Neuroprotective effects of genistein in VSC4.1 motoneurons exposed to activated microglial cytokines

Pro-inflammatory cytokines released from activated microglia may be responsible for neuronal damage and resulting motor deficits associated with CNS disorders such as spinal cord injury, Parkinson’s disease, and multiple sclerosis. Estrogen (17β-estradiol) is capable of ameliorating motoneuron death following spinal cord injury, but has a number of deleterious side effects. Genistein (GEN), an estrogen receptor beta agonist and potent antioxidant, may represent an alternative to estrogen in treating neurodegenerative disorders. However, little is known about the neuroprotective effects of GEN. We therefore tested whether GEN would prevent apoptosis in cultured motoneurons following exposure to pro-inflammatory cytokines released from IFN-γ activated microglia. Exposure of ventral spinal cord 4.1 motoneurons to microglial cytokine supernatant in vitro caused significant apoptosis and reduced mitochondrial membrane potential. An increase in reactive oxygen species, intracellular Ca²⁺, calpain, caspases, cytochrome c, and the bax:bcl-2 ratio were also noted. GEN treatment reversed apoptotic death and cellular changes following cytokine exposure and was associated with increased expression of estrogen receptor β suggesting that GEN may promote neuroprotection via receptor-mediated pathways. The addition of ICI 182, 780, an estrogen receptor antagonist following GEN treatment attenuated neuroprotection, suggesting that GEN may act mainly via estrogen receptor β to protect VSC4.1 motoneurons. We conclude that GEN protects cultured ventral spinal cord 4.1 cells from inflammatory insult and thus may represent a potential beneficial therapy in the treatment of neurodegenerative disorders.     

Culture models for the study of estradiol-induced synaptic plasticity

Estrogen, which classically affects areas of the brain related to reproduction, has also been found to affect brain regions important in learning and memory. Additionally, it has been suggested that estrogen exerts protective effects against neurodegenerative diseases such as Alzheimer's disease. Important mechanisms by which estrogen may confer protection are through the maintenance or modulation of existing synapses, or by the production of new ones. It has now been demonstrated that estrogen can increase synaptogenesis and spine production in the hippocampus, both in vivo as well as in primary hippocampal neurons in culture. The latter model system is the primary focus of this review. Synaptogenesis and spine production have been well characterized in developing and adult animals, and parallels between the synaptic morphology reflecting these processes can be readily observed in high-density primary hippocampal cultures. Moreover, in culture, estrogen induces a variety of ultrastructural modifications, many of which occur in vivo, that have been linked to various in vivo models of learning and memory. For these reasons, high-density hippocampal culture systems should be regarded as valuable tools with which to predict in vivo physiology, and may well be particularly useful for studies of the neuroprotective effects of estrogen.     

Differential effects of sex steroids in young and aged female mRen2.Lewis rats: A model of estrogen and salt-sensitive hypertension

Background: Male-female differences in the expression of hypertension and in end-organ damage are evident in both experimental models and human subjects, with males exhibiting a more rapid onset of cardiovascular disease and mortality than do females. The basis for these male-female differences is probably the balance of the complex effects of sex steroids (androgens, estrogen, progesterone) and their metabolites on the multiple regulatory systems that influence blood pressure (BP). A key target of estrogen and other steroids is likely to be the different components of the renin-angiotensin-aldosterone system (RAAS).Objective: The aim of this study was to review the current experimental evidence on the protective effects of estrogen in hypertensive models.Methods: The search terms estrogen , renin-aangiotensin-aldosterone system, renin receptor, salt-sensitivity, endorgan damage, hypertension, kidney, mRen2.Lewis, and injury markers were used to identify relevant publications in the PubMed database (restricted to the English language) from January 1990 to October 2007.Results: In a new congenic model that expresses the mouse renin 2 gene (mRen2.Lewis), estrogen depletion (via ovariectomy [OVX ]) in young rats was found to have a marked stimulatory effect on the progression of increased BP and cardiac dysfunction. Moreover, estrogen depletion exacerbated salt-sensitive hypertension and the extent of salt-induced cardiac and renal injury in young mRen2.Lewis rats, which probably reflected the inability to appropriately regulate various components of the RAAS. However, OVX in aged mRen2.Lewis rats conveyed renal protective effects from a high-salt diet compared with intact hypertensive littermates (64 weeks), and these effects were independent of changes in BP.Conclusion: These studies in hypertensive mRen2.Lewis rats underscored the influence of ovarian hormones on BP and tissue injury, as well as the plasticity of this response, apparently due to age and salt status.     

High-Altitude Pulmonary Hypertension and Signal Transduction in the Cardiovascular System

Estrogen plays a cardioprotective role in female rat hearts subjected to ischemia/reperfusion injury. The its effects are, at least partially, associated with decreased cardiomyocyte contraction and increased expression of β₂-adrenoceptor (β₂-AR). We tested whether β₂-AR could be involved in cardioprotection against ischemic damage and whether the roles of β₂-AR were dependent on estrogenic environment. We first determined the effects of hypoxia/reoxygenation (H/R) on cardiomyocyte shortening in female rats. We then determined the roles of β₂-AR in cardiomyocyte shortening, lactate dehydrogenase (LDH) release in culture medium, and cell death during hypoxia in isolated myocytes from female rats. We further determined the effects of estrogen on the roles of β₂-AR during hypoxia. H/R induced short-term hibernation and stunning at the level of ventricular myocytes from normal female rats. Inhibition of β₂-AR with ICI118,551 significantly elevated adrenergic contractile reserve, myocardial injury, and cell death in normal female rats during hypoxia, whereas ovariectomy (OVX) prominently enhanced myocyte contraction, myocardial injury, and cell death, and deprived the alternations in normal female rats. These changes were restored to normal by estrogen replacement (OVX+E₂). These data suggest that β₂-AR may be involved in the cardioprotection against ischemic damage, and the cardioprotection may depend on estrogenic environment.     

Novel effects of diosgenin on skin aging

Extracts of Dioscorea coomposita or Dioscorea villosa are consumed as supplemental health foods at the time of climacteric. The extracts contain large amounts of the plant steroid, diosgenin. Here, we studied the safety and efficacy of diosgenin against skin aging at the time of climacteric. In vitro, diosgenin enhanced DNA synthesis in a human 3D skin equivalent model, and increased bromodeoxyuridine uptake and intracellular cAMP level in adult human keratinocytes. The increase of bromodeoxyuridine uptake by diosgenin was blocked by an adenylate cyclase inhibitor, but not by antisense oligonucleotides against estrogen receptor α, estrogen receptor β or an orphan G-protein-coupled receptor, GPR30, indicating the involvement of cAMP but not estrogen receptor α, estrogen receptor β or GPR30. In vivo, administration of diosgenin improved the epidermal thickness in the ovariectomized mice, a climacteric model, without altering the degree of fat accumulation. In order to examine the safety of diosgenin, diosgenin and 17β-estradiol were administered to breast cancer-burdened mice. The results revealed that while 17β-estradiol accelerated the tumor growth, diosgenin did not show this effect. Our finding, a restoration of keratinocyte proliferation in aged skin, suggests that diosgenin may have potential as a safe health food for climacteric.     

Efficacy of curcumin in the treatment of experimental vulvovaginal candidiasis

BackgroundCandida albicans is the main agent that causes vulvovaginal candidiasis. Resistance among isolates to azole antifungal agents has been reported.AimsDue to the well-known antifungal potential of curcumin, the purpose of this work was to evaluate the in vitro anticandidal activity of curcumin and its effect in the treatment of experimental vulvovaginal candidiasis.MethodsThe anticandidal activity of curcumin was investigated against eight Candida strains by the broth microdilution assay, and its mechanism of action was evaluated by testing the binding to ergosterol. Then, the effect of curcumin in the treatment of vulvovaginal candidiasis was evaluated in an immunosuppressed, estrogen treated rat model.ResultsCurcumin showed minimum inhibitory concentration values of 125–1000 μg/ml, and the best result was observed against Candida glabrata. The compound was shown to be able to bind to the ergosterol present in the membrane, event that may be the mechanism of action. In addition, in the in vivo model of vulvovaginal candidiasis with C. albicans, treatments reduced the vaginal fungal burden in infected rats after seven days of treatment with different doses.ConclusionsCurcumin could be considered a promising effective antifungal agent in the treatment of vulvovaginal candidiasis.     

Genetic and pharmacologic strategies to determine the function of estrogen receptor α and estrogen receptor β in cardiovascular system

Background: The biological functions of estrogens extend beyond the female and male reproductive tract, affecting the cardiovascular and renal systems. Traditional views on the role of postmenopausal hormone therapy (HT) in protecting against heart disease, which were challenged by clinical end point studies that found adverse effects of combined HT, are now being replaced by more differentiated concepts suggesting a beneficial role of early and unopposed HT that does not include a progestin.Objective: We reviewed recent insights, concepts, and research results on the biology of both estrogen receptor (ER) subtypes, ERα and ERβ, in cardiac and vascular tissues. Knowledge of these ER subtypes is crucial to understanding gender and estrogen effects and to developing novel, exciting strategies that may have a profound clinical impact.Methods: This review focuses on in vivo studies and includes data presented at the August 2007 meeting of the American Physiological Society as well as data from a search of the MEDLINE and Ovid databases from January 1986 to November 2007. Search results were restricted to English-language publications, using the following search terms: estrogen, estrogen receptor α, estrogen receptor β, estrogen receptor α agonist, estrogen receptor α antagonist, estrogen receptor β agonist, estrogen receptor β antagonist, PPT, DPN, heart, vasculature, ERKO mice, BERKO mice, transgenic mice, and knockout mice.Results: Genetic mouse models and pharmacologic studies that employed selective as well as nonselective ER agonists support the concept that both ER subtypes confer protective effects in experimental models of human heart disease, including hypertension, cardiac hypertrophy, and chronic heart failure.Conclusions: Genetic models and novel ligands hold the promise of further improving our understanding of estrogen action in multiple tissues and organs. These efforts will ultimately enhance the safety and efficacy of HT and may also result in new applications for synthetic female sex hormone analogues.     

Peroxynitrite induced formation of the neurotoxins 5-S-cysteinyl-dopamine and DHBT-1: implications for Parkinson's disease and protection by polyphenols

Mechanisms of nigral cell injury in Parkinson’s disease remain unclear, although a combination of increased oxidative stress, the formation of catecholamine-quinones and the subsequent formation of neurotoxic cysteinyl-catecholamine conjugates may contribute. In the present study, peroxynitrite was observed to generate both 2-S- and 5-S-cysteinyl-dopamine and a dihydrobenzothiazine species, DHBT-1, following the reaction of dopamine with l-cysteine. The formation of 5-S-cysteinyl-dopamine and DHBT-1 in the presence of peroxynitrite induced significant neuronal injury. Pre-treatment of cortical neurons with pelargonidin, quercetin, hesperetin, caffeic acid, the 4′-O-Me derivatives of catechin and epicatechin (0.1-3.0 μM) resulted in concentration dependant protection against 5-S-cysteinyl-dopamine-induced neurotoxicity. These data suggest that polyphenols may protect against neuronal injury induced by endogenous neurotoxins relevant to the aetiology of the Parkinson disease.     

Sex hormones and aortic wall remodeling in an arteriovenous fistula

Background: An arteriovenous fistula (AVF) creates high blood flow through the artery and fistula. With this high flow, there is flow-induced remodeling and an increase in diameter, but no intimal hyperplasia. Estrogen has been shown to modify vascular remodeling, decreasing intimal hyperplasia after endothelial injury.Objective: These experiments tested the hypothesis that estrogen administration would decrease wall thickness in an AVE model. Because estrogen may decrease wall thickness, we also tested the hypothesis that testosterone would increase wall thickness.Methods: A fistula was created between the abdominal aorta and the inferior vena cava in Sprague-Dawley rats to generate high blood flow conditions in the aorta. Four groups of female animals were examined: sham, control with AVE ovariectomized (OVX) with AVE and OVX plus testosterone with AVE Four groups of male animals were also examined: sham, control with AVE castrated with AVE and castrated plus estrogen with AVE Five weeks after creation of the AVF, the aortas were collected and fixed; wall thickness was measured both proximal and distal to the AVEResults: Ovariectomy resulted in a significant decrease in estrogen levels (P < 0.01). Testosterone administration tended to increase testosterone levels in the OVX females, but values did not approach levels observed in the control males. No difference was noted in the proximal wall thickness between the control and the OVX animals. The OVX females receiving testosterone exhibited a significant increase in both proximal and distal wall thickness compared with control females (P < 0.001). In the male animals, there was no significant change in aortic wall thickness in the castrated rats compared with the controls. Estrogen administration in the castrated males resulted in a significant decrease in wall thickness in the proximal and distal aorta (P < 0.05).Conclusion: These studies suggest that, in a model of vascular remodeling, estrogen administration decreases wall thickness, and testosterone administration increases wall thickness.     

The Novel Role of Platelet-Activating Factor in Protecting Mice against Lipopolysaccharide-Induced Endotoxic Shock

Background

Platelet-activating factor (PAF) has been long believed to be associated with many pathophysiological processes during septic shock. Here we present novel activities for PAF in protecting mice against LPS-mediated endotoxic shock.

Principal Findings

In vivo PAF treatment immediately after LPS challenge markedly improved the survival rate against mortality from endotoxic shock. Administration of PAF prominently attenuated LPS-induced organ injury, including profound hypotension, excessive polymorphonuclear neutrophil infiltration, and severe multiple organ failure. In addition, PAF treatment protects against LPS-induced lymphocytes apoptosis. These protective effects of PAF was correlated with significantly decreases in the production of the inflammatory mediators such as TNF-α, IL-1β, IL-12, and IFN-γ, while increasing production of the anti-inflammatory cytokine IL-10 in vivo and in vitro.

Conclusions

Taken together, these results suggest that PAF may protect mice against endotoxic shock via a complex mechanism involving modulation of inflammatory and anti-inflammatory mediators.     

Immunoreactivities of AR,ERα, ERβ and aromatase in the nuptial pad of Chinese brown frog (Rana dybowskii) during pre-hibernation and the breeding period

There is a prominent local raised pad called nuptial pad on the forelimb of Chinese brown frog (Rana dybowskii), which is hypothetically concluded as an enhancement of the grip and a spreader of pheromone during the amplexus. In this study, we investigated the immunolocalization and protein expression levels of androgen receptors (AR), estrogen receptor α (ERα), ERβ and aromatase in the nuptial pad of R. dybowskii during pre-hibernation and the breeding period. Histologically, the annual development of the nuptial pad in R. dybowskii is manifested as the larger area of specialized mucous gland and the longer length of papillary epidermal projection during the breeding period. AR, ERα, ERβ and aromatase are present in the stratum granulosum, stratum spinosum, stratum basale and the secretory portion of specialized mucous glands during both periods. Western blotting results confirmed that AR, ERα and ERβ protein levels are higher during pre-hibernation than those during the breeding season. These results suggest that nuptial pad is the direct target organ of androgen and estrogen. Androgen may participate in the regulation of annual development and glandular function of nuptial pad, and estrogen may play an endocrine, autocrine or paracrine role during pre-hibernation and the breeding period.Key words: Androgen receptor, aromatase, estrogen receptor, nuptial pad, Rana dybowskii.     

Microarray Analysis of Perinatal-Estrogen-Induced Changes in Gene Expression Related to Brain Sexual Differentiation in Mice

Sexual dimorphism of the behaviors or physiological functions in mammals is mainly due to the sex difference of the brain. A number of studies have suggested that the brain is masculinized or defeminized by estradiol converted from testicular androgens in perinatal period in rodents. However, the mechanisms of estrogen action resulting in masculinization/defeminization of the brain have not been clarified yet. The large-scale analysis with microarray in the present study is an attempt to obtain the candidate gene(s) mediating the perinatal estrogen effect causing the brain sexual differentiation. Female mice were injected with estradiol benzoate (EB) or vehicle on the day of birth, and the hypothalamus was collected at either 1, 3, 6, 12, or 24 h after the EB injection. More than one hundred genes down-regulated by the EB treatment in a biphasic manner peaked at 3 h and 12-24 h after the EB treatment, while forty to seventy genes were constantly up-regulated after it. Twelve genes, including Ptgds, Hcrt, Tmed2, Klc1, and Nedd4, whose mRNA expressions were down-regulated by the neonatal EB treatment, were chosen for further examination by semiquantitative RT-PCR in the hypothalamus of perinatal intact male and female mice. We selected the genes based on the known profiles of their potential roles in brain development. mRNA expression levels of Ptgds, Hcrt, Tmed2, and Nedd4 were significantly lower in male mice than females at the day of birth, suggesting that the genes are down-regulated by estrogen converted from testicular androgen in perinatal male mice. Some genes, such as Ptgds encoding prostaglandin D2 production enzyme and Hcrt encording orexin, have been reported to have a role in neuroprotection. Thus, Ptgds and Hcrt could be possible candidate genes, which may mediate the effect of perinatal estrogen responsible for brain sexual differentiation.     

Cerebral venous sinus thrombosis after gender reassignment surgery

Background: Many factors have been implicated in the etiology of cerebral venous sinus thrombosis (CVT). These include head injury, cancer, infections (sepsis, sinusitis, and mastoiditis), coagulopathies, pregnancy, systemic lupus erythematosus, and dehydration.Case summary: We present the case of a patient who received long-term estrogen therapy for ~15 years after feminizing genitoplasty. The patient experienced a CVT with an excellent clinical outcome. A similar case has not been reported in the literature.Conclusion: Because CVT may be associated with morbidity, mortality, and risks from the complications and treatments of the condition, further research is needed to clarify the factors that may contribute to the long-term risk of CVT in patients receiving long-term estrogen therapy after feminizing genitoplasty.     

Exopolysaccharide-peptide complex from oyster mushroom (Pleurotus ostreatus) protects against hepatotoxicity in rats

Liver damage involves oxidative stress and a progression from chronic hepatitis to hepatocellular carcinoma (HCC). The increased incidence of liver disease in Egypt and other countries in the last decade, coupled with poor prognosis, justify the critical need to introduce alternative chemopreventive agents that may protect against liver damage. The aim of this study was to evaluate the efficacy of exopolysaccharide-peptide (PSP) complex extracted from Pleurotus ostreatus as a hepatoprotective agent against diethylnitrosamine (DEN)/carbon tetrachloride (CCL₄)-induced hepatocellular damage in rats. The levels of liver injury markers (ALT, AST and ALP) were substantially increased following DEN/CCl₄ treatment. DEN/CCl₄ - induced oxidative stress was confirmed by elevated levels of lipid peroxidation and decreased levels of superoxide dismutase, glutathione-S-transferase, and reduced glutathione. PSP reversed these alterations in the liver and serum, and provided protection evidenced by reversal of histopathological changes in the liver. The present study demonstrated that PSP extract from P. ostreatus exhibited hepatoprotective and antioxidant effects against DEN/CCl₄-induced hepatocellular damage in rats. Given the high prevalence of HCV-related liver damage in Egypt, our results suggest further clinical evaluation of P. ostreatus extracts and their potential hepatoprotective effects in patients with liver disease.     

Protective effects of L-pGlu-(2-propyl)-L-His-L-ProNH2, a newer thyrotropin releasing hormone analog in in vitro and in vivo models of cerebral ischemia

In the present study, the newly synthesized TRH analog (l-pGlu-(2-propyl)-l-His-l-ProNH₂; NP-647) was evaluated for its effects in in vitro (oxygen glucose deprivation (OGD)-, glutamate- and H₂O₂-induced injury in PC-12 cells) and in vivo (transient global ischemia) models of cerebral ischemic injury. PC-12 cells were subjected to oxygen and glucose deprivation for 6 h. Exposure of NP-647 was given before and during OGD. In glutamate and H₂O₂ induced injury, exposure of NP-647 was given 1, 6 and 24 h prior to exposure of glutamate and H₂O₂ exposure. NP-647, per se found to be non-toxic in 1-100 μM concentrations. NP-647 showed protection against OGD at the 1 and 10 μM. The concentration-dependent protection was observed in H₂O₂- and glutamate-induced cellular injury. In in vivo studies, NP-647 treatment showed protection of hippocampal (CA1) neuronal damage in transient global ischemia in mice and subsequent improvement in memory retention was observed using passive avoidance retention test. Moreover, administration of NP-647 resulted in decrease in inflammatory cytokines TNF-α and IL-6 as well as lipid peroxidation. These results suggest potential of NP-647 in the treatment of cerebral ischemia and its neuroprotective effect may be attributed to reduction of excitotoxicity, oxidative stress and inflammation.     

Transcriptional regulation of Fhl1 by estradiol in rat myoblastocytes

Fhl1 (Four and a Half LIM domain 1) regulates muscle growth and development. In addition, skeletal myoblast growth is significantly affected by gender differences, implicating estrogen in the regulation of muscle development. We sought to determine if estrogen influences Fhl1 gene expression levels in rat L6GNR4 myoblastocytes that express the estrogen receptor β (ERβ), while luciferase assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay were employed to confirm the interaction between ERβ and Fhl1. Treatment of L6GNR4 cells with physiological levels of 17β-estradiol (E2) results in markedly decreased endogenous Fhl1 expression. Tamoxifen, an ER antagonist, partially reverses E2-mediated Fhl1 down-regulation in L6GNR4 cells. Furthermore, luciferase assay and EMSA identified a novel promoter region of Fhl1 that directly interacts with ERβ. ChIP of the ERβ-Fhl1 promoter complex from L6GNR4 cells confirmed that endogenous ERβ interacts with this region. These data indicate that E2 down-regulates Fhl1 expression through its binding to the ERβ. This is the first report of a small molecule that can affect Fhl1 expression. E2 may therefore be useful in developing new strategies for regulating Fhl1 expression and understanding the influence of estrogen on muscle growth and development.     

Sex-specific alterations in estrogen receptor interactions following induced androgen imbalance in vivo

Androgens have been shown, under in vitro conditions, to be capable of impeding the rate of formation of estrogen-receptor complexes in target tissues of the rat. The present study was designed to investigate effects of abnormal androgen levels in vivo on various estrogen receptor systems. Serum levels of testosterone (T) and 5α-DHT were measured in adult neonatally-androgenized rats. The T/DHT ratio in the androgenized animal was 0.70, compared to 4.37 in the normal adult rat, and this was unaccompanied by any change in the sum of the 2 androgens. Estradiol levels were equivalent to those of normal rats in estrus. In addition to this animal model, castrate rats of both sexes which had been administered chronic high dosages of various androgens were examined. Equilibrium binding studies of cytosol from uterus, anterior putuitary and hypothalamus showed that estrogen receptors were not modified in either of these animal models, with specific reference to affinity of estradiol binding or concentration of binding sites. The association rate kinetics for estradiol-receptor complex formation in uterus and pituitary were unaffected by androgen administration to ovariectomized animals; however, in the corresponding male castrate model, interaction between estradiol and its pituitary cytosol receptor was accelerated by in vivo exposure to androgens, and the effect was dependent on the nature and level of the androgen used. Neonatally-androgenized rats also manifested an initial rate of estradiol-receptor interaction which was appreciably higher than control values. The reversibility of the androgen effect on the estrogen receptor was demonstrated in an in vitro protocol. The results indicate that the in vivo effects of androgens on estrogen receptor kinetics are sex-dependent and, where observed, any influence was of a stimulatory nature. Moreover, it appears that the nature of the androgen present in vivo is at least as important a determinant as the total androgen concentration, and that androgens do not engender permanent changes in the ability of the estrogen receptor to interact with estradiol.     

Reciprocal regulation of estrogen and NGF receptors by their ligands in PC12 cells

Recent work has shown that estrogen receptor mRNA and protein co-localize with neurotrophin receptor systems in the developing basal forebrain. In the present study we examined the potential for reciprocal regulation of estrogen and neurotrophin receptor systems by their ligands in a prototypical neurotrophin target, the PC12 cell. using in situ hybridization histochemistry, RT-PCR and a modified nuclear exchange assay, we found both estrogen receptor mRNA and estrogen binding in PC12 cells. Moreover, while estrogen binding was relatively low in naive PC12 cells, long-term exposure to NGF enhanced estrogen binding in these cells by sixfold. Furthermore, concurrent exposure to estrogen and NGF receptor mRNAs deifferentially regulated the expression of the two NGF receptor mRNAs. The expression of trkA mRNA was up-regulated, while p75^NGFR mRNA was down-regulated transiently. The present data indicate that NGF may increase neuronal sensitivity to estrogen, and that estrogen, by differentially regulating p75^NGFR and trkA mRNA, may alter the ratio fo the two NGF receptors, and, conseuqnetly, neurotrophin responsivity. In view of the widespread co-localization of estrogen and neurotrophin receptor systems in the developing CNS, the reciprocal regulation of these receptor systems by NGF and estrogen may have important implications for processes governing neural maturation and the maintenance of neural funciton. 1994 John Wiley & Sons, Inc.