Luis Miguel Garcia-Segura

The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis.

The gastrointestinal peptide hormone ghrelin stimulates appetite in rodents and humans via hypothalamic actions. We discovered expression of ghrelin in a previously uncharacterized group of neurons adjacent to the third ventricle between the dorsal, ventral, paraventricular, and arcuate hypothalamic nuclei. These neurons send efferents onto key hypothalamic circuits, including those producing neuropeptide Y (NPY), Agouti-related protein (AGRP), proopiomelanocortin (POMC) products, and corticotropin-releasing hormone (CRH). Within the hypothalamus, ghrelin bound mostly on presynaptic terminals of NPY neurons. Using electrophysiological recordings, we found that ghrelin stimulated the activity of arcuate NPY neurons and mimicked the effect of NPY in the paraventricular nucleus of the hypothalamus (PVH). We propose that at these sites, release of ghrelin may stimulate the release of orexigenic peptides and neurotransmitters, thus representing a novel regulatory circuit controlling energy homeostasis.

Neuroprotection by estradiol

This review highlights recent evidence from clinical and basic science studies supporting a role for estrogen in neuroprotection. Accumulated clinical evidence suggests that estrogen exposure decreases the risk and delays the onset and progression of Alzheimers disease and schizophrenia, and may also enhance recovery from traumatic neurological injury such as stroke. Recent basic science studies show that not only does exogenous estradiol decrease the response to various forms of insult, but the brain itself upregulates both estrogen synthesis and estrogen receptor expression at sites of injury. Thus, our view of the role of estrogen in neural function must be broadened to include not only its function in neuroendocrine regulation and reproductive behaviors, but also to include a direct protective role in response to degenerative disease or injury. Estrogen may play this protective role through several routes. Key among these are estrogen dependent alterations in cell survival, axonal sprouting, regenerative responses, enhanced synaptic transmission and enhanced neurogenesis. Some of the mechanisms underlying these effects are independent of the classically defined nuclear estrogen receptors and involve unidentified membrane receptors, direct modulation of neurotransmitter receptor function, or the known anti-oxidant activities of estrogen. Other neuroprotective effects of estrogen do depend on the classical nuclear estrogen receptor, through which estrogen alters expression of estrogen responsive genes that play a role in apoptosis, axonal regeneration, or general trophic support. Yet another possibility is that estrogen receptors in the membrane or cytoplasm alter phosphorylation cascades through direct interactions with protein kinases or that estrogen receptor signaling may converge with signaling by other trophic molecules to confer resistance to injury. Although there is clear evidence that estradiol exposure can be deleterious to some neuronal populations, the potential clinical benefits of estrogen treatment for enhancing cognitive function may outweigh the associated central and peripheral risks. Exciting and important avenues for future investigation into the protective effects of estrogen include the optimal ligand and doses that can be used clinically to confer benefit without undue risk, modulation of neurotrophin and neurotrophin receptor expression, interaction of estrogen with regulated cofactors and coactivators that couple estrogen receptors to basal transcriptional machinery, interactions of estrogen with other survival and regeneration promoting factors, potential estrogenic effects on neuronal replenishment, and modulation of phenotypic choices by neural stem cells.

Aromatase expression by astrocytes after brain injury: implications for local estrogen formation in brain repair.

Recent evidence indicates that 17beta-estradiol may have neuroprotective and neuroregenerative properties. Estradiol is formed locally in neural tissue from precursor androgens. The expression of aromatase, the enzyme that catalyses the conversion of androgens to estrogens, is restricted, under normal circumstances, to specific neuronal populations. These neurons are located in brain areas in which local estrogen formation may be involved in neuroendocrine control and in the modulation of reproductive or sex dimorphic behaviours. In this study the distribution of aromatase immunoreactivity has been assessed in the brain of mice and rats after a neurotoxic lesion induced by the systemic administration of kainic acid. This treatment resulted in the induction of aromatase expression by reactive glia in the hippocampus and in other brain areas that are affected by kainic acid. The reactive glia were identified as astrocytes by co-localization of aromatase with glial fibrillary acidic protein and by ultrastructural analysis. No immunoreactive astrocytes were detected in control animals. The same result, the de novo induction of aromatase expression in reactive astrocytes on the hippocampus, was observed after a penetrating brain injury. Furthermore, using a 3H2O assay, aromatase activity was found to increase significantly in the injured hippocampus. These findings indicate that although astrocytes do not normally express aromatase, the enzyme expression is induced in these glial cells by different forms of brain injury. The results suggest a role for local astroglial estrogen formation in brain repair.

Immunohistochemical mapping of calcium-binding protein immunoreactivity in the rat central nervous system

A complete mapping of immunoreactive sites for vitamin D-dependent calcium-binding protein (CaBP) was performed on serial sections from the rat central nervous system. CaBP immunoreactivity was found in the perikarya, dendrites and axons of some neurons from the limbic system, from many neurosecretory nuclei, from most sensory nuclei and from the cerebral and cerebellar cortex. In contrast, no CaBP antigenic sites were detectable in the motoneurons of the spinal cord and in those of the cranial nerve nuclei, nor in the neurons from the cerebellar nuclei. A quantitative evaluation revealed a great variability in the number of CaBP-immunoreactive neurons among different areas of the central nervous system. Positive cells represented less than 1% of the neurons in the frontal cortex, whereas 74% of the Purkinje cells from the cerebellar cortex showed immunoreactive staining for CaBP. In addition, 45% of the ependymal cells of the telencephalic ventricles were positive. These data show that CaBP is widely distributed in neurons and ependymal cells from the rat central nervous system although it is more concentrated in some specific areas.

Gonadal hormones as promoters of structural synaptic plasticity: Cellular mechanisms

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.

Role of astroglia in estrogen regulation of synaptic plasticity and brain repair

Astroglia are targets for estrogen and testosterone and are apparently involved in the action of sex steroids on the brain. Sex hormones induce changes in the expression of glial fibrillary acidic protein, the growth of astrocytic processes, and the degree of apposition of astroglial processes to neuronal membranes in the rat hypothalamus. These changes are linked to modifications in the number of synaptic inputs to hypothalamic neurons. These findings suggest that astrocytes may participate in the genesis of androgen-induced sex differences in synaptic connectivity and in estrogen-induced synaptic plasticity in the adult brain. Astrocytes and tanycytes may also participate in the cellular effects of sex steroids by releasing neuroactive substances and by regulating the local accumulation of specific growth factors, such as insulin-like growth factor-I, that are involved in estrogen-induced synaptic plasticity and estrogen-mediated neuroendocrine control. Astroglia may also be involved in regenerative and neuroprotective effects of sex steroids, since astroglia formation after brain injury or after peripheral nerve axotomy is regulated by sex hormones. Furthermore, the expression of aromatase, the enzyme that produces estrogen, is induced de novo in astrocytes in lesioned brain areas of adult male and female rodents. Since astroglia do not express aromatase under normal circumstances, the induction of this enzyme may be part of the program of glial activation to cope with the new conditions of the neural tissue after injury. Given the neuroprotective and growth-promoting effects of estrogen after injury, the local production of this steroid may be a relevant component of the reparative process.

Steroid hormones and neurosteroids in normal and pathological aging of the nervous system

Without medical progress, dementing diseases such as Alzheimers disease will become one of the main causes of disability. Preventing or delaying them has thus become a real challenge for biomedical research. Steroids offer interesting therapeutical opportunities for promoting successful aging because of their pleiotropic effects in the nervous system: they regulate main neurotransmitter systems, promote the viability of neurons, play an important role in myelination and influence cognitive processes, in particular learning and memory. Preclinical research has provided evidence that the normally aging nervous system maintains some capacity for regeneration and that age-dependent changes in the nervous system and cognitive dysfunctions can be reversed to some extent by the administration of steroids. The aging nervous system also remains sensitive to the neuroprotective effects of steroids. In contrast to the large number of studies documenting beneficial effects of steroids on the nervous system in young and aged animals, the results from hormone replacement studies in the elderly are so far not conclusive. There is also little information concerning changes of steroid levels in the aging human brain. As steroids present in nervous tissues originate from the endocrine glands (steroid hormones) and from local synthesis (neurosteroids), changes in blood levels of steroids with age do not necessarily reflect changes in their brain levels. There is indeed strong evidence that neurosteroids are also synthesized in human brain and peripheral nerves. The development of a very sensitive and precise method for the analysis of steroids by gas chromatography/mass spectrometry (GC/MS) offers new possibilities for the study of neurosteroids. The concentrations of a range of neurosteroids have recently been measured in various brain regions of aged Alzheimers disease patients and aged non-demented controls by GC/MS, providing reference values. In Alzheimers patients, there was a general trend toward lower levels of neurosteroids in different brain regions, and neurosteroid levels were negatively correlated with two biochemical markers of Alzheimers disease, the phosphorylated tau protein and the beta-amyloid peptides. The metabolism of dehydroepiandrosterone has also been analyzed for the first time in the aging brain from Alzheimer patients and non-demented controls. The conversion of dehydroepiandrosterone to Delta5-androstene-3beta,17beta-diol and to 7alpha-OH-dehydroepiandrosterone occurred in frontal cortex, hippocampus, amygdala, cerebellum and striatum of both Alzheimers patients and controls. The formation of these metabolites within distinct brain regions negatively correlated with the density of beta-amyloid deposits.

Estradiol upregulates Bcl-2 expression in adult brain neurons

BCL-2, a protein which negatively modulates apoptosis, is up-regulated by estrogen in several tissues. To determine the effect of estradiol on Bcl-2 in the adult brain, its immunoreactive distribution was examined in the hypothalamic arcuate nucleus of female rats under different endocrine conditions. The number of Bcl-2–immunoreactive neurons was significantly increased (p< 0.001) on the day of estrus compared with proestrus, diestrus and metestrus, was decreased by ovariectomy and showed a dose–response increase after estradiol administration to ovariectomized rats. Progesterone, when injected simultaneously with estradiol, reduced the effect of estradiol. These findings indicate that ovarian hormones regulate Bcl-2 in hypothalamic neurons and suggest that this protein may be involved in the neuro-protective effects of estrogen.

Localization of estrogen receptor β-immunoreactivity in astrocytes of the adult rat brain

Estrogen receptors are direct regulators of transcription that function by binding to specific DNA sequences in promoters of target genes. The two cloned forms of estrogen receptors, α and β, are expressed in the central nervous system by different neuronal populations. Astrocytes in vitro are also reported to express estrogen receptor α; however, this expression has not been confirmed in the rat brain in vivo. The apparent absence of estrogen receptors in glia in vivo contrasts with the well‐known effects of this hormone on astrocytes of different brain areas, including the hippocampal formation. In this study, the expression of estrogen receptors in the hippocampal formation of adult male rats has been assessed by confocal microscopy. Estrogen receptor α‐immunoreactivity was localized in neuronal nuclei in the pyramidal cell layer of CA1‐CA3 fields. Estrogen receptor β‐immunoreactivity was observed in the perikarya, apical dendrites, and cell nuclei of pyramidal neurons in CA1 and CA2. Furthermore, estrogen receptor β‐immunoreactive glia were observed in CA1, CA2, CA3, and in the hilus of the dentate gyrus of male and female rats. Estrogen receptor β‐immunoreactivity was localized in glial processes and perikarya and, in some cases, in glial cell nuclei. Double immunocytochemical labeling of estrogen receptor β and the specific astroglial marker glial fibrillary acidic protein revealed that estrogen receptor β‐immunoreactive glial cells were astrocytes. Estrogen receptor α was not co‐localized with glial fibrillary acidic protein. The presence of estrogen receptor β in astrocytes of adult male and female rats demonstrates a possible mechanism by which estrogen can directly modulate gene expression in these cells. GLIA 26:260–267, 1999.

Glial expression of estrogen and androgen receptors after rat brain injury

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. ERα was expressed in astrocytes immunoreactive (ERα‐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. ERα‐ir astrocytes and AR‐ir microglia were observed 3 days after lesion. The number of ERα‐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 ERβ 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. J. Comp. Neurol. 450:256–271, 2002.