Estradiol-induced modulation of estrogen receptor-beta and GABA within the adult neocortex: a potential transsynaptic mechanism for estrogen modulation of BDNF

Estrogen influences brain-derived neurotrophic factor (BDNF) expression in the neocortex. However, BDNF-producing cortical neurons do not express detectable levels of nuclear estrogen receptors; instead, the most abundant cortical nuclear estrogen receptor, ER-beta, is present in GABAergic neurons, prompting us to test the hypothesis that estrogen effects on BDNF are mediated via cortical inhibitory interneurons. Adult female ovariectomized rats were provided acute estrogen replacement and the number of cortical GABA, ER-beta, and ER-beta/GABA double-labeled neurons was examined. Within 48 hours of injection of 17-beta-estradiol, the number of perirhinal neurons double-labeled for ER-beta/GABA was reduced by 28% (P<0.01 compared to vehicle-treated ovariectomized controls), and all cells expressing detectable levels of GABA were reduced by 19% (P<0.01). To investigate potential relationships between estrogen receptors, GABAergic neurons, and BDNF-expressing cells, brain sections were double- or triple-labeled for ER-beta, GABAergic, and BDNF immunomarkers. The findings indicated that ER-beta-bearing inhibitory neurons project onto other GABAergic neurons that lack nuclear estrogen receptors; these inhibitory neurons in turn innervate BDNF-expressing excitatory cells. High estrogen states reduce cortical GABA levels, presumably releasing inhibition on BDNF-expressing neurons. This identifies a putative two-step transsynaptic mechanism whereby estrogen availability modulates expression of inhibitory transmitters, resulting in increased BDNF expression.     

Estrogen is more than just a "sex hormone": novel sites for estrogen action in the hippocampus and cerebral cortex

For decades estrogen was thought of only as a "sex hormone," as it plays a fundamental role in regulating behavioral and physiological events essential for successful procreation. In recent years, estrogen has been shown to exert effects on the structure and function of the hippocampus and cortex. The discovery of a new estrogen receptor (ER-beta) and localization of ER-alpha and ER-beta mRNAs in the pyramidal cells of the rat hippocampus and ER-beta mRNA in rat cortex have provided new insight into how estrogen may directly modulate the structure and function of these neurons. Moreover, recent in vivo (125)I-estrogen binding studies have shown that nuclear estrogen binding sites are widely distributed in the pyramidal cells throughout CA1-3 of the hippocampus and laminae II-VI of the isocortex, demonstrating that ER mRNAs are translated into biologically active protein. The functional impact of estrogen receptor localization in the cortex and hippocampus may prove relevant to the emerging role for estrogen as a protective factor in neurodegenerative injury. This potential role is further highlighted by the recent findings that the expression of ER-alpha and ER-beta changes following ischemic brain injury and that these changes correlate with the hormonal modulation of protective factors. These data provide the first evidence that the expression of ERs in the adult cortex is not static, but instead, responsive to neuronal injury and perhaps additional factors that influence the cortical environment and status of these neurons. Together, these data indicate that estrogen has a far greater effect on the hippocampus and isocortex than previously thought. Furthermore, these new findings challenge our current thinking about steroid hormones and their mechanism(s) of action in regions associated with learning and memory and affected by the neurodegenerative conditions of aging.     

Levels of estrogen receptors alpha and beta in frontal cortex of patients with Alzheimer's disease: relationship to Mini-Mental State Examination scores

Estrogen exerts beneficial effects on the brain throughout life. Studies demonstrate that estrogen is neuroprotective and that reduced brain estrogen activity may influence the clinical course of Alzheimer's disease (AD). Changes in levels of estrogen receptors have been detected in postmortem brain tissue of AD patients. Very little is known about the relationship between clinical stage and levels of estrogen receptors in postmortem brain. We hypothesized that estrogen receptor levels would be related to severity of cognitive impairment assessed proximate to death. Western blotting was used to quantify ER-alpha and ER-beta in nuclear, cytosolic, and crude membrane fractions of superior frontal cortex from 25 AD patients. Multiple linear regression analyses adjusted for age, sex, and education showed a significant linear relationship between Mini-Mental State Examination score (MMSE) and wild-type nuclear ER-alpha (a = 5.463, p = 0.03), but none between MMSE and wild-type nuclear ER-beta (a = 2.29, p = 0.36). We incidentally observed additional higher and lower molecular mass bands for ER-alpha in study subjects. Additional experiments performed on frontal cortex nuclear fractions prepared from subjects enrolled in a different study confirmed that these same bands are present in female and males with and without AD. Together our data show a relationship between wild-type ER-alpha and level of cognitive impairment in AD, and also suggest the possibility that variant isoforms of ER-alpha may be present in frontal cortex of patients with and without AD.     

Estrogen receptor-beta, but not estrogen receptor-alpha, is expressed in prolactin neurons of the female rat paraventricular and supraoptic nuclei: comparison with other neuropeptides

Estrogen receptor-alpha (ER-alpha) and ER-beta exhibit fine differences in their distributions in the rodent forebrain, and one such difference is observed in the paraventricular (PVN) and supraoptic (SON) nuclei. To investigate the functional significance of ER in these brain areas, we examined the neuropeptide characteristics of ER-expressing neurons in the PVN and SON of female rats by using dual-label immunocytochemistry. The distributions of ER-alpha immunoreactivity (ir) and ER-beta ir were nonoverlapping in the PVN and SON. Nuclear ER-alpha ir was found in a population of thyrotropin-releasing hormone (TRH)-expressing neurons in the PVN (5.93% +/- 1.20% SEM), but not in any other identified cell phenotype of the PVN and SON. The phenotype of neurons with the highest percentage expressing ER-beta was found to be prolactin (PRL) immunoreactive in both the parvocellular (84.95% +/- 4.11%) and the magnocellular (84.76% +/- 3.40%) parts of the PVN as well as the SON (87.57% +/- 4.64%). Similarly, most vasopressin-immunoreactive neurons were also ER-beta positive in the PVN (66.14% +/- 2.47%) and SON (72.42% +/- 4.51%). In contrast, although a high percentage of oxytocin (OXY) neurons coexpressed ER-beta in the PVN (84.39% +/- 2.99%), there was very little ER-beta/OXY colocalization in the SON. Low levels of corticotropin-releasing hormone neurons also expressed ER-beta ir in the PVN (12.57% +/- 1.99%), but there was no ER-beta colocalization with TRH. In summary, these findings further support the possibility of direct effects of estrogen on neuropeptide expression and implicate estrogen involvement in the regulation of various aspects of neuroendocrine function.     

The Distribution of Brain-derived Neurotrophic Factor and its Receptor trkB in Parvlbumin-containing Neurons of the Rat Visual Cortex

We analysed the distribution of brain-derived neurotrophic factor (BDNF) and its receptor trkB in the adult rat visual cortex, paying particular attention to a GABAergic neuronal subpopulation—the parvalburnin-positive cells. We found expression of trkB in the cell body and apical dendrite of pyramidal neurons and in the cell body of non-pyramidal neurons. Double labelling experiments revealed extensive colocalization of parvalbumin and trkB immunoreactivity in non-pyramidal neurons. Interestingly, the trkB-positive pyramidal neurons appeared surrounded by parvalbumin-labelled boutons. The use of double immunohistochemistry and in situ hybridization histochemistry showed that parvalbumin-positive neurons express trkB mRNA. BDNF rnRNA was found in several cells. Coexpression of BDNF mRNA and parvalbumin immunoreactivity was extremely rare. These data strongly suggest that BDNF synthesized by cortical neurons acts as a postsynaptically derived factor for parvalbumin-positive neurons in the adult rat visual cortex.     

Distribution patterns of estrogen receptor alpha and beta in the human cortex and hippocampus during development and adulthood

The expression of estrogen receptors (ERs) in the developing and adult human brain has not been clearly established, although estrogens are crucial for neuronal differentiation, synapse formation, and cognitive functions. By using immunohistochemistry, we have studied the distribution of ER alpha and ER beta in human cerebral cortex and hippocampus from early prenatal stages to adult life. ER alpha was detected in the cortex at 9 gestational weeks (GW), with a high expression in proliferating zones and the cortical plate. The staining intensity decreased gradually during prenatal development but increased again from birth to adulthood. In contrast, ER beta was first detected at 15 GW in proliferating zones, and at 16/17 GW, numerous ER beta immunopositive cells were also observed in the cortical plate. ER beta expression persisted in the adult cortex, being widely distributed throughout cortical layers II-VI. In addition, from around 15 GW to adulthood, ER alpha and ER beta were expressed in human hippocampus mainly in pyramidal cells of Ammon's horn and in the dentate gyrus. Western blotting and immunohistochemistry in the adult cerebral cortex and hippocampus revealed lower protein expression of ER alpha compared with ER beta. Double immunostaining showed that during fetal life both ERs are expressed in neurons as well as in radial glia, although only ER alpha is expressed in the Cajal-Retzius neurons of the marginal zone. These observations demonstrate that the expression of ER alpha and ER beta displays different spatial-temporal patterns during human cortical and hippocampal development and suggest that both ERs may play distinct roles in several processes related to prenatal brain development.     

Estrogen receptor immunoreactivity in the adult primate brain: neuronal distribution and association with p75, trkA, and choline acetyltransferase

The neuroactive steroid hormone, estrogen, has been implicated in both the prevention and treatment of Alzheimer's disease. Interactions between estrogen and neurotrophic systems may partially explain the beneficial effects of estrogen therapy. Previous studies have identified estrogen binding sites colocalized with neurotrophin-related proteins and mRNA within the rodent brain. Extending these studies to a model more relevant to human systems, we have mapped the distribution of estrogen receptor alpha (ER-alpha)-immunoreactive neurons in adult nonhuman primate brains. In addition, we used double-label immunohistochemistry to examine colocalization of ER-alpha with the low- and high-affinity neurotrophin receptors, p75 and trkA, and with the cholinergic marker choline acetyltransferase. Large numbers of ER-alpha-immunoreactive cells were detected in several amygdaloid and hypothalamic nuclei. ER-alpha-labeled cells were also found in the lateral septum, nucleus of the stria terminals, subfornical organ, and periaqueductal gray. Only rare, scattered ER-alpha-immunoreactive cells were noted in the cholinergic basal forebrain. In contrast to rodents, no cells exhibited ER-alpha and p75 or ER-alpha and trkA double-labeling. However, ER-labeled neurons in the amygdala, a region containing putative nerve growth factor-producing cells and exhibiting a role in memory, were densely and specifically invested with cholinergic terminals projecting from the basal forebrain. Estrogen-labeled neurons were also present in the lateral septal nucleus, a system that receives hippocampal inputs and projects to the neurotrophin-sensitive medial septum. Thus, interactions between neurotrophin-sensitive neurons and ER-bearing neurons exist in the primate brain, providing a potential paracrine basis for estrogen-state modulation of vulnerability to Alzheimer's disease.     

Regulation of the content of progesterone and estrogen receptors, and their cofactors SRC-1 and SMRT by the 26S proteasome in the rat brain during the estrous cycle

In this work we have determined the role of the 26S proteasome in the regulation of the content of progesterone receptors (PR-A and PR-B), estrogen receptors (ER-alpha and ER-beta), the coactivator SRC-1 and the corepressor SMRT in the rat brain during the estrous cycle. The 26S proteasome inhibitor MG132 was injected once into the lateral ventricle on proestrous day; and 24h later, on estrous day we evaluated the content of PR and ER isoforms, SRC-1 and SMRT in the hypothalamus, the preoptic area and the hippocampus by Western blot. A significant increase in the content of both PR isoforms, ER-beta and SRC-1 was observed after the administration of MG132 in the three studied cerebral regions. SMRT content was increased in the hypothalamus and the preoptic area and a significant increase in ER-alpha content was only observed in the preoptic area. These results suggest that essential proteins that participate in progesterone and estrogen actions in the brain should be regulated by the 26S proteasome in a tissue-specific manner in physiological conditions.     

Region- and peptide-specific regulation of the neurotrophins by estrogen

We have previously shown that estrogen increases the expression of brain-derived neurotrophic factor (BDNF) mRNA in the olfactory bulb and cingulate cortex. Here we report that estrogen regulation of BDNF protein and the structurally related peptides nerve growth factor (NGF) and neurotrophin (NT)-4 is region- and peptide-specific. The olfactory bulb and cingulate cortex are both estrogen-sensitive targets and each receives a separate projection from neurons in the horizontal limb of the diagonal band of Broca (hlDBB). Furthermore, neurotrophins are retrogradely transported from the bulbar and cortical targets to the hlDBB. Four weeks of estrogen replacement to ovariectomized animals increased BDNF expression in the olfactory bulb, but decreased BDNF in the cingulate cortex. On the other hand, estrogen increased NT-4 expression in the cingulate cortex, but not in the olfactory bulb. NGF expression was not affected by estrogen in either region studied. In the hlDBB, estrogen increased BDNF but decreased NT-4, suggesting that estrogen differentially affects retrograde accumulation of these peptides. While both estrogen receptor alpha and beta have been identified in the olfactory bulb and cingulate cortex, our results indicate that estrogen receptor alpha expression is relatively higher in the olfactory bulb as compared to the cortex. Since the two estrogen receptors have been shown to stimulate different signaling pathways, we hypothesize that estrogen acting through specific receptors may differentially influence the extent and direction of neurotrophin expression.     

Estrogen receptor-beta regulates tryptophan hydroxylase-1 expression in the murine midbrain raphe.

BACKGROUND: Distinct expression patterns of estrogen receptor (ER)-alpha and ER-beta are displayed in the murine central nervous system. ER-beta is the predominant form of the receptor expressed in the murine midbrain dorsal raphe nucleus (DRN). Tryptophan hydroxylase (TPH) is abundantly expressed in the serotonergic neurons of the DRN and is regulated by estrogen in both the monkey and the guinea pig. METHODS: In this study we used immunocytochemistry to show that ER-beta and TPH are colocalized in the serotonergic cells of the murine DRN. We utilized the ER-alpha and ER-beta gene deletion mouse models and in situ hybridization to demonstrate that ER-beta is responsible for regulating TPH1 mRNA expression. RESULTS: Estrogen increased TPH1 mRNA expression in the DRN of wild type and ER-alpha knockout mice (alpha-ERKO) but not ER-beta knockouts (beta-ERKO). CONCLUSIONS: These data indicate that ER-beta is responsible for mediating estrogen regulated TPH1 expression in the murine DRN.     

Brain-derived neurotrophic factor acutely depresses excitatory synaptic transmission to GABAergic neurons in visual cortical slices

Brain-derived neurotrophic factor (BDNF) acutely modulates synaptic transmission to excitatory neurons in hippocampus and neocortex. The question of whether BDNF acts similarly on excitatory synaptic transmission to GABAergic neurons was eluded in previous studies using cortical slices. To address this question, we used transgenic mice in which expression of green fluorescence protein (GFP) is regulated by glutamic acid decarboxylase 67 (GAD67) promoter. In cortical slices prepared from these GAD67-GFP knock-in mice, we could detect GABAergic neurons under a fluorescent microscope. An application of BDNF rapidly depressed excitatory postsynaptic currents (EPSCs) evoked by layer IV stimulation in most GFP-positive neurons in layer II/III of the cortex. This effect was seen at synapses activated during the BDNF application and blocked by anti-TrkB IgG, indicating that the acute inhibitory action of BDNF is activity-dependent and mediated through TrkB. Paired-pulse ratios of the amplitude of EPSCs to paired stimulation at intervals of 10-100 ms were not significantly changed after BDNF application, suggesting that the site of depression may be postsynaptic. Responses to directly applied glutamate were also depressed by BDNF in most of neurons, being consistent with the interpretation of postsynaptic action of BDNF. The depressive action of BDNF was blocked by an intracellular injection of a Ca(2+) chelator, suggesting that a rise in Ca(2+) is involved in the acute depression of EPSCs. This action of BDNF was seen in 67% of parvalbumin (PV)-positive neurons, but in only 19% of PV-negative neurons, indicating that the depressive action is biased to PV-positive GABAergic neurons.     

Synaptic and nonsynaptic localization of GABAA receptors containing the alpha5 subunit in the rat brain

The alpha5 subunit of the GABA(A) receptors (GABA(A)Rs) has a restricted expression in the brain. Maximum expression of this subunit occurs in the hippocampus, cerebral cortex, and olfactory bulb. Hippocampal pyramidal cells show high expression of alpha5 subunit-containing GABA(A)Rs (alpha5-GABA(A)Rs) both in culture and in the intact brain. A large pool of alpha5-GABA(A)Rs is extrasynaptic and it has been proposed to be involved in the tonic GABAergic inhibition of the hippocampus. Nevertheless, there are no studies on the localization of the alpha5-GABA(A)Rs at the electron microscope (EM) level. By using both immunofluorescence of cultured hippocampal pyramidal cells and EM postembedding immunogold of the intact hippocampus we show that, in addition to the extrasynaptic pool, there is a pool of alpha5-GABA(A)Rs that concentrates at the GABAergic synapses in dendrites of hippocampal pyramidal cells. The results suggest that the synaptic alpha5-GABA(A)Rs might play a role in the phasic GABAergic inhibition of pyramidal neurons in hippocampus and cerebral cortex.     

Brain-derived neurotrophic factor in Huntington disease

Trophic factors, administered systemically or delivered via genetically-modified cells grafted into target regions, have been proposed as putative therapeutic agents in human neurodegenerative disorders. In parallel to the study of the beneficial effects in experimental models of particular diseases, a crucial aspect of the study of trophic factors is the gathering of information about the actual trophic factor expression in human diseased states. Brain-derived neurotrophic factor (BDNF) promotes survival and growth of various nerve cell populations during normal development and following various insults in the developing and adult brain. In particular, BDNF prevents cell death of certain striatal populations in excitotoxic models of Huntington disease (HD) following intrastriatal injection of quinolinic acid to the adult rodent brain. The present study examines BDNF expression, by gel electrophoresis and Western blotting, and immunohistochemistry, in the brains of patients who had suffered from HD. Reduced BDNF expression, ranging from 53 to 82%, has been found in the caudate and putamen in HD when compared with age-matched controls. No modifications in BDNF expression levels have been seen in the parietal cortex, temporal cortex and hippocampus. Furthermore, immunohistochemistry has shown reduced BDNF immunoreactivity in caudate neurons, but not in cortical neurons in HD when compared with controls. These data demonstrate selective BDNF decay in regions that are vulnerable to HD, and suggest, in combination with results in experimental models, that a BDNF surplus may have beneficial effects in the treatment of HD.     

TrkB-like immunoreactivity is present on geniculocortical afferents in layer IV of kitten primary visual cortex.

Exogenous administration of the neurotrophins brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-4/5), or blockade of their endogenous actions, have been reported to affect the anatomic organization and physiological responses of neurons in developing mammalian primary visual cortex. Experimental alteration of levels of these neurotrophic factors can also influence the morphology of the geniculocortical afferents that project from the lateral geniculate nucleus (LGN) to primary visual cortex. BDNF and NT-4/5 are ligands of the TrkB tyrosine kinase receptor. Although multiple populations of cortical neurons express TrkB, it is not known whether geniculocortical afferents express this receptor on their axon branches in visual cortex. We have anatomically labeled geniculocortical afferents of postnatal day 40 kittens with the anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin (PHA-L) and performed double-label immunofluorescence with a panel of anti-TrkB antibodies. Confocal microscopy and object-based colocalization analysis were used to measure levels of TrkB-like immunoreactivity (IR) on geniculocortical afferents in layer IV of primary visual cortex. By using a conservative analysis involving a comparison of measured colocalization with the amount of colocalization expected based on random overlap of TrkB puncta and PHA-L--labeled afferents, 3 of 5 anti-TrkB antibodies tested showed significant colocalization with the geniculocortical axons. Results for the other two antibodies were indeterminate. The indices obtained for colocalization of TrkB and geniculocortical afferents were also compared with the equivalent index obtained for GAD65, a protein that has a similar overall expression pattern to that of TrkB but is not expressed on geniculocortical axons. This analysis indicated that TrkB was present on geniculocortical axons for all five TrkB antibodies tested. TrkB-like IR was also observed on neuronal somata in the LGN. These results indicate that TrkB receptors on geniculocortical afferents are potential mediators of the actions of BDNF and NT-4/5 in developing visual cortex.     

Pro-brain-derived neurotrophic factor is decreased in parietal cortex in Alzheimer's disease

Brain-derived neurotrophic factor (BDNF) promotes the function and survival of the major neuronal types affected in Alzheimer disease, such as hippocampal, cortical and basal forebrain cholinergic neurons. We and others have demonstrated a reduction in BDNF mRNA expression in Alzheimer's disease hippocampus and cortex, which may help to explain the selective vulnerability of these neurons. Several studies have also shown decreased BDNF protein in Alzheimer's disease. BDNF protein is synthesized as a precursor, proBDNF, which is cleaved to the mature 14-kDa form. We demonstrate here that BDNF exists as a mixture of proBDNF and mature BDNF in all regions tested of human brain. Using Western blotting, we observe a 40% reduction in proBDNF levels in Alzheimer's disease parietal cortex compared to controls. Thus, decreased BDNF protein measured by ELISA and immunohistochemistry likely represents a mixture of the two BDNF forms, and previously reported decreases in BDNF protein may be due, at least in part, to a significant reduction in proBDNF levels. Although the biological activity of proBDNF is unknown, reduced proBDNF may have functional consequences for the selective neuronal degeneration in Alzheimer's disease brain.     

Brain-derived neurotrophic factor and tyrosine kinase receptor TrkB in rat brain are significantly altered after haloperidol and risperidone administration

The antipsychotics haloperidol and risperidone are widely used in the therapy of schizophrenia. The former drug mainly acts on the dopamine (DA) D(2) receptor whereas risperidone binds to both DA and serotonin (5HT) receptors, particularly in the neurons of striatal and limbic structures. Recent evidence suggests that neurotrophins might also be involved in antipsychotic action in the central nervous system (CNS). We have previously reported that haloperidol and risperidone significantly affect brain nerve growth factor (NGF) level suggesting that these drugs influence the turnover of endogenous growth factors. Brain-derived neurotrophic factor (BDNF) supports survival and differentiation of developing and mature brain DA neurons. We hypothesized that treatments with haloperidol or risperidone will affect synthesis/release of brain BDNF and tested this hypothesis by measuring BDNF and TrkB in rat brain regions after a 29-day-treatment with haloperidol or risperidone added to chow. Drug treatments had no effects on weight of brain regions. Chronic administration of these drugs, however, altered BDNF synthesis or release and expression of TrkB-immunoreactivity within the brain. Both haloperidol and risperidone significantly decreased BDNF concentrations in frontal cortex, occipital cortex and hippocampus and decreased or increased TrkB receptors in selected brain structures. Because BDNF can act on a variety of CNS neurons, it is reasonable to hypothesize that alteration of brain level of this neurotrophin could constitute one of the mechanisms of action of antipsychotic drugs. These observations also support the possibility that neurotrophic factors play a role in altered brain function in schizophrenic disorders.     

Brain-derived neurotrophic factor upregulates and maintains AMPA receptor currents in neocortical GABAergic neurons

The regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors is implicated in synaptic plasticity. Although we have found that brain-derived neurotrophic factor (BDNF) triggers surface translocation of AMPA receptor proteins, the physiological significance of the BDNF effect remained to be determined. The present immunohistochemical studies revealed that cortical GABAergic neurons exhibited the most striking response to BDNF. Accordingly, we monitored AMPA-triggered currents through GABAergic neurons: Chronic BDNF treatment increased the AMPA-triggered currents but not NMDA-triggered currents in culture. In parallel, the amplitude, but not frequency, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) was elevated in GABAergic neurons. In agreement, BDNF enhanced GABA release triggered by AMPA compared to the amount triggered by high potassium. Conversely, there was a significant decrease in the mEPSC amplitude of GABAergic neurons in heterozygous BDNF-knockout mice. These findings indicate that the neurotrophin enhances the input sensitivity of GABAergic neurons to facilitate their inhibitory function in the neocortex.