiple conditions and neurodegenerative diseases because they couple membrane depolarization to the influx of calcium-and other processes such as gene expression-in excitable cells. L-type calcium channels, one of the three major classes and probably the best characterized of the voltage-gated calcium channels, act as an essential calcium binding proteins with a significant biological relevance. It is well known that estradiol can activate rapidly brain signaling pathways and modulatory/regulatory proteins through non-genomic (or non-transcriptional) mechanisms, which lead to an increase of intracellular calcium that activate multiple kinases and signaling cascades, in the same way as L-type calcium channels responses. In this context, estrogens-L-type calcium channels signaling raises intracellular calcium levels and activates the same signaling cascades in the brain probably through estrogen receptor-independent modulatory mechanisms. In this review, we discuss the available literature on this area, which seems to suggest that estradiol exerts dual effects/modulation on these channels in a concentration-dependent manner (as a potentiator of these channels in pM concentrations and as an inhibitor in nM concentrations). Indeed, estradiol may orchestrate multiple neurotrophic responses, which open a new avenue for the development of novel estrogen-based therapies to alleviate different neuropathologies. We also highlight that it is essential to determine through computational and/or experimental approaches the interaction between estradiol and L-type calcium channels to assist these developments, which is an interesting area of research that deserves a closer look in future biomedical research.

Although the brain is composed of numerous cell types, neurons have received the vast majority of attention in the attempt to understand how this organ functions. Neurons are indeed fundamental but, in order for them to function correctly, they rely on the surrounding "non-neuronal" cells. These different cell types, which include glia, epithelial cells, pericytes, and endothelia, supply essential substances to neurons, in addition to protecting them from dangerous substances and situations. Moreover, it is now clear that non-neuronal cells can also actively participate in determining neuronal signaling outcomes. Due to the increasing problem of obesity in industrialized countries, investigation of the central control of energy balance has greatly increased in attempts to identify new therapeutic targets. This has led to interesting advances in our understanding of how appetite and systemic metabolism are modulated by non-neuronal cells. For example, not only are nutrients and hormones transported into the brain by non-neuronal cells, but these cells can also metabolize these metabolic factors, thus modifying the signals reaching the neurons. The hypothalamus is the main integrating center of incoming metabolic and hormonal signals and interprets this information in order to control appetite and systemic metabolism. Hence, the factors transported and released from surrounding non-neuronal cells will undoubtedly influence metabolic homeostasis. This review focuses on what is known to date regarding the involvement of different cell types in the transport and metabolism of nutrients and hormones in the hypothalamus. The possible involvement of non-neuronal cells, in particular glial cells, in physiopathological outcomes of poor dietary habits and excess weight gain are also discussed.

Microglial cells are extremely important for homeostasis of the CNS. Upon brain damage, microglia become reactive in response to inflammatory stimuli and lead to the secretion of inflammatory cytokines. Because microglia have the ability of adjusting their steady state to an active phenotype that modulates the CNS environment, chronic activation of microglia has an important role in mediating neuroinflammatory brain diseases. Depending upon the nature and degree of the injury stimulus, microglial activity may alternate, either to acute and mild responses -sometimes beneficial- or chronic and severe that may result in neurodegeneration. In this context, proper and controlled activation of microglia should be considered as a potential neuroprotective strategy against neurodegeneration. More recently, the use of estrogenic compounds to regulate microgliosis has shown promising results, and is currently being investigated due to their potential pharmacologic ability in the regulation of inflammation. In this review, we highlight the role of microgliamediated damage and discuss the effect of neurosteroids in reducing the adverse impact of inflammation in the brain.

Imbalances between excitatory and inhibitory transmissions in the brain anticipate the neuronal damage and death that occur in the neurodegenerative diseases like Alzheimer's disease (AD). We previously showed that amyloid-β (Aß), a natural peptide involved in the onset and development of AD, counteracts the neurotrophic activity of the nerve growth factor (NGF) by dampening the γ-aminobutyric acid (GABA)ergic connectivity of cultured hippocampal neurons. Neuronal plasticity is partly controlled by the NGF-promoted expression of the homologue of enhancer-of-split 1 (Hes1), a transcription factor that regulates the formation of GABAergic synapses. We now show that Hes1 controls the expression of cerebellin 4 (Cbln4), a member of a small family of secreted synaptic proteins, and we present the evidence that Cbln4 plays an essential role in the formation and maintenance of inhibitory GABAergic connections. Cbln4 immunoreactivity was found in the hippocampus, mostly in the dendrites and somata of pyramidal neurons. In the CA1, the hippocampal region where the first neurons degenerate in AD, Cbln4 immunoreactivity was associated with GABAergic synapses (detected by vesicular inhibitory amino acid transporter [VGAT] immunostaining), which appear to surround and embrace the somata of CA1 pyramidal neurons (basket cells). Moreover, significant decreases of Hes1, Cbln4, and VGAT immunoreactivities and messenger RNA expression were found in the hippocampus of a mouse model of AD. We also found that either the overexpression of Cbln4 in cultured hippocampal neurons or the application of recombinant Cbln4 to the cultures increased the number of GABAergic varicosities, rescuing neurons from Aß-induced death. In contrast, knockdown of Cbln4 gene in cultured neurons was followed by a large reduction of GABAergic connections. Such an effect was reverted by exogenously added Cbln4. These findings suggest a therapeutic potential for Cbln4 in the treatment of AD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The blood-brain barrier (BBB) is a tightly regulated interface in the Central Nervous System (CNS) that regulates the exchange of molecules in and out from the brain thus maintaining the CNS homeostasis. It is mainly composed of endothelial cells (ECs), pericytes and astrocytes that create a neurovascular unit (NVU) with the adjacent neurons. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted factors that lead to the adequate association between the cells of the BBB and the formation of strong tight junctions. Under neurological disorders, such as chronic cerebral ischemia, brain trauma, Epilepsy, Alzheimer and Parkinson's Diseases, a disruption of the BBB takes place, involving a lost in the permeability of the barrier and phenotypical changes in both the ECs and astrocytes. In this aspect, it has been established that the process of reactive gliosis is a common feature of astrocytes during BBB disruption, which has a detrimental effect on the barrier function and a subsequent damage in neuronal survival. In this review we discuss the implications of astrocyte functions in the protection of the BBB, and in the development of Parkinson's disease (PD) and related disorders. Additionally, we highlight the current and future strategies in astrocyte protection aimed at the development of restorative therapies for the BBB in pathological conditions.

Cortical spreading depression (CSD) is a presumably pathophysiological phenomenon that interrupts local cortical function for periods of minutes to hours. This phenomenon is important due to its association with different neurological disorders such as migraine, malignant stroke and traumatic brain injury (TBI). Glial cells, especially astrocytes, play an important role in the regulation of CSD and in the protection of neurons under brain trauma. The correlation of TBI with CSD and the astrocytic function under these conditions remain unclear. This review discusses the possible link of TBI and CSD and its implication for neuronal survival. Additionally, we highlight the importance of astrocytic function for brain protection, and suggest possible therapeutic strategies targeting astrocytes to improve the outcome following TBI-associated CSD., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

GRP78 participates in multiple functions in the cell during normal and pathological conditions, controlling calcium homeostasis, protein folding and Unfolded Protein Response. GRP78 is located in the endoplasmic reticulum, but it can change its location under stress, hypoxic and apoptotic conditions. NF-κB represents the keystone of the inflammatory process and regulates the transcription of several genes related with apoptosis, differentiation, and cell growth. The possible relationship between GRP78-NF-κB could support and explain several mechanisms that may regulate a variety of cell functions, especially following brain injuries. Although several reports show interactions between NF-κB and Heat Shock Proteins family members, there is a lack of information on how GRP78 may be interacting with NF-κB, and possibly regulating its downstream activation. Therefore, we assessed the computational predictions of the GRP78 (Chain A) and NF-κB complex (IkB alpha and p65) protein-protein interactions. The interaction interface of the docking model showed that the amino acids ASN 47, GLU 215, GLY 403 of GRP78 and THR 54, ASN 182 and HIS 184 of NF-κB are key residues involved in the docking. The electrostatic field between GRP78-NF-κB interfaces and Molecular Dynamic simulations support the possible interaction between the proteins. In conclusion, this work shed some light in the possible GRP78-NF-κB complex indicating key residues in this crosstalk, which may be used as an input for better drug design strategy targeting NF-κB downstream signaling as a new therapeutic approach following brain injuries., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Much attention has been drawn to the possible involvement of hypothalamic inflammation in the pathogenesis of metabolic disorders, especially in response to a high-fat diet. Microglia, the macrophages of the central nervous system, can be activated by proinflammatory signals resulting in the local production of specific interleukins and cytokines, which in turn could exacerbate the pathogenic process. Because obesity itself is considered to be a state of chronic inflammation, we evaluated whether being overweight results in microglial activation in the hypothalamus of rats on a normal diet. Accordingly, we used a model of neonatal overnutrition that entailed adjustment of litter size at birth (small litters: four pups/dam versus normal litters: 12 pups/dam) and resulted in a 15% increase in bodyweight and increased circulating leptin levels at postnatal day 60. Rats that were overnourished during neonatal life had an increased number of activated microglia in specific hypothalamic areas such as the ventromedial hypothalamus, which is an important site for metabolic control. However, this effect was not confined to the hypothalamus because significant microglial activation was also observed in the cerebellar white matter. There was no change in circulating tumour necrosis factor (TNF) α levels or TNFα mRNA levels in either the hypothalamus or cerebellum. Interleukin (IL)6 protein levels were higher in both the hypothalamus and cerebellum, with no change in IL6 mRNA levels. Because circulating IL6 levels were elevated, this rise in central IL6 could be a result of increased uptake. Thus, activation of microglia occurs in adult rats exposed to neonatal overnutrition and a moderate increase in weight gain on a normal diet, possibly representing a secondary response to systemic inflammation. Moreover, this activation could result in local changes in specific hypothalamic nuclei that in turn further deregulate metabolic homeostasis., (© 2011 The Authors. Journal of Neuroendocrinology © 2011 Blackwell Publishing Ltd.)

Stress during pregnancy can impair biological and behavioral responses in the adult offspring and some of these effects are associated with structural changes in specific brain regions. Furthermore, these outcomes can vary according to strain, gender, and type and duration of the maternal stress. Indeed, early stress can induce sexually dimorphic long-term effects on diverse endocrine axes, including subsequent responses to stress. However, whether hypothalamic structural modifications are associated with these endocrine disruptions has not been reported. Thus, we examined the gender differences in the long-term effects of prenatal and adult immobilization stress on the hypothalamic-pituitary-adrenocortical (HPA) axis and the associated changes in hypothalamic structural proteins. Pregnant Wistar rats were subjected to immobilization stress three times daily (45 min each) during the last week of gestation. One half of the offspring were subjected to the same regimen of stress on 10 consecutive days starting at postnatal day (PND) 90. At sacrifice (PND 180), serum corticosterone levels were significantly higher in females compared to males and increased significantly in females subjected to both stresses with no change in males. Prenatal stress increased pituitary ACTH content in males, with no effect in females. Hypothalamic CRH mRNA levels were significantly increased by prenatal stress in females, but decreased in male rats. In females neither stress affected hypothalamic cell death, as determined by cytoplasmic histone-associated DNA fragment levels or proliferation, determined by proliferating cell nuclear antigen levels (PCNA); however, in males there was a significant decrease in cell death in response to prenatal stress and a decrease in PCNA levels with both prenatal and adult stress. In all groups BrdU immunoreactivity colocalized in glial fibrillary acidic protein (GFAP) positive cells, with few BrdU/NeuN labelled cells found. Furthermore, in males the astrocyte marker S100β increased with prenatal stress and decreased with adult stress, suggesting affectation of astrocytes. Synapsin-1 levels were increased by adult stress in females and by prenatal stress in males, while, PSD95 levels were increased in females and decreased in males by both prenatal and adult stress. In conclusion, hypothalamic structural rearrangement appears to be involved in the long-term endocrine outcomes observed after both chronic prenatal and adult stresses. Furthermore, many of these changes are not only different between males and females, but opposite, which could underlie the gender differences in the long-term sequelae of chronic stress, including subsequent responses to stress., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is three times more prevalent in women than men, and epidemiological studies have shown that estrogen replacement in aging women forestalls the onset of AD. Hyperphosphorylation of the tau protein that forms the neurofibrillary tangles found in AD brains might be responsible for the breakdown of microtubules in affected neurons. The mechanisms by which tau protein is phosphorylated in the AD brain are not fully understood. Using a human neuroblastoma cell line (SH-SY5Y) and primary cultures of newborn male or female rat cerebral cortical neurons, we investigated the effect of 17beta-estradiol on tau protein expression and phosphorylation. We found that estradiol increased total tau and induced dephosphorylation at the proline-directed site of the molecule. Further, estradiol prevented okadaic acid-induced hyperphosphorylation of tau in both proline- and non-proline-directed sites, and antiestrogens blocked this effect. To our knowledge, this is the first report of an effect of estradiol on naturally occurring and induced tau phosphorylation. This assumes special significance because the estrogen action was found to be sexually dimorphic in rat cortical neurons and differentiation-sensitive in human neuroblastoma cells.

Introduction: The nervous system is a target for steroid hormones as well as a steroidogenic tissue, and it produces steroids that have a paracrine or autocrine effect on neurons and glial cells. Steroids formed in nervous tissue are called neurosteroids in order to differentiate them, in terms of their origin, from the peripheral steroids, although they both share the same molecular structure., Development: We analyse the capacity of neurons and glial cells to synthesise steroids and describe the role played in steroidogenesis by certain key molecules, such as steroidogenic acute regulatory protein, peripheral benzodiazepine receptor and aromatase enzyme, which acts as a catalyst in the conversion of testosterone into estradiol. We also provide a description of the different mechanisms of action of the hormonal steroids and neurosteroids in the nervous system. These include both the regulation of protein synthesis by neurons and glial cells, by acting on nuclear receptors, and rapid effects mediated by membrane receptors or the allosteric modulation of neurotransmitter receptors. We review the clinical and experimental evidence for the neuroprotective effects of sex steroids and neurosteroids, and the limitations of hormone replacement therapy following menopause., Conclusions: Given the restraints involved in the systemic use of hormones as neuroprotective therapy, alternative strategies that take advantage of the neuroprotective properties of steroids must be sought. These could involve locally increasing their synthesis inside the brain or developing molecules that activate the steroid receptors in the nervous system and not in the peripheral organs.

Biodegradable nerve guides have represented new treatment alternatives for nerve repairing. They are gradually biodegradable, exert biological effects directly to the injured nerve, and act as drug- or cell-delivery devices. Furthermore, progesterone (PROG) has been demonstrated to promote injured peripheral nerve regeneration. In this study, it was hypothesized that PROG delivered from chitosan prostheses provides better facial nerve regenerative response than chitosan prostheses with no PROG. As there are no reports on the use of the former as nerve-guide material in the regeneration of injured nerves, this is the main objective of the present work. Chitosan prostheses containing PROG were used to bridge 10-mm gaps in rabbit facial nerves. The regenerated nerves were evaluated 45 days after implantation in animals with the use of light microscopy and morphometric analysis. Gas chromatography was used in order to quantify PROG content in prosthesis prior to and after implantation in subcutaneous tissue at different periods of up to 60 days. In addition, the prosthesis walls were evaluated with histological techniques in order to assess their integrity and the surrounding tissue reaction. Chitosan prostheses allowed PROG release during the time needed for nerve regeneration. At 45 days myelinated nerve fibers were observed in both the proximal and distal stumps. This parameter and the N ratio were higher in the progesterone-treated group when compared to that of the vehicle control. Findings indicate that chitosan prostheses were useful in nerve regeneration, acting as a long-lasting PROG delivery device a faster nerve regeneration., (Copyright 2003 Wiley Periodicals, Inc.)

Previous studies have shown the existence of proliferating cells in explants from bovine (Bos Taurus) lateral ventricle walls that were maintained for several days in vitro in the absence of serum and growth factors. In this study we have characterized the nature of new cells and have assessed whether the insulin-like growth factor-1 (IGF-1) receptor regulates their survival and/or proliferation. The explants were composed of the ependymal layer and attached subependymal cells. Ependymal cells in culture were labelled with glial markers (S-100, vimentin, GFAP, BLBP, 3A7 and 3CB2) and did not incorporate bromodeoxiuridine when this molecule was added to the culture media. Most subependymal cells were immunoreactive for beta III-tubulin, a neuronal marker, and did incorporate bromodeoxiuridine. Subependymal neurons displayed immunoreactivity for IGF-1 and its receptor and expressed IGF-1 mRNA, indicating that IGF-1 is produced in the explants and may act on new neurons. Addition to the culture media of an IGF-1 receptor antagonist, the peptide JB1, did not affect the incorporation of bromodeoxiuridine to proliferating subependymal cells. However, JB1 significantly increased the number of TUNEL positive cells in the subependymal zone, suggesting that IGF-1 receptor is involved in the survival of subependymal neurons. In conclusion, these findings indicate that neurogenesis is maintained in explants from the lateral cerebral ventricle of adult bovine brains and that IGF-1 is locally produced in the explants and may regulate the survival of the proliferating neurons.

Objective: In malnutrition both the GH-IGF and reproductive axes are greatly affected. Because the actions of IGF and sex steroids are inter-dependent in many tissues, we have examined how ovariectomy modulates the response of the systemic IGF system to undernutrition., Design and Methods: Peripubertal (30 days of age) female rats were either sham operated or ovariectomized. Five days later half of each group was submitted to a protein-caloric restriction diet. All rats were killed at 60 days of age., Results: Growth was decreased in all rats submitted to calorie restriction and this was consistent with a decrease in circulating IGF-I concentrations and liver IGF-I mRNA expression. While in normally fed rats ovariectomy had no significant effect on serum IGF-I concentrations, ovariectomized and underfed rats had significantly higher levels than intact underfed rats. In undernourished rats, serum IGF-binding proteins (IGFBP)-1, -2 and -3 concentrations were significantly reduced and this was not modified by ovariectomy. In contrast, liver mRNA concentrations of IGFBP-1 and -2 were increased and IGFBP-3 unchanged in intact undernourished animals, suggesting that undernutrition could be affecting the proteolysis of these binding proteins, and this response was significantly modulated by ovariectomy., Conclusion: These results indicate that the presence of circulating ovarian hormones significantly affects the response of the IGF system to undernutrition.

Estrogens and androgens can protect neurons from death caused by injury to the central nervous system. Astrocytes and microglia are major players in events triggered by neural lesions. To determine whether glia are direct targets of estrogens or androgens after neural insults, steroid receptor expression in glial cells was assessed in two different lesion models. An excitotoxic injury to the hippocampus or a stab wound to the parietal cortex and hippocampus was performed in male rats, and the resultant expression of steroid receptors in glial cells was assessed using double-label immunohistochemistry. Both lesions induced the expression of estrogen receptors (ERs) and androgen receptors (ARs) in glial cells. ERalpha was expressed in astrocytes immunoreactive (ERalpha-ir) for glial fibrillary acidic protein or vimentin. AR immunoreactivity colocalized with microglial markers, such as Griffonia simplicifolia lectin-1 or OX-6. The time course of ER and AR expression in glia was studied in the stab wound model. ERalpha-ir astrocytes and AR-ir microglia were observed 3 days after lesion. The number of ERalpha-ir and AR-ir glial cells reached a maximum 7 days after lesion and returned to low levels by 28 days postinjury. The studies of ERbeta expression in glia were inconclusive; different results were obtained with different antibodies. In sum, these results suggest that reactive astrocytes and reactive microglia are a direct target for estrogens and androgens, respectively., (Copyright 2002 Wiley-Liss, Inc.)

The ability of cannabinoid CB(1) receptors to influence glutamatergic excitatory neurotransmission has fueled interest in how these receptors and their endogenous ligands may interact in conditions of excitotoxic insults. The present study characterized the impact of stimulated and inhibited CB(1) receptor function on NMDA-induced excitotoxicity. Neonatal (6-day-old) rat pups received a systemic injection of a mixed CB(1) /CB(2) receptor agonist (WIN55,212-2) or their respective antagonists (SR141716A for CB(1) and SR144528 for CB(2) ) prior to an unilateral intrastriatal microinjection of NMDA. The NMDA-induced excitotoxic damage in the ipsilateral forebrain was not influenced by agonist-stimulated CB(1) receptor function. In contrast, blockade of CB(1), but not CB(2), receptor activity evoked a robust neuroprotective response by reducing the infarct area and the number of cortical degenerating neurons. These results suggest a critical involvement of CB(1) receptor tonus on neuronal survival following NMDA receptor-induced excitotoxicity in vivo.

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.

Effects of microgravity on postural control and volume of extracellular fluids as well as stress associated with space flight may affect the function of hypothalamic neurosecretory neurons. Since environmental modifications in young animals may result in permanent alterations in neuroendocrine function, the present study was designed to determine the effect of a space flight on oxytocinergic and vasopressinergic magnocellular hypothalamic neurons of prepuberal rats. Fifteen-day-old Sprague-Dawley female rats were flown aboard the Space Shuttle Columbia (STS-90, Neurolab mission, experiment 150) for 16 days. Age-matched litters remained on the ground in cages similar to those of the flight animals. Six animals from each group were killed on the day of landing and eight animals from each group were maintained under standard vivarium conditions and killed 18 weeks after landing. Several signs of enhanced transcriptional and biosynthetic activity were observed in magnocellular supraoptic neurons of flight animals on the day of landing compared to control animals. These include increased c-Fos expression, larger nucleoli and cytoplasm, and higher volume occupied in the neuronal perikaryon by mitochondriae, endoplasmic reticulum, Golgi apparatus, lysosomes and cytoplasmic inclusions known as nematosomes. In contrast, the volume occupied by neurosecretory vesicles in the supraoptic neuronal perikarya was significantly decreased in flight rats. This decrease was associated with a significant decrease in oxytocin and vasopressin immunoreactive levels, suggestive of an increased hormonal release. Vasopressin levels, cytoplasmic volume and c-Fos expression returned to control levels by 18 weeks after landing. These reversible effects were probably associated to osmotic stimuli resulting from modifications in the volume and distribution of extracellular fluids and plasma during flight and landing. However, oxytocin levels were still reduced at 18 weeks after landing in flight animals compared to controls. This indicates that space flight during prepuberal age may induce irreversible modifications in the regulation of oxytocinergic neurons, which in turn may result in permanent endocrine and behavioral impairments.

The Na,K-ATPase, which catalyzes the active transport of Na(+) and K(+), has two principal subunits (alpha and beta) that have several genetically distinct isoforms. Most of these isoforms are expressed in the nervous system, but certain ones are preferentially expressed in glia and others in neurons. Of the beta isoforms, beta1 predominates in neurons and beta2 in astrocytes, although there are some exceptions. Here we demonstrate that beta3 is expressed in rat and mouse white matter oligodendrocytes. Immunofluorescence microscopy identified beta3 in oligodendrocytes of rat brain white matter in typical linear arrays of cell bodies between fascicles of axons. The intensity of stain peaked at 20 postnatal days. beta3 was identified in cortical oligodendrocytes grown in culture, where it was expressed in processes and colocalized with antibody to galactocerebroside. In the mouse and rat optic nerve, beta3 stain was seen in oligodendrocytes, where it colocalized with carbonic anhydrase II. For comparison, optic nerve was stained for the beta1 and beta2 subunits, showing distinct patterns of labelling of axons (beta1) and astrocytes (beta2). The C6 glioma cell line was also found to express the beta3 isoform preferentially. Since beta3 was not found at detectable levels in astrocytes, this suggests that C6 is closer to oligodendrocytes than astrocytes in the glial cell lineage., (Copyright 2000 Wiley-Liss, Inc.)

The adult female pituitary has significantly more lactotrophs than that of the male, while the later has a higher percent of somatotrophs. It is clear that GH and prolactin (PRL) gene expression and somatotroph and lactotroph proliferation are modulated by the postpubertal hormone environment; however, the role of the neonatal steroid environment in this process is not known. 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, in response to neonatal and adult testosterone treatment. Female rats exposed to testosterone during the neonatal period, adulthood or both periods, as well as normal females and males were used. Exposure to testosterone during the neonatal period significantly increased the percentage of somatotrophs (ANOVA: P<0. 005) and decreased that of lactotrophs in the adult female rat (ANOVA: P<0.001). Adult testosterone treatment had no significant effect on the percentage of somatotrophs. The percentage of lactotrophs was significantly increased by adult testosterone only in those rats also exposed to neonatal testosterone. PRL mRNA concentrations, as reflected by silver grains/cell, were reduced by neonatal testosterone and increased by adult testosterone treatment (ANOVA: P<0.0001). Overall PRL mRNA levels, measured by densitometry, were also reduced by neonatal testosterone exposure, but adult testosterone had no effect (ANOVA: P<0.001). GH mRNA levels per cell, as reflected by silver grains/cell, were increased by adult testosterone, while neonatal testosterone treatment had no effect. Overall GH mRNA levels per unit area, determined by densitometry measurements, were increased by both neonatal and adult testosterone treatment, with the combination of these two treatments resulting in adult females having levels indistinguishable from intact males (ANOVA: P<0.003). These results suggest that, in combination with postpubertal sex steroids, the neonatal gonadal steroid environment plays an important role in determining anterior pituitary hormone synthesis and cellular composition.

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.

Glucagon-like peptide-1 (GLP-1)(7-36) amide, a member of the glucagon and related peptides family, and its receptor have an anatomically specific expression in the brain. Furthermore, the GLP-1 receptor is expressed in both neurons and glia. Because after a penetrating injury a large population of astrocytes become activated and augment their expression of numerous substances, we have used in situ hybridization to determine whether the expression of the GLP-1 receptor increases in response to a penetrating injury. We have found that GLP-1 receptor expression increases dramatically along the border of the injury. Furthermore, this expression can be colocalized to glial fibrillary acidic protein (GFAP) and non-GFAP mRNA containing cells, suggesting that at least part of this increase is due to an increase in GLP-1 receptor expression in glial cells., (Copyright 1999 Harcourt Publishers Ltd.)

alpha-Haemolysin is an extracellular protein toxin (approximately 107 kDa) secreted by Escherichia coli that acts at the level of the plasma membranes of target eukaryotic cells. The nature of the toxin interaction with the membrane is not known at present, although it has been established that receptor-mediated binding is not essential. In this work, we have studied the perturbation produced by purified alpha-haemolysin on pure phosphatidylcholine bilayers in the form of large unilamellar vesicles, under conditions in which the toxin has been shown to induce vesicle leakage. The bilayer systems containing bound protein have been examined by differential scanning calorimetry, fluorescence spectroscopy, differential solubilization by Triton X-114, and freeze-fracture electron microscopy. All the data concur in indicating that alpha-haemolysin, under conditions leading to cell lysis, becomes inserted in the target membrane in the way of intrinsic or integral proteins. In addition, the experimental results support the idea that inserted alpha-haemolysin occupies only one of the membrane phospholipid monolayers, i.e. it is not a transmembrane protein. The experimental data are complemented by structure prediction studies according to which as many as ten amphipathic alpha-helices, appropriate for protein-lipid interaction, but no hydrophobic transmembrane helices are predicted in alpha-haemolysin. These observations and predictions have important consequences for the mechanism of cell lysis by alpha-haemolysin; in particular, a non-transmembrane arrangement of the toxin in the target membrane is not compatible with the concept of alpha-haemolysin as a pore-forming toxin.

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.

Anterior pituitary hormone secretion is sexually dimorphic due partially to gender differences in the postpubertal hormone environment; however, differences in the pituitary's responsiveness to these signals may also play a role. We have used simple and double in situ hybridization to determine whether lactotrophs and somatotrophs from male and female rats respond differently in vitro to growth hormone-releasing hormone (GHRH), somatostatin (SS) or insulin-like growth factor (IGF)-I and whether sex steroids modulate these responses. Cultures were treated with either 17 beta-estradiol (E; 10(-9)M), testosterone (T; 10(-7)M), dihydrotestosterone (DHT; 10(-7) M) or vehicle in combination with either GHRH (10(-7)M), SS (10(-7)M), IGF-I (10(-7)M) or vehicle. Basal mRNA levels of GH, prolactin (PRL) and pituitary transcription factor-1 (Pit-1) did not differ between the sexes. The responses to peptide hormones alone were similar between the sexes, but not in the presence of gonadal steroids. In females, DHT reduced and E increased the stimulatory effect of GHRH and inhibitory effect of SS on GH mRNA levels (two-way ANOVA: P < 0.05), while having no effect in males. An additive effect of E and GHRH on PRL mRNA levels was seen only in males. The E induced rise in PRL mRNA levels was completely inhibited by SS in females, but only partially so in males (two-way ANOVA: P < 0.001). IGF-I inhibited the E induced rise in PRL and lactotroph Pit-1 mRNA levels only in females. These results suggest that sex steroids modulate the pituitary's response to hypothalamic and circulating factors differently in males and females and that this may play a role in generating the sexually dimorphic patterns of pituitary hormone secretion.

1. The secretory pattern of growth hormone (GH) is sexually dimorphic in the adult rat. However, this difference between the sexes does not become apparent until after the onset of puberty, suggesting that pubertal sex steroids play an important role in the manifestation of this phenomenon. 2. We have addressed the question as to whether there exists a sexual dimorphism in the hypothalamic neuropeptides that regulate GH release from the anterior pituitary, i.e., somatostatin (SS) and growth hormone-releasing hormone (GHRH). In addition, we have investigated whether the developmental changes in the GH secretory pattern are correlated with changes in these neuropeptides. The effect of testosterone treatment on SS and GHRH neurons during both the neonatal period and adulthood have also been studied. 3. We have found that the synthetic capacity, as reflected in relative messenger RNA (mRNA) levels, of both SS and GHRH neurons changes throughout development in both male and female rats. These mRNA levels are sexually dimorphic at certain times during maturation and can be modulated by changes in testosterone levels, suggesting that sex steroid modulation of these two neuropeptide systems could at least partially account for the sexual dimorphism seen in the adult GH secretory pattern. 4. The neonatal steroid environment has also been suggested to be involved in the generation of the final adult GH secretory pattern, although the mechanisms underlying this effect are even less well understood. In support of the hypothesis that the neonatal steroid environment plays an important role in organizing the GH axis, we have found that the number of GHRH neurons in the adult brain, as well as their sensitivity to adult steroids, is modulated by neonatal testosterone treatment. The number of SS neurons in the periventricular and paraventricular nuclei were not modulated by neonatal steroids; however, the synthetic capacity of these neurons does appear to be influenced by the neonatal steroid environment. 5. These studies suggest that both the neonatal and adult sex steroid environments influence the adult GH secretory pattern by modulating GHRH and SS neurons.

During normal pregnancy, the levels of placental GH in the maternal circulation increase significantly until 35 wk of gestation. We have previously shown that these levels are significantly reduced in cases of intrauterine growth retardation (IUGR). To better understand the basis of this observation, we have studied the expression of placental GH in placentas from normal births (n = 6) and births with IUGR (n = 5). In situ hybridization histochemistry was used to determine the mean number of cells per area expressing this message, as well as the mean level of specific mRNA per cell. We have found that the mean mRNA signal level per cell of placental GH did not differ between normal or IUGR placentas. However, the mean number of cells/ area expressing this mRNA was significantly greater in normal placentas compared with IUGR placentas (normal 12.8 +/- 0.9 cells/unit area, IUGR 4.9 +/- 2.4 cells/unit area, analysis of variance: p < 0.004). These data suggest that the decreased levels of placental GH in the maternal circulation in IUGR are not due only to the reduced size of the placenta, but also to changes in the placental tissue which result in a reduced number of cells per area that are capable of producing this peptide.

Growth hormone (GH)-releasing peptides (GHRPs), a family of synthetic oligopeptides which stimulate GH release, were identified more than a decade ago. The effects of these peptides on GH release have been described in vivo and in vitro, in both animals and humans, using various doses and administration routes. It is generally accepted that GHRPs stimulate the release of GH by acting at the level of the pituitary through a receptor different to that for the endogenous GH-releasing hormone (GHRH). In addition, it has been reported that there are specific binding sites for these peptides in the hypothalamus and that systemic administration of GHRPs increases the expression of the immediate early gene c-fos in a subpopulation of hypothalamic neurons. However, the identity of these hypothalamic neurons and the mechanism of action of GHRPs at both the hypothalamic and pituitary levels remain unknown. One interesting aspect of GHRPs is that they are orally active and this phenomenon has been demonstrated in both animals and humans. Furthermore, these drugs stimulate GH secretion in humans dose-dependently with the magnitude and duration of this response being comparable to that seen with an intravenous peptide bolus. We have studied the oral activity of GHRP-2 on GH release in normal children. In addition, we have analyzed the response to GHRP-2 of obese adolescents, as well as the effects of an intravenous bolus of GHRH alone and GHRH plus GHRP-2. Orally administered GHRP-2 stimulates GH secretion in normal children and, although it seems that this drug is more potent in girls, there were no statistical differences between the groups. Characteristically, GH levels started to increase by 15 min, peaked at 60 min and returned to basal concentrations by 180 min. The effect of GHRP-2 was synergistic with GHRH 1-29 NH2. In addition, obese subjects appeared to have a greater response to this peptide than did normal controls. To study the effects of GHRPs on hypothalamic GHRH and somatostatin neurons, female dwarf rats (dw/dw) were treated continuously with GHRP-6 (1 mg/kg per 24 h) for 14 days. In situ hybridization for GHRH and SS was performed. We found that GHRP-6 stimulated GHRH mRNA levels in the posterior arcuate nucleus (ARC), with no significant effect in the anterior ARC or ventromedial hypothalamic neurons. SS mRNA levels in the posterior periventricular nucleus (PeN) were decreased after GHRP-6 treatment, while no effect was seen in the anterior PeN, ARC, or lateral paraventricular nucleus. These results suggest that GHRP-6 treatment modulates hypothalamic neurons controlling GH secretion; however, whether this effect is direct or mediated through another factor remains to be elucidated.

Pituitary transcription factor-1 (Pit-1 or GHF-1) is a transcription factor specific to the anterior pituitary and is involved in the expression and regulation of the growth hormone (GH), prolactin (PRL) and thyroid-stimulating hormone (TSH) beta-subunit genes. The expression of these three genes can be modulated by changes in the hormone environment and it is thought that some of these effects are mediated through Pit-1, but little is known about the physiological regulation of this transcription factor. Therefore, we first asked whether Pit-1 gene expression is modified as a result of changes in the in vivo gonadal steroid environment and if this could be correlated with changes in GH and/or PRL mRNA levels. Secondly, we sought to determine if sex steroids affect the mRNA levels of these three peptides by acting at the level of the pituitary and whether these effects are androgen or estrogen mediated. Finally, how sex steroids modulate the response of these three genes to the hypothalamic neuropeptides growth hormone-releasing hormone (GHRH) and somatostatin (SS) was analyzed. To this end, we compared Pit-1, GH and PRL mRNA levels in the anterior pituitary of intact, castrated, and castrated testosterone-replaced adult male rats. In addition, primary cultures of adult male pituitaries were used to study the direct effects of both androgens and estrogens on Pit-1, GH, and PRL mRNA levels. In situ hybridization histochemistry was used to compare relative levels of Pit-1, GH and PRL mRNA. Densitometric analysis of the in vivo studies showed that castration resulted in a 57, 40 and 55% decline in Pit-1, GH and PRL mRNA signal levels, respectively. Furthermore, replacement with testosterone (T) at the time of castration completely prevented the decline in all three mRNA species (ANOVA: Pit-1 mRNA, p < 0.0001; GH mRNA, p < 0.0001; PRL mRNA, p < 0.0001). In vivo, both T (10(-7) M) and estradiol (10(-9) M) were capable of stimulating Pit-1 mRNA and PRL mRNA levels, while dihydrotestosterone (DHT; 10(-7) M) had no effect. There was no effect of any of these steroid treatments on GH mRNA levels in vitro. Addition of GHRH to the cultures increased GH mRNA levels, as well as those of Pit-1 and PRL, and SS had the opposite effect on GH mRNA levels. Whereas the GH response to GHRH was not significantly modified by exposure to sex steroids, the effect of SS was. The presence of sex steroids was capable of modifying the Pit-1 and PRL responses to both GHRH and SS. These results clearly indicate that changes in circulating levels of sex steroids modulate the expression of Pit-1 in the anterior pituitary and that these changes can be correlated with commensurate modifications in GH and PRL mRNA levels. Furthermore, the effect on both Pit-1 and PRL mRNA levels occurs, at least in part, at the level of the anterior pituitary and is an estrogen-receptor-mediated event. In contrast, the effects of gonadal steroids on GH mRNA levels are less direct and are most likely mediated at the level of the hypothalamus, as well as through modulation of the response of the somatotroph to hypothalamic factors. We conclude that the transcription factor Pit-1 is actively regulated physiologically and may be involved in mediating some of the effects of sex steroids and hypothalamic factors on the synthesis of certain anterior pituitary hormones.

We have used isoform-specific antisera against the Na,K-ATPase beta 1 (SpETb1) and beta 2(AMOG) (SpETb2) subunit isoforms in order to establish their specific cellular and subcellular localization in several developmental stages of the rat central nervous system. Immunocytochemical preparations revealed beta 1 Isoform protein in most neural cells, being predominantly located in the soma of neurons and astrocytes, with no appreciable developmental variations. In the newborn rat, beta 2(AMOG) immunoreactivity was present in cellular processes of astroglia and in the somas of neurons and decreasing in intensity with maturation until adulthood, where no beta 2 isoform was detected in neurons. The differential location of these isoforms, both developmentally and at the cellular level suggest a complex regulation of their genes expression and mechanisms of subcellular distribution, as well as functional differences.

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.

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.

This study has analysed by immunocytochemistry the expression pattern of histone H1 zero after the osmotically induced activation of transcription in supraoptic nucleus neurons of the rat. In control rats, histone H1 zero was constitutively expressed in neuronal and glial cell nuclei of supraoptic nucleus. After chronic neuronal stimulation by intermittent salt-loading, the majority of neuronal cell nuclei exhibited a marked reduction of immunostaining, which was confirmed by densitometric analysis of immunoreactivity. This effect was reversible, since optical density values returned to control levels when the stimulation of supraoptic neurons was suppressed by rehydration. Ultrastructural immunocytochemistry of histone H1 zero showed that immunogold particles specifically decorated chromatin fibers, with the highest accumulation of particles being on the condensed inactive chromatin. These results indicate that transcriptional activation in supraoptic neurons is accompanied by a depletion of the chromatin-associated histone H1 zero, and also suggest that this transcription-dependent expression of histone H1 zero may be involved in regulating chromatin condensation and gene expression in mature neurons that constitutively express this protein.

It is now obvious that the CNS is capable of undergoing a variety of plastic changes at all stages of development. Although the magnitude and distribution of these changes may be more dramatic in the immature animal, the adult brain retains a remarkable capacity for undergoing morphological and functional modifications. Throughout development, as well as in the postpubertal animal, gonadal steroids exert an important influence over the architecture of specific sex steroid-responsive areas, resulting in sexual dimorphisms at both morphological and physiological levels. We are only now beginning to gain insight into the mechanisms involved in gonadal steroid-induced synaptic changes. The number of synaptic inputs to specific neuronal populations is sexually dimorphic and this can be modulated by changes in the sex steroid environment. These modifications can be correlated with other morphological changes, such as glial cell activation, that are occurring simultaneously in the same anatomical area. Indeed, the close physical relationship between glial cells and neuronal synaptic contacts makes them an ideal candidate for participating in this process. Interestingly, not only can the morphology and immunoreactivity of glial cells be modulated by gonadal steroids, but a close negative correlation between the number of synapses and the amount of glial ensheathing of a neuron has been demonstrated, suggesting an active participation of these cells in this process. Glia have sex steroid receptors, are capable of producing and metabolizing steroids, and can produce other neuronal trophic factors in response to sex steroids. Hence, their role in gonadal steroid-induced synaptic plasticity is becoming more apparent. In addition, there is recent evidence that this process may involve certain cell surface molecules, such as the N-CAMs, since a specific isoform of this molecule, previously referred to as the embryonic form, is found in those areas of the brain which maintain the capacity to undergo synaptic remodelling. However, there is much work to be done in order to fully understand this phenomenon and before bringing it into a clinical setting in hopes of treating neurodegenerative diseases or injuries to the nervous system.

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.

The secretory pattern of GH is markedly sexually dimorphic in the adult rat, a phenomenon that becomes manifest around the time of pubertal development. This event is due partially to the pubertal rise in gonadal steroids. However, the fetal and neonatal sex steroid environments also play an important role in generating this sexual dimorphism. Hypothalamic mRNA levels of GH-releasing hormone (GHRH) and somatostatin (SS), two neuropeptides implicated in the control of GH release, are sexually dimorphic in both neonatal and adult animals and, at least in the adult animal, are responsive to modulation by sex steroids. In this study, we examined the effects of neonatal testosterone on the number of GHRH and SS neurons in the adult hypothalamus as well as its effects on the responsivity of these neurons to later increases in sex steroids. To address these questions, male rats were either castrated or sham castrated on the day of birth (P0); these animals, along with intact females, received an injection of either testosterone or vehicle. At 60 days of age, half of each group received a Silastic capsule containing testosterone, and half received a sham implant. Growth rates were monitored throughout the study. At 75 days of age, animals were killed, and in situ hybridization to detect GHRH and SS mRNA containing neurons was performed. The number of GHRH neurons in the arcuate nucleus and ventromedial hypothalamus and the number of SS neurons in the periventricular nucleus and paraventricular nucleus were counted. Using a computerized image analysis system, GHRH and SS mRNA signal levels in individual neurons were also measured. Both neonatal and adult steroid treatments significantly increased growth rates. Those animals exposed to neonatal testosterone had significantly more detectable GHRH neurons than those that received only vehicle [P < 0.0001, by analysis of variance (ANOVA)]. Neonatal testosterone treatment had no effect on GHRH mRNA levels. Adult testosterone treatment, while having no effect on GHRH neuron numbers, stimulated GHRH mRNA levels in both males and females (P < 0.0001, ANOVA), but the magnitude of the increase depended upon whether the animal had been exposed to testosterone during the neonatal period. In contrast, the number of SS neurons was not affected by either steroid treatment. However, both treatments modulated SS mRNA levels (P < 0.0001, by ANOVA), with neonatal testosterone treatment alone resulting in significantly higher levels of SS mRNA in the adult animal. Adult testosterone treatment also significantly increased SS mRNA levels, and this was independent of previous exposure to sex steroids.(ABSTRACT TRUNCATED AT 400 WORDS)

The growth hormone (GH) secretory pattern changes significantly throughout development in both male and female rats, becoming markedly sexually dimorphic after pubertal onset. This observation suggests that pubertal sex steroids play a role in the manifestation of this phenomenon. The neonatal steroid environment has also been shown to be intricately involved in the generation of the final adult GH secretory pattern, but the mechanisms underlying this effect remain unknown. We have addressed the question as to whether the developmental changes in the GH secretory pattern are correlated with changes in the hypothalamic neuropeptides that regulate its release from the anterior pituitary, i.e., somatostatin (SS) and growth hormone-releasing hormone (GHRH). The effects of neonatal testosterone and adult testosterone treatments on these two neuropeptide systems have also been studied. We have found that the synthetic capacity, as reflected in relative messenger RNA (mRNA) levels, of both SS and GHRH neurons changes throughout development in both male and female rats. These mRNA levels are also sexually dimorphic at certain times during maturation and, at least in the adult male, can be modulated by changes in testosterone levels. In support of the hypothesis that sex steroids play a role in the organization of the developing hypothalamus, we have shown that both estradiol and testosterone promote the survival of hypothalamic neurons in vitro. Preliminary in vivo studies indicate that the neonatal sex steroid environment may influence the number of GHRH neurons that are found in the adult brain, as well as their sensitivity to adult steroids.(ABSTRACT TRUNCATED AT 250 WORDS)