RESUMEN
Synaptic alterations concomitant with neuroinflammation have been described in patients and experimental models of autism spectrum disorder (ASD). However, the role of microglia and astroglia in relation to synaptic changes is poorly understood. Male Wistar rats prenatally exposed to valproic acid (VPA, 450 mg/kg, i.p.) or saline (control) at embryonic day 10.5 were used to study synapses, microglia, and astroglia in the prefrontal cortex (PFC) at postnatal days 3 and 35 (PND3 and PND35). Primary cultures of cortical neurons, microglia, and astroglia isolated from control and VPA animals were used to study each cell type individually, neuron-microglia and microglia-astroglia crosstalk. In the PFC of VPA rats, synaptic changes characterized by an increase in the number of excitatory synapses were evidenced at PND3 and persisted until PND35. At PND3, microglia and astroglia from VPA animals were morphologically similar to those of age-matched controls, whereas at PND35, reactive microgliosis and astrogliosis were observed in the PFC of VPA animals. Cortical neurons isolated from VPA rats mimicked in vitro the synaptic pattern seen in vivo. Cortical microglia and astroglia isolated from VPA animals exhibited reactive morphology, increased pro-inflammatory cytokines, and a compromised miRNA processing machinery. Microglia from VPA animals also showed resistance to a phagocytic challenge. In the presence of neurons from VPA animals, microglia isolated from VPA rats revealed a non-reactive morphology and promoted neurite outgrowth, while microglia from control animals displayed a reactive profile and promoted dendritic retraction. In microglia-astroglia co-cultures, microglia from VPA animals displayed a reactive profile and exacerbated astrocyte reactivity. Our study indicates that cortical microglia from VPA animals are insensitive or adapted to neuronal cues expressed by neurons from VPA animals. Further, long-term in vivo microgliosis could be the result of altered microglia-astroglia crosstalk in VPA animals. Thus, our study highlights cortical microglia-astroglia communication as a new mechanism implicated in neuroinflammation in ASD; consequently, we propose that this crosstalk is a potential target for interventions in this disorder.
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Atypical connectivity between brain regions and altered structure of the corpus callosum (CC) in imaging studies supports the long-distance hypoconnectivity hypothesis proposed for autism spectrum disorder (ASD). The aim of this study was to unveil the CC ultrastructural and cellular changes employing the valproic acid (VPA) rat model of ASD. Male Wistar rats were exposed to VPA (450 mg/kg i.p.) or saline (control) during gestation (embryonic day 10.5), and maturation, exploration, and social behavior were subsequently tested. Myelin content, ultrastructure, and oligodendroglial lineage were studied in the CC at post-natal days 15 (infant) and 36 (juvenile). As a functional outcome, brain metabolic activity was determined by positron emission tomography. Concomitantly with behavioral deficits in juvenile VPA rats, the CC showed reduced myelin basic protein, conserved total number of axons, reduced percentage of myelinated axons, and aberrant and less compact arrangements of myelin sheath ultrastructure. Mature oligodendrocytes decreased and oligodendrocyte precursors increased in the absence of astrogliosis or microgliosis. In medial prefrontal and somatosensory cortices of juvenile VPA rats, myelin ultrastructure and oligodendroglial lineage were preserved. VPA animals exhibited global brain hypometabolism and local hypermetabolism in brain regions relevant for ASD. In turn, the CC of infant VPA rats showed reduced myelin content but preserved oligodendroglial lineage. Our findings indicate that CC hypomyelination is established during infancy and prior to oligodendroglial pattern alterations, which suggests that axon-oligodendroglia communication could be compromised in VPA animals. Thus, CC hypomyelination may underlie white matter alterations and contribute to atypical patterns of connectivity and metabolism found in ASD.
Asunto(s)
Trastorno del Espectro Autista/metabolismo , Cuerpo Calloso/metabolismo , Red Nerviosa/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Conducta Social , Ácido Valproico/toxicidad , Animales , Trastorno del Espectro Autista/inducido químicamente , Trastorno del Espectro Autista/patología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Cuerpo Calloso/efectos de los fármacos , Cuerpo Calloso/patología , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Femenino , Masculino , Red Nerviosa/efectos de los fármacos , Red Nerviosa/patología , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/patología , Ratas , Ratas Wistar , Tomografía Computarizada de Emisión de Fotón Único/métodosRESUMEN
BACKGROUND: Autism spectrum disorders (ASD) are synaptopathies characterized by area-specific synaptic alterations and neuroinflammation. Structural and adhesive features of hippocampal synapses have been described in the valproic acid (VPA) model. However, neuronal and microglial contribution to hippocampal synaptic pattern and its time-course of appearance is still unknown. METHODS: Male pups born from pregnant rats injected at embryonic day 10.5 with VPA (450 mg/kg, i.p.) or saline (control) were used. Maturation, exploratory activity and social interaction were assessed as autistic-like traits. Synaptic, cell adhesion and microglial markers were evaluated in the CA3 hippocampal region at postnatal day (PND) 3 and 35. Primary cultures of hippocampal neurons from control and VPA animals were used to study synaptic features and glutamate-induced structural remodeling. Basal and stimuli-mediated reactivity was assessed on microglia primary cultures isolated from control and VPA animals. RESULTS: At PND3, before VPA behavioral deficits were evident, synaptophysin immunoreactivity and the balance between the neuronal cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) were preserved in the hippocampus of VPA animals along with the absence of microgliosis. At PND35, concomitantly with the establishment of behavioral deficits, the hippocampus of VPA rats showed fewer excitatory synapses and increased NCAM/PSA-NCAM balance without microgliosis. Hippocampal neurons from VPA animals in culture exhibited a preserved synaptic puncta number at the beginning of the synaptogenic period in vitro but showed fewer excitatory synapses as well as increased NCAM/PSA-NCAM balance and resistance to glutamate-induced structural synaptic remodeling after active synaptogenesis. Microglial cells isolated from VPA animals and cultured in the absence of neurons showed similar basal and stimuli-induced reactivity to the control group. Results indicate that in the absence of glia, hippocampal neurons from VPA animals mirrored the in vivo synaptic pattern and suggest that while neurons are primed during the prenatal period, hippocampal microglia are not intrinsically altered. CONCLUSIONS: Our study suggests microglial role is not determinant for developing neuronal alterations or counteracting neuronal outcome in the hippocampus and highlights the crucial role of hippocampal neurons and structural plasticity in the establishment of the synaptic alterations in the VPA rat model.
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Anticonvulsivantes , Trastorno del Espectro Autista/inducido químicamente , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Sinapsis/efectos de los fármacos , Ácido Valproico , Animales , Trastorno del Espectro Autista/metabolismo , Conducta Animal/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Hipocampo/ultraestructura , Masculino , Microglía/efectos de los fármacos , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Neuronas/metabolismo , Neuronas/ultraestructura , Fosfoproteínas Fosfatasas/metabolismo , Embarazo , Ratas WistarRESUMEN
Temporal Lobe Epilepsy (TLE) is the most common form of human epilepsy and available treatments with antiepileptic drugs are not disease-modifying therapies. The neuroinflammation, neuronal death and exacerbated plasticity that occur during the silent period, following the initial precipitating event (IPE), seem to be crucial for epileptogenesis. Damage Associated Molecular Patterns (DAMP) such as HMGB-1, are released early during this period concomitantly with a phenomenon of reactive gliosis and neurodegeneration. Here, using a combination of primary neuronal and glial cell cultures, we show that exposure to HMGB-1 induces dendrite loss and neurodegeneration in a glial-dependent manner. In glial cells, loss of function studies showed that HMGB-1 exposure induces NF-κB activation by engaging a signaling pathway that involves TLR2, TLR4, and RAGE. In the absence of glial cells, HMGB-1 failed to induce neurodegeneration of primary cultured cortical neurons. Moreover, purified astrocytes were unable to fully respond to HMGB-1 with NF-κB activation and required microglial cooperation. In agreement, in vivo HMGB-1 blockage with glycyrrhizin, immediately after pilocarpine-induced status epilepticus (SE), reduced neuronal degeneration, reactive astrogliosis and microgliosis in the long term. We conclude that microglial-astroglial cooperation is required for astrocytes to respond to HMGB-1 and to induce neurodegeneration. Disruption of this HMGB-1 mediated signaling pathway shows beneficial effects by reducing neuroinflammation and neurodegeneration after SE. Thus, early treatment strategies during the latency period aimed at blocking downstream signaling pathways activated by HMGB-1 are likely to have a significant effect in the neuroinflammation and neurodegeneration that are proposed as key factors in epileptogenesis.
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Astroglial cells are crucial for central nervous system (CNS) homeostasis. They undergo complex morpho-functional changes during aging and in response to hormonal milieu. Ovarian hormones positively affect different astroglia parameters, including regulation of cell morphology and release of neurotrophic and neuroprotective factors. Thus, ovarian hormone loss during menopause has profound impact in astroglial pathophysilogy and has been widely associated to the process of brain aging. Humanin (HN) is a secreted mitochondrial-encoded peptide with neuroprotective effects. It is localized in several tissues with high metabolic rate and its expression decreases with age. In the brain, humanin has been found in glial cells in physiological conditions. We previously reported that surgical menopause induces hippocampal mitochondrial dysfunction that mimics an aging phenotype. However, the effect of ovarian hormone deprivation on humanin expression in this area has not been studied. Also, whether astrocytes express and release humanin and the regulation of such processes by ovarian hormones remain elusive. Although humanin has also proven to be beneficial in ameliorating cognitive impairment induced by different insults, its putative actions on structural synaptic plasticity have not been fully addressed. In a model of surgical menopause in rats, we studied hippocampal humanin expression and localization by real-time quantitative polymerase chain reaction (RT-qPCR) and double immunohistochemistry, respectively. Humanin production and release and ovarian hormone regulation of such processes were studied in cultured astrocytes by flow cytometry and ELISA, respectively. Humanin effects on glutamate-induced structural synaptic alterations were determined in primary cultures of hippocampal neurons by immunocytochemistry. Humanin expression was lower in the hippocampus of ovariectomized rats and its immunoreactivity colocalized with astroglial markers. Chronic ovariectomy also promoted the presence of less complex astrocytes in this area. Ovarian hormones increased humanin intracellular content and release by cultured astrocytes. Humanin prevented glutamate-induced dendritic atrophy and reduction in puncta number and total puncta area for pre-synaptic marker synaptophysin in cultured hippocampal neurons. In conclusion, astroglial functional and morphological alterations induced by chronic ovariectomy resemble an aging phenotype and could affect astroglial support to neuronal function by altering synaptic connectivity and functionality. Reduced astroglial-derived humanin may represent an underlying mechanism for synaptic dysfunction and cognitive decline after menopause.
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Prenatal iron deficiency (pID) has been described to increase the risk for neurodevelopmental disorders such as autism and schizophrenia; however, the precise molecular mechanisms are still unknown. Here, we utilized high-throughput MS to examine the proteomic effects of pID in adulthood on the rat frontal cortex area (FCA). In addition, the FCA proteome was examined in adulthood following risperidone treatment in adolescence to see if these effects could be prevented. We identified 1501 proteins of which 100 were significantly differentially expressed in the FCA at postnatal day 90. Pathway analysis of proteins affected by pID revealed changes in metabolic processes, including the tricyclic acid cycle, mitochondrial dysfunction, and P13K/Akt signaling. Interestingly, most of these protein changes were not present in the adult pID offspring who received risperidone in adolescence. Considering the link between pID and several neurodevelopmental disorders such as autism and schizophrenia these presented results bring new perspectives to understand the role of iron in metabolic pathways and provide novel biomarkers for future studies of pID.
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Antipsicóticos/farmacología , Lóbulo Frontal/metabolismo , Deficiencias de Hierro , Efectos Tardíos de la Exposición Prenatal/tratamiento farmacológico , Proteoma/análisis , Risperidona/farmacología , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Lóbulo Frontal/efectos de los fármacos , Hierro/metabolismo , Espectrometría de Masas , Embarazo , Proteómica , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacosRESUMEN
Mitochondrial dysfunction is a common hallmark in aging. In the female, reproductive senescence is characterized by loss of ovarian hormones, many of whose neuroprotective effects converge upon mitochondria. The functional integrity of mitochondria is dependent on membrane fatty acid and phospholipid composition, which are also affected during aging. The effect of long-term ovarian hormone deprivation upon mitochondrial function and its putative association with changes in mitochondrial membrane lipid profile in the hippocampus, an area primarily affected during aging and highly responsive to ovarian hormones, is unknown. To this aim, Wistar adult female rats were ovariectomized or sham-operated. Twelve weeks later, different parameters of mitochondrial function (O2 uptake, ATP production, membrane potential and respiratory complex activities) as well as membrane phospholipid content and composition were evaluated in hippocampal mitochondria. Chronic ovariectomy reduced mitochondrial O2 uptake and ATP production rates and induced membrane depolarization during active respiration without altering the activity of respiratory complexes. Mitochondrial membrane lipid profile showed no changes in cholesterol levels but higher levels of unsaturated fatty acids and a higher peroxidizability index in mitochondria from ovariectomized rats. Interestingly, ovariectomy also reduced cardiolipin content and altered cardiolipin fatty acid profile leading to a lower peroxidizability index. In conclusion, chronic ovarian hormone deprivation induces mitochondrial dysfunction and changes in the mitochondrial membrane lipid profile comparable to an aging phenotype. Our study provides insights into ovarian hormone loss-induced early lipidomic changes with bioenergetic deficits in the hippocampus that may contribute to the increased risk of Alzheimer's disease and other age-associated disorders observed in postmenopause.
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Ácidos Grasos/fisiología , Hormonas Esteroides Gonadales/deficiencia , Hipocampo/metabolismo , Mitocondrias/metabolismo , Fosfolípidos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Femenino , Potencial de la Membrana Mitocondrial/fisiología , Ovariectomía , Consumo de Oxígeno/fisiología , Ratas , Ratas WistarRESUMEN
Autism spectrum disorders (ASD) are a group of developmental disabilities characterized by impaired social interaction, communication deficit and repetitive and stereotyped behaviors. Neuroinflammation and synaptic alterations in several brain areas have been suggested to contribute to the physiopathology of ASD. Although the limbic system plays an important role in the functions found impaired in ASD, reports on these areas are scarce and results controversial. In the present study we searched in the medial prefrontal cortex (mPFC) and hippocampus of rats exposed to the valproic acid (VPA) model of ASD for early structural and molecular changes, coincident in time with the behavioral alterations. After confirming delayed growth and maturation in VPA rats, we were able to detect decreased exploratory activity and social interaction at an early time point (postnatal day 35). In mPFC, although typical cortical column organization was preserved in VPA animals, we found that interneuronal space was wider than in controls. Hippocampal CA3 (cornu ammonis 3) pyramidal layer and the granular layer of the dentate gyrus both showed a disorganized spatial arrangement in VPA animals. Neuronal alterations were accompanied with increased tomato lectin and glial fibrillary acidic protein (GFAP) immunostainings both in the mPFC and hippocampus. In the latter region, the increased GFAP immunoreactivity was CA3 specific. At the synaptic level, while mPFC from VPA animals showed increased synaptophysin (SYN) immunostaining, a SYN deficit was found in all hippocampal subfields. Additionally, both the mPFC and the hippocampus of VPA rats showed increased neuronal cell adhesion molecule (NCAM) immunostaining together with decreased levels of its polysialylated form (PSA-NCAM). Interestingly, these changes were more robust in the CA3 hippocampal subfield. Our results indicate that exploratory and social deficits correlate with region-dependent neuronal disorganization and reactive gliosis in the mPFC and hippocampus of VPA rats. While microgliosis is spread in these two limbic areas, astrogliosis, although extended in the mPFC, is circumscribed to the CA3 hippocampal subfield. Our work indicates that neuroinflammation and synaptic alterations do coexist in VPA rats, making this model suitable for studying novel aspects of neuron-glia interactions. Moreover, it suggests that the mPFC and hippocampus might behave differently in the context of the local hyperconnectivity and synaptic hypotheses of autism. © 2015 S. Karger AG, Basel.
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Trastorno Autístico/patología , Conducta Animal/fisiología , GABAérgicos/farmacología , Hipocampo/patología , Neuronas/patología , Corteza Prefrontal/patología , Ácido Valproico/farmacología , Animales , Trastorno Autístico/inducido químicamente , Trastorno Autístico/inmunología , Modelos Animales de Enfermedad , Conducta Exploratoria/fisiología , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/inmunología , Masculino , Neuronas/inmunología , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/inmunología , Embarazo , Efectos Tardíos de la Exposición Prenatal/patología , Ratas , Ratas Wistar , Conducta SocialRESUMEN
Dendritic and synapse remodeling are forms of structural plasticity that play a critical role in normal hippocampal function. Neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) participate in neurite outgrowth and synapse formation and plasticity. However, it remains unclear whether they contribute to dendritic retraction and synaptic disassembly. Cultured hippocampal neurons exposed to glutamate (5 µM) showed a reduced MAP-2 (+) area in the absence of neuronal death 24 h after the insult. Concomitantly, synapse loss, revealed by decreased synaptophysin and post-synaptic density-95 cluster number and area, together with changes in NCAM and PSA-NCAM levels were found. Dendritic atrophy and PSA-NCAM reduction proved NMDA-receptor dependent. Live-imaging experiments evidenced dendritic atrophy 4 h after the insult; this effect was preceded by smaller NCAM clusters (1 h) and decreased surface and total PSA-NCAM levels (3 h). Simultaneously, total NCAM cluster number and area remained unchanged. The subsequent synapse disassembly (6 h) was accompanied by reductions in total NCAM cluster number and area. A PSA mimetic peptide prevented both the dendritic atrophy and the subsequent synaptic changes (6 h) but had no effect on the earliest synaptic remodeling (3 h). Thus, NCAM-synaptic reorganization and PSA-NCAM level decrease precede glutamate-induced dendritic atrophy, whereas the NCAM level reduction is a delayed event related to synapse loss. Consequently, distinctive stages in PSA-NCAM/NCAM balance seem to accompany glutamate-induced dendritic atrophy and synapse loss.
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Dendritas/metabolismo , Ácido Glutámico/farmacología , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Ácidos Siálicos/metabolismo , Sinapsis/metabolismo , Animales , Atrofia/inducido químicamente , Atrofia/metabolismo , Atrofia/patología , Dendritas/efectos de los fármacos , Dendritas/patología , Hipocampo/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Wistar , Sinapsis/efectos de los fármacos , Sinapsis/patología , Sinaptofisina/metabolismoRESUMEN
We previously reported that combined fluoxetine administration at antidepressant doses renders additive antidepressant effects, whereas non-antidepressant doses potentiate the omega-3 fatty acid antidepressant effect. In the present study, we aimed to evaluate putative pharmacokinetic and brain omega-3 fatty acid-related aspects for fluoxetine potentiation of omega-3 fatty acid antidepressant effect in rats. Coadministration of omega-3 fatty acids with a non-antidepressant dose of fluoxetine (1 mg/kg day) failed to affect both brain fluoxetine concentration and norfluoxetine plasma concentration profile. Fluoxetine plasma concentrations remained below the sensitivity limit of the detection method. Either antidepressant (10 mg/kg day) or non-antidepressant (1 mg/kg day) doses of fluoxetine in combination with omega-3 fatty acids increased hippocampal docosapentaenoic acid (DPA, 22:5 omega-3) levels. Although individual treatments had no effects on DPA concentration, DPA increase was higher when omega-3 were combined with the non-antidepressant dose of fluoxetine. Chronic DPA administration exerted antidepressant-like effects in the forced swimming test while increasing hippocampal docosahexaenoic (22:6 omega-3) and DPA levels. Our results suggest no pharmacokinetic interaction and reveal specific hippocampal DPA changes after fluoxetine and omega-3 combined treatments in our experimental conditions. The DPA role in the synergistic effect of fluoxetine and omega-3 combined treatments will be for sure the focus of future studies. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3316-3325, 2014.
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Encéfalo/efectos de los fármacos , Ácidos Grasos Omega-3/farmacología , Fluoxetina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Administración Oral , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Sinergismo Farmacológico , Ácidos Grasos Omega-3/administración & dosificación , Ácidos Grasos Omega-3/farmacocinética , Fluoxetina/análogos & derivados , Fluoxetina/sangre , Masculino , Ratas , Ratas Wistar , Inhibidores Selectivos de la Recaptación de Serotonina/farmacocinéticaRESUMEN
The aim of this study was to determine whether early demyelination can impact behavior in young adulthood. For this purpose, albino Wistar rats of either sex were exposed to cuprizone (CPZ) in two different intoxication protocols: one group was intoxicated before weaning (CPZ-BW), from postnatal day 7 (P7) to P21, through maternal milk, whereas the other group was intoxicated after weaning (CPZ-AW), from P21 to P35. After treatment, rats were returned to a normal diet until P90 when behavioral studies were performed. Both treatments produced marked demyelination in the corpus callosum and retraction of cortical myelin fibers. The subsequent normal diet allowed for effective remyelination at P90. Interestingly, CPZ-AW correlated with significant behavioral and neurochemical changes in a gender-dependent manner. CPZ-AW treatment altered both the number of social activities and the latency to the first social interaction in males, while also highly compromising recognition-related activities. In addition, only P90 males treated AW showed a hyperdopaminergic striatum, confirmed by an increase in tyrosine hydroxylase expression and in dopamine levels. Our results suggest that the timing of demyelination significantly influences the development of altered behavior, particularly in adult males.
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Encéfalo/crecimiento & desarrollo , Encéfalo/fisiopatología , Enfermedades Desmielinizantes/fisiopatología , Esquizofrenia/fisiopatología , Caracteres Sexuales , Animales , Astrocitos/patología , Astrocitos/fisiología , Encéfalo/patología , Cuprizona , Enfermedades Desmielinizantes/patología , Dieta , Modelos Animales de Enfermedad , Dopamina/metabolismo , Femenino , Masculino , Microglía/patología , Microglía/fisiología , Actividad Motora/fisiología , Ratas Wistar , Reconocimiento en Psicología/fisiología , Esquizofrenia/patología , Serotonina/metabolismo , Conducta Social , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Classical cadherins, which are adhesion molecules functioning at the CNS synapse, are synthesized as adhesively inactive precursor proteins in the endoplasmic reticulum (ER). Signal sequence and prodomain cleavage in the ER and Golgi apparatus, respectively, activates their adhesive properties. Here, we provide the first evidence for sorting of nonadhesive precursor N-cadherin (ProN) to the neuronal surface, where it coexists with adhesively competent mature N-cadherin (N-cad), generating a spectrum of adhesive strengths. In cultured hippocampal neurons, a high ProN/N-cad ratio downregulates synapse formation. Neurons expressing genetically engineered uncleavable ProN make markedly fewer synapses. The synapse number can be rescued to normality by depleting surface ProN levels through prodomain cleavage by an exogenous protease. Finally, prodomain processing is developmentally regulated in the rat hippocampus. We conclude that it is the ProN/N-cad ratio and not mature N-cad alone that is critical for regulation of adhesion during synaptogenesis.
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Cadherinas/metabolismo , Sinapsis/fisiología , Sinapsis/ultraestructura , Animales , Células Cultivadas , Neurogénesis/fisiología , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-DawleyRESUMEN
S100B is a soluble protein secreted by astrocytes that exerts pro-survival or pro-apoptotic effects depending on the concentration reached in the extracellular millieu. The S100B receptor termed RAGE (for receptor for advanced end glycation products) is highly expressed in the developing brain but is undetectable in normal adult brain. In this study, we show that RAGE expression is induced in cortical neurons of the ischemic penumbra. Increased RAGE expression was also observed in primary cortical neurons exposed to excitotoxic glutamate (EG). S100B exerts effects on survival pathways and neurite extension when the cortical neurons have been previously exposed to EG and these S100B effects were prevented by anti-RAGE blocking antibodies. Furthermore, nuclear factor kappa B (NF-κB) is activated by S100B in a dose- and RAGE-dependent manner and neuronal death induced by NF-κB inhibition was prevented by S100B that restored NF-κB activation levels. Together, these findings suggest that excitotoxic damage can induce RAGE expression in neurons from ischemic penumbra and demonstrate that cortical neurons respond to S100B through engagement of RAGE followed by activation of NF-κB signaling. In addition, basal NF-κB activity in neurons is crucial to modulate the extent of pro-survival or pro-death S100B effects.
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Dendritas/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , FN-kappa B/metabolismo , Neuronas/patología , Receptores Inmunológicos/metabolismo , Proteínas S100/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Anticuerpos/farmacología , Isquemia Encefálica/patología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/patología , Interacciones Farmacológicas , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Glutámico/toxicidad , Masculino , Neuronas/efectos de los fármacos , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Wistar , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/inmunología , Proteínas S100/metabolismo , Transducción de Señal/fisiología , Sulfadiazina/farmacología , Factores de TiempoRESUMEN
Despite the advances in psychopharmacology, the treatment of depressive disorders is still not satisfactory. Side effects and resistance to antidepressant drugs are the greatest complications during treatment. Based on recent evidence, omega-3 fatty acids may influence vulnerability and outcome in depressive disorders. The aim of this study was to further characterize the omega-3 antidepressant-like effect in rats in terms of its behavioral features in the depression model forced swimming test either alone or in combination with antidepressants fluoxetine or mirtazapine. Ultimately, we prompted to determine the lowest dose at which omega-3 fatty acids and antidepressant drugs may still represent a pharmacological advantage when employed in combined treatments. Chronic diet supplementation with omega-3 fatty acids produced concentration-dependent antidepressant-like effects in the forced swimming test displaying a behavioral profile similar to fluoxetine but different from mirtazapine. Fluoxetine or mirtazapine at antidepressant doses (10 and 20 mg/kg/day, respectively) rendered additive effects in combination with omega-3 fatty acid supplementation (720 mg/kg/day). Beneficial effects of combined treatment were also observed at sub-effective doses (1 mg/kg/day) of fluoxetine or mirtazapine, since in combination with omega-3 fatty acids (720 mg/kg/day), antidepressants potentiated omega-3 antidepressant-like effects. The antidepressant-like effects occurred in the absence of changes in brain phospholipid classes. The therapeutic approach of combining omega-3 fatty acids with low ineffective doses of antidepressants might represent benefits in the treatment of depression, especially in patients with depression resistant to conventional treatments and even may contribute to patient compliance by decreasing the magnitude of some antidepressant dose-dependent side effects.
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Antidepresivos/farmacología , Ácidos Grasos Omega-3/farmacología , Fluoxetina/farmacología , Mianserina/análogos & derivados , Animales , Conducta Animal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Masculino , Mianserina/farmacología , Mirtazapina , Ratas , Ratas Wistar , Natación , Factores de TiempoRESUMEN
The p75 neurotrophin receptor (p75(NTR)) is involved in neuronal functions ranging from induction of apoptosis and growth inhibition to the promotion of survival. p75(NTR) expression is induced in the central nervous system (CNS) by a range of pathological conditions, where it seems to have a role in neuronal death and axonal growth inhibition. The cellular mechanisms driving p75(NTR) expression in cell lines and primary neurons is Sp1 dependent (Ramos et al. [2007] J. Neurosci. 27:1498). In this study, we analyzed the spatiotemporal profile of p75(NTR) expression after an ischemic lesion induced by cortical devascularization (CD). Our results show that p75(NTR) expression occurs in isolated neurons of the ischemic lesion site. The p75(NTR+) neurons presented morphological alterations and active caspase-3 staining. Some p75(NTR+) neurons were also positive for sortilin. The peak of p75(NTR) expression was localized 3 days postlesion (3DPL) in the penumbra. Sp1 transcription factor nuclear localization was observed in p75(NTR+) neurons. The overall level of Sp1 expression was increased until 14DPL on the ipsilateral hemisphere. With primary cortical neurons, we demonstrated that p75(NTR) expression is induced by excitotoxic stress and correlated with increased Sp1 abundance. We conclude that p75(NTR) expression is localized in selected neurons of the ischemic lesion and that these neurons are probably condemned to apoptotic cell death. In primary neuronal culture, it is clear that excitotoxicity and Sp1 are involved in induction of p75(NTR) expression, although, in vivo, some additional mechanisms are likely to be involved in the control of p75(NTR) expression in specific neurons in vivo.
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Isquemia Encefálica/metabolismo , Corteza Cerebral/metabolismo , Trastornos Cerebrovasculares/metabolismo , Neuronas/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Apoptosis/fisiología , Infarto Encefálico/metabolismo , Infarto Encefálico/patología , Infarto Encefálico/fisiopatología , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Caspasa 3/metabolismo , Células Cultivadas , Arterias Cerebrales/patología , Arterias Cerebrales/fisiopatología , Arterias Cerebrales/cirugía , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/fisiopatología , Trastornos Cerebrovasculares/patología , Trastornos Cerebrovasculares/fisiopatología , Modelos Animales de Enfermedad , Masculino , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso , Neuronas/patología , Neurotoxinas/metabolismo , Ratas , Ratas Wistar , Receptores de Factores de Crecimiento , Factor de Transcripción Sp1/metabolismo , Estrés Fisiológico/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Dysfunction of hippocampal plasticity has been proposed to play a critical role in the pathophysiology of depression. However, antidepressant drug effects on synaptic plasticity and cytoskeletal remodeling remain controversial. The aim of the present study was to evaluate in animals exposed to the learned helplessness (LH) paradigm, an accepted experimental model of depression, the effect of chronic treatment with fluoxetine (FLX) on synaptic and cytoskeletal proteins known to undergo plastic changes. Synaptophysin (SYN), postsynaptic density 95 (PSD-95), axon growth-associated protein 43 (GAP-43), and cytoskeletal proteins (intermediate neurofilaments and MAP-2) were studied in the hippocampus by immunohistochemistry. Whereas LH animals treated 21 days with saline (LH-S group) displayed diminished SYN and PSD-95 immunostainings in the CA3 but not in the DG, chronic treatment with FLX not only reversed the despaired behavior induced by exposure to LH paradigm, but also fully recovered SYN and PSD-95 labeling to control values. Similar results were obtained for the axonal remodeling marker GAP-43. FLX treatment did not modify either the decreased light neurofilament subunit (NFL) observed in the hippocampus of LH animals or any other cytoskeletal protein studied. When FLX treatment was withdrawn for 90 days in those LH-FLX animals in which reversion of despair had been observed at day 25, recurrence of despaired behavior was found accompanied by decreased SYN, PSD-95, and NFL labelings. Results indicate that the synapse remodeling induced by FLX in the CA3 region could underlie its behavioral efficacy despite the absence of cytoskeletal remodeling and that the stability of synaptic changes would depend on the continuous administration of the drug.
Asunto(s)
Antidepresivos de Segunda Generación/uso terapéutico , Conducta Animal/efectos de los fármacos , Trastorno Depresivo/tratamiento farmacológico , Trastorno Depresivo/psicología , Fluoxetina/uso terapéutico , Desamparo Adquirido , Sinapsis/metabolismo , Animales , Antidepresivos de Segunda Generación/administración & dosificación , Axones/fisiología , Biomarcadores , Citoesqueleto/efectos de los fármacos , Citoesqueleto/patología , Interpretación Estadística de Datos , Trastorno Depresivo/metabolismo , Fluoxetina/administración & dosificación , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Masculino , Plasticidad Neuronal/efectos de los fármacos , Ratas , Ratas Wistar , Recurrencia , Sinapsis/efectos de los fármacos , Fijación del TejidoRESUMEN
BACKGROUND: Atrophy of pyramidal hippocampal neurons and of the entire hippocampus has been reported in experimental models of depression and in depressive patients respectively. We investigated the efficacy of valproic acid (VPA) for reversing a depressive-like behaviour and a cytoskeletal alteration in the hippocampus, the loss of the light neurofilament subunit (NF-L). METHODS: Depressive-like behaviour was induced by inescapable stress. Animals were divided into four groups: two to assess the response to 21 days of treatment with 200 mg/kg (I.P.) of valproic acid, and two in which the treatment was interrupted and the effects of VPA were evaluated 90 days later. Depressive-like behaviour was evaluated by the quantification of escape movements in a swimming test. NF-L was quantified by immunohistochemistry in dentate gyrus and CA3 of hippocampus. RESULTS: VPA corrected the depressive-like behaviour and reversed the diminution of NF-L in the hippocampus. Ninety days after the end of the treatment, and in contrast to the results previously obtained with fluoxetine, no recurrence of the depressive-like behaviour was observed. CONCLUSIONS: Despite interruption of the treatment, a long-lasting effect of VPA was observed. A possible relationship between the effect on NF-L and the prevention of depressive-like behaviour recurrence could be suggested.
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Antidepresivos , Citoesqueleto/efectos de los fármacos , Depresión/tratamiento farmacológico , Hipocampo/citología , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Ácido Valproico/farmacología , Animales , Atrofia , Conducta Animal/efectos de los fármacos , Depresión/psicología , Inmunohistoquímica , Masculino , Proteínas de Neurofilamentos/metabolismo , Ratas , Ratas Wistar , Estrés Psicológico/complicaciones , Estrés Psicológico/psicología , Natación/psicología , Fijación del TejidoRESUMEN
Hypercortisolism is a common trait of Cushing's disease and depression. These two disorders also share hippocampal volume decrease and cognitive deficits. However, experimentally induced hypercortisolism induces neuronal atrophy, which has been proposed to be the phenomenon underlying the hippocampal shrinkage. We hypothesized that the above-mentioned atrophy is due to a deleterious effect of high concentrations of glucocorticoids on cytoskeletal proteins. One or two pellets (100 mg each) of corticosterone were subcutaneously implanted in adult rats. Twenty-one days later, light, medium and heavy subunits of intermediate neurofilaments (NFL, NFM and NFH) and the microtubule-associated protein 2 (MAP2) were quantified by immunohistochemistry in Ammon's horn and dentate gyrus. We also evaluated the in vitro glutamate release in hippocampal slices. Both doses of corticosterone induced a decrement of NFL, NFM and NFH in both hippocampal areas but only 200 mg decreased MAP2. This dose also diminished the potassium-stimulated glutamate release. All of these changes seemed not to be due to neuron loss, as no decrement in neuron-specific nuclear protein-positive cells was found. With the exception of NFL, the above-mentioned diminution was not observed in the globus pallidus, one of the brain regions with the lowest glucocorticoid receptor density. These results provide a subcellular insight into the trophic changes found in experimental models of hypercortisolism. The coincidence between decrements in MAP2 and glutamate release suggests possible links between high glucocorticoid levels, dendritic atrophy and the cognitive impairment reported in patients suffering from Cushing's disease and depression.
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Antiinflamatorios/administración & dosificación , Corticosterona/administración & dosificación , Proteínas del Citoesqueleto/metabolismo , Regulación de la Expresión Génica/genética , Hipocampo/efectos de los fármacos , Animales , Antiinflamatorios/sangre , Recuento de Células/métodos , Corticosterona/sangre , Relación Dosis-Respuesta a Droga , Ácido Glutámico/metabolismo , Hipocampo/anatomía & histología , Inmunohistoquímica/métodos , Técnicas In Vitro , Masculino , Fosfopiruvato Hidratasa/metabolismo , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
The proconvulsive effect of the new generation of antidepressants remains controversial. The authors investigated in naïve rats the effect of chronic treatment with fluoxetine (FLX) on the convulsive threshold and on two parameters of the hippocampal glutamatergic neurotransmission: the in vitro glutamate release and the binding of [3H] MK801 to NMDA receptors. While the acute treatment with FLX provoked no change either in seizure susceptibility or in the glutamate release, the chronic treatment decreased the convulsive threshold in coincidence with an increment in the in vitro glutamate release. No significant effects on the binding of [3H] MK801 to NMDA receptors were found to be attributable to the FLX treatment. We also assessed the effect of the chronic treatment with FLX on the seizure threshold in rats exposed to an experimental model of depression, the learned helplessness paradigm (LH). While a decrease in the K+-stimulated glutamate release was observed in non treated LH animals, when they were chronically injected with FLX, no changes in the epileptic susceptibility and no increments in the glutamate release were found. Our results indicate that chronic treatment with FLX decreases the epileptic threshold in naïve but not in LH rats and that this effect correlates with the levels of the hippocampal glutamate release.
Asunto(s)
Antidepresivos de Segunda Generación/farmacología , Fluoxetina/farmacología , Ácido Glutámico/metabolismo , Desamparo Adquirido , Hipocampo/metabolismo , Convulsiones/fisiopatología , Animales , Conducta Animal/efectos de los fármacos , Convulsivantes , Maleato de Dizocilpina/metabolismo , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Masculino , Pentilenotetrazol , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
An endogenous Na(+), K(+)-ATPase inhibitor, termed endobain E, has been isolated from rat brain and proved to decrease [3H]dizocilpine binding to cerebral cortex N-methyl-D-aspartate (NMDA) receptor, an effect independent of sodium pump activity. The purpose of this study was to disclose the mechanism of [3H]dizocilpine binding reduction by endobain E by performing saturation, kinetic and competitive assays. In saturation binding assays, endobain E increased K(d) without modifying B(max) value. To determine whether competitive or allosteric interaction was involved, kinetics of [3H]dizocilpine binding to cerebral cortex membranes was studied. Endobain E increased [3H]dizocilpine dissociation rate constant and induced an initial fast phase, without modifying association rate constant, indicating an allosteric interaction. In competitive [3H]dizocilpine binding assays, no additive effect was observed with endobain E plus competitive antagonists for glutamate or glycine sites (2-amino-5-phosphonopentanoic acid (AP-5) and 7-chlorokynurenic acid, respectively), indicating that coagonist site blockade interferes with endobain E effect. However, the higher glutamate and glycine concentration, the greater its effect. Endobain E binding reduction was partially additive with that induced by ketamine or Mg(2+) (receptor-associated channel blockers). Results suggest that the greater the channel activation by glutamate and glycine, the greater endobain E allosteric effect. Furthermore, as ketamine and Mg(2+) interfere with endobain E effect, this factor most likely binds to the inner surface of the NMDA associated channel.