RESUMEN
Hippocampal pyramidal neurons are important for encoding and retrieval of spatial maps and episodic memories. While previous work has shown that Zbtb20 is a cell fate determinant for CA1 pyramidal neurons, the regulatory mechanisms governing this process are not known. In this study, we demonstrate that Zbtb20 binds to genes that control neuronal subtype specification in the developing isocortex, including Cux1, Cux2, Fezf2, Foxp2, Mef2c, Rorb, Satb2, Sox5, Tbr1, Tle4, and Zfpm2. We show that Zbtb20 represses these genes during ectopic CA1 pyramidal neuron development in transgenic mice. These data reveal a novel regulatory mechanism by which Zbtb20 suppresses the acquisition of an isocortical fate during archicortical neurogenesis to ensure commitment to a CA1 pyramidal neuron fate. We further show that the expression pattern of Zbtb20 is evolutionary conserved in the fetal human hippocampus, where it is complementary to the expression pattern of the Zbtb20 target gene Tbr1. Therefore, the disclosed Zbtb20-mediated transcriptional repressor mechanism may be involved in development of the human archicortex.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Red Nerviosa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células Piramidales/fisiología , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Inmunoprecipitación de Cromatina , Feto , Perfilación de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción/genéticaRESUMEN
Expression of the transcriptional repressor Zbtb20 is confined to the hippocampal primordium of the developing dorsal midline cortex in mice. Here, we show that misexpression of Zbtb20 converts projection neurons of the subiculum and postsubiculum (dorsal presubiculum) to CA1 pyramidal neurons that are innervated by Schaffer collateral projections in ectopic strata oriens and radiatum. The Zbtb20-transformed neurons express Bcl11B, Satb2, and Calbindin-D28k, which are markers of adult CA1 pyramidal neurons. Downregulation of Zbtb20 expression by RNA interference impairs the normal maturation of CA1 pyramidal neurons resulting in deficiencies in Calbindin-D28k expression and in reduced apical dendritic arborizations in stratum lacunosum moleculare. Overall, the results show that Zbtb20 is required for various aspects of CA1 pyramidal neuron development such as the postnatal extension of apical dendritic arbors in the distal target zone and the subtype differentiation of Calbindin-D28k-positive subsets. They further suggest that Zbtb20 plays a role in arealization of the midline cortex.
Asunto(s)
Región CA1 Hipocampal/crecimiento & desarrollo , Corteza Cerebral/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Neurogénesis/genética , Células Piramidales/metabolismo , Factores de Transcripción/fisiología , Animales , Biomarcadores/metabolismo , Región CA1 Hipocampal/patología , Calbindina 1 , Calbindinas , Diferenciación Celular/genética , Forma de la Célula/genética , Corteza Cerebral/patología , Dendritas/metabolismo , Dendritas/patología , Regulación hacia Abajo/genética , Hipertrofia , Ratones , Células Piramidales/patología , Proteína G de Unión al Calcio S100/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Dedos de Zinc/genéticaRESUMEN
There is great interest in testing neuroprotectants which inhibit the neurodegeneration that results from excessive activation of N-methyl-D-aspartate (NMDA) receptors. As an alternative to in vivo testing in animal models, we demonstrate here the use of a complex in vitro model to compare the efficacy and toxicity of NMDA receptor inhibitors. Organotypic hippocampal slice cultures were used to compare the effectiveness of the Alzheimer's disease drug, memantine, the Parkinson's disease drug, procyclidine, and the novel neuroprotectant, gacyclidine (GK11), against NMDA-induced toxicity. All three drugs are non-competitive NMDA receptor open-channel blockers that inhibit excitotoxic injury, and their neuroprotective capacities have been extensively investigated in vivo in animal models. They have also been evaluated as potential countermeasure agents against organophosphate poisoning. Quantitative densitometric image analysis of propidium iodide uptake in hippocampal regions CA1, CA3 and DG, showed that, after exposure to 10microM NMDA for 24 hours, GK11 was the most potent of the three drugs, with an IC50 of about 50nM and complete protection at 250nM. When applied at high doses, GK11 was still the more potent neuroprotectant, and also the least cytotoxic. These findings are consistent with those from in vivo tests in rodents. We conclude that the slice culture model provides valuable pre-clinical data, and that applying the model to the screening of neuroprotectants might significantly limit the use of in vivo tests in animals.
Asunto(s)
Ciclohexenos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Piperidinas/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Ensayos Clínicos como Asunto , Antagonistas de Aminoácidos Excitadores/metabolismo , Técnicas In Vitro , Propidio/metabolismo , Ratas , Ratas WistarRESUMEN
PNQX (9-methyl-amino-6-nitro-hexahydro-benzo(F)quinoxalinedione) is a selective AMPA antagonist with demonstrated neuroprotective effects in focal ischemia in rats. Here we report corresponding effects in mouse hippocampal slice cultures subjected to oxygen and glucose deprivation (OGD) and in transient global cerebral ischemia in gerbils. For in vitro studies, hippocampal slice cultures derived from 7-day-old mice and grown for 14 days, were submersed in oxygen-glucose deprived medium for 30 min and exposed to PNQX for 24 h, starting together with OGD, immediately after OGD, or 2 h after OGD. For comparison, other cultures were exposed to the NMDA antagonist MK-801 using the same protocol. Both PNQX and MK-801 displayed significant neuroprotective effects in all hippocampal subfields when present during and after OGD. When added just after OGD, only PNQX retained some neuroprotective effect. When added 2 h after OGD neither PNQX nor MK-801 had an effect. Transient global cerebral ischemia was induced in Mongolian gerbils by occlusion of both common carotid arteries for 4.5 min, with PNQX (10 mg/kg) being injected i.p. 30, 60 and 90 min after the insult. Subsequent analysis of brain sections stained for the neurodegeneration marker Fluoro-Jade B and immunostained for the astroglial marker glial fibrillary acidic protein revealed a significant PNQX-induced decrease in neuronal cell death and astroglial activation. We conclude that, PNQX provided neuroprotection against both global cerebral ischemia in gerbils in vivo and oxygen-glucose deprivation in mouse hippocampal slice cultures.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/farmacología , Glucosa/deficiencia , Hipocampo/patología , Hipoxia Encefálica/tratamiento farmacológico , Ataque Isquémico Transitorio/tratamiento farmacológico , Ataque Isquémico Transitorio/patología , Fármacos Neuroprotectores/farmacología , Quinoxalinas/farmacología , Receptores AMPA/antagonistas & inhibidores , Animales , Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/fisiología , Muerte Celular/efectos de los fármacos , Colorantes , Maleato de Dizocilpina/farmacología , Fluoresceínas , Gerbillinae , Hipoxia Encefálica/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/biosíntesis , Degeneración Nerviosa/patología , Técnicas de Cultivo de Órganos , Compuestos Orgánicos , Cloruro de TolonioRESUMEN
Most in vitro models are only used to assess short-term effects of test compounds. However, as demonstrated here, hippocampal slice cultures can be used for long-term studies. The test compound used was the metabotropic glutamate receptor antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3), which is known to be toxic in vivo after subchronic, but not acute, administration. Degenerative effects were monitored by measuring the cellular uptake of propidium iodide (PI; continuously present in the medium) and lactate dehydrogenase (LDH) leakage, and by using a panel of histological stains. Hippocampal slices, derived from 2-3 day old rats and grown for 3 weeks, were subsequently exposed for the next 3 weeks to 0, 10 or 100microM L-AP3, with PI (2microM) in the culture medium. Exposure to 100microM L-AP3 induced severe toxicity after 4-6 days, shown by massive PI uptake, LDH leakage, changes in MAP2 and GFAP immunostaining, and in Nissl and Timm staining. In contrast, 10microM L-AP3 did not induce detectable neuronal degeneration. Treatment with the NMDA receptor antagonist, MK-801, or the AMPA/KA receptor antagonist NBQX, together with 100microM L-AP3, reduced neurodegeneration down to close to control values. It is concluded that continuous incubation of hippocampal slice cultures with PI is technically feasible for use in studies of inducible neuronal degeneration over time.
Asunto(s)
Alanina/análogos & derivados , Colorantes , Antagonistas de Aminoácidos Excitadores/toxicidad , Hipocampo/efectos de los fármacos , Propidio , Técnicas de Cultivo de Tejidos , Alanina/toxicidad , Alternativas a las Pruebas en Animales , Animales , Maleato de Dizocilpina , Hipocampo/enzimología , L-Lactato Deshidrogenasa/metabolismo , Quinoxalinas , Ratas , Coloración y EtiquetadoRESUMEN
Transgenic mice, expressing fluorescent proteins in neurons and glia, provide new opportunities for real-time microscopic monitoring of degenerative and regenerative structural changes. We have previously validated and compared a number of quantifiable markers for neuronal damage and cell death in organotypic brain slice cultures, such as cellular uptake of propidium iodide (PI), loss of microtubule-associated protein 2 (MAP2), Fluoro-Jade (FJ) cell staining, and the release of cytosolic lactate dehydrogenase (LDH). An important supplement to these markers would be data on corresponding morphological changes, as well as the opportunity to monitor reversible changes or long-term effects in the event of minor damage. As a first step, we present: a) the developmental expression in organotypic hippocampal brain slice cultures of transgenic fluorescent proteins, useful for the visualisation of neuronal subpopulations and astroglial cells; and b) examples of excitotoxic, glutamate receptor-induced degeneration of hippocampal CA1 pyramidal cells, with corresponding astroglial reactivity in such cultures. The slice cultures were set up according to standard techniques, by using one-week old pups from four transgenic mouse strains which express fluorescent proteins in their neurons and/or astroglial cells. From the time of explantation, and subsequently for up to nine weeks in culture, the transgenic neuronal fluorescence displayed the expected characteristics of a developmental, in vivo-like increase, including both the number and localisation of cells, as well as the intensity of fluorescence. At that stage and later, the transgenic fluorescence clearly permitted the visualisation of cell bodies, larger and smaller dendritic branches, spines and axons. In separate experiments, with a 24-hour exposure of matured sliced cultures to 100 microM of the glutamate agonist, N-methyl-D-aspartate (NMDA), we observed, by time-lapse recording, a gradual, but rapid loss of fluorescent CA1 pyramidal cells, accompanied by astrogliosis of transgene fluorescent astroglial cells. Based on these results, we consider that organotypic brain slice cultures from transgenic mice, with fluorescent neurons and glia, combined with detailed visualisation by time-lapse fluorescence microscopy, have great potential for investigating both major irreversible and minor reversible structural changes in neurons and glia, induced by neurotoxins and other neurodegenerative compounds and conditions.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/metabolismo , N-Metilaspartato/farmacología , Degeneración Nerviosa/metabolismo , Técnicas de Cultivo de Tejidos/métodos , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Biomarcadores/metabolismo , Células Cultivadas , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Factores de TiempoRESUMEN
Slices of developing brain tissue can be grown for several weeks as so-called organotypic slice cultures. Here we summarize and review studies using hippocampal slice cultures to investigate mechanisms and treatment strategies for the neurodegenerative disorders like stroke (cerebral ischemia), Alzheimer's disease (AD) and epilepsia. Studies of non-excitotoxic neurotoxic compounds and the experimental use of slice cultures in studies of HIV neurotoxicity, traumatic brain injury (TBI) and neurogenesis are included. For cerebral ischemia, experimental models with oxygen-glucose deprivation (OGD) and exposure to glutamate receptor agonists (excitotoxins) are reviewed. For epilepsia, focus is on induction of seizures with effects on neuronal loss, axonal sprouting and neurogenesis. For Alzheimer's disease, the review centers on the use of beta-amyloid (Abeta) in different models, while the section on repair is focused on neurogenesis and cell migration. The culturing techniques, set-up of models, and analytical tools, including markers for neurodegeneration, like the fluorescent dye propidium iodide (PI), are reviewed and discussed. Comparisons are made between hippocampal slice cultures and other in vitro models using dispersed cell cultures, experimental in vivo models, and in some instances, clinical trials. New techniques including slice culturing of hippocampal tissue from transgenic mice as well as more mature brain tissue, and slice cultures coupled to microelectrode arrays (MEAs), on-line biosensor monitoring, and time-lapse fluorescence microscopy are also presented.
Asunto(s)
Daño Encefálico Crónico/patología , Hipocampo/patología , Degeneración Nerviosa/patología , Enfermedades Neurodegenerativas/patología , Técnicas de Cultivo de Órganos/métodos , Enfermedad de Alzheimer/patología , Animales , Calcio/metabolismo , Modelos Animales de Enfermedad , Epilepsia/patología , Ratones , Ratas , Transducción de Señal/fisiología , Accidente Cerebrovascular/patologíaRESUMEN
In vitro electrophysiological techniques for the assessment of neurotoxicity could have several advantages over other methods in current use, including the ability to detect damage at a very early stage, and could further assist in replacing animal experimentation in vivo. We investigated how an electrophysiological parameter, the extracellularly-recorded compound action potential ("population spike", PS) could be used as a marker of in vitro neurotoxicity in the case of two well-known toxic compounds, kainic acid (KA) and trimethyltin (TMT). We compared the use of this electrophysiological endpoint with changes in immunoreactivity for microtubule-associated protein 2 (MAP2), a standard histological test for neurotoxicity. We found that both toxic compounds reliably caused disappearance of the PS, and that such disappearance occurred after only 1 hour of exposure to the drug. By contrast, densitometric measurements of MAP2 immunoreactivity were unaffected by both KA and TMT after such a short exposure time. We conclude that, in the case of KA and TMT, the extracellular PS was abolished at a very early time-point, when MAP2 immunoreactivity levels were still comparable to those of the untreated controls. Electrophysiology could be a reliable and early indicator of neurotoxicity, which could improve our ability to test for neurotoxicity in vitro, thus further replacing the need for in vivo experimentation.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Hipocampo/efectos de los fármacos , Ácido Kaínico/toxicidad , Proteínas Asociadas a Microtúbulos/metabolismo , Compuestos de Trimetilestaño/toxicidad , Potenciales de Acción/fisiología , Animales , Relación Dosis-Respuesta a Droga , Agonistas de Aminoácidos Excitadores/toxicidad , Femenino , Hipocampo/fisiología , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Coloración y Etiquetado , Factores de TiempoRESUMEN
The potential neuroprotective effects of the GABA(A) receptor agonists THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and muscimol, and the selective GluR5 kainate receptor agonist ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid), which activates GABAergic interneurons, were examined in hippocampal slice cultures exposed to N-methyl-D-aspartate (NMDA). The NMDA-induced excitotoxicity was quantified by densitometric measurements of propidium iodide (PI) uptake. THIP (100-1000 microM) was neuroprotective in slice cultures co-exposed to NMDA (10 microM) for 48 h, while muscimol (100-1000 microM) and ATPA (1-3 microM) were without effect. The results demonstrate that direct GABA(A) agonism can mediate neuroprotection in the hippocampus in vitro as previously suggested in vivo.
Asunto(s)
Hipocampo/efectos de los fármacos , Isoxazoles/farmacología , Muscimol/farmacología , Fármacos Neuroprotectores/farmacología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Relación Dosis-Respuesta a Droga , Glutamato Descarboxilasa/metabolismo , Hipocampo/lesiones , Hipocampo/metabolismo , Ácido Kaínico/efectos adversos , N-Metilaspartato/efectos adversos , Técnicas de Cultivo de Órganos , Propidio/metabolismo , Células Piramidales/efectos de los fármacos , Células Piramidales/lesiones , Células Piramidales/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The microtubule-disrupting agent colchicine is known to be particular toxic for certain types of neurons, including the granule cells of the dentate gyrus. In this study we investigated whether colchicine could induce such neuron-specific degeneration in developing (1 week in vitro) and mature (3 weeks in vitro) organotypic hippocampal slice cultures and whether the induced cell death was apoptotic and/or necrotic. When applied to 1-week-old cultures for 48 h, colchicine induced primarily apoptotic, but also a minor degree of necrotic cell death in the dentate granule cells, as investigated by cellular uptake of the fluorescent dye propidium iodide (PI), immunostaining for active caspase 3 and c-Jun/AP-1 (N) and fragmentation of nuclei as seen in Hoechst 33342 staining. All four markers appeared after 12 h of colchicine exposure. Two of them, active caspase 3 and c-Jun/AP-1 (N) displayed a similar time course and reached a maximum after 24 h of exposure, 24 h ahead of both PI uptake and Hoechst 33342 staining, which together displayed similar time profiles and a close correlation. In 3-week-old cultures, colchicine did not induce apoptotic or necrotic cell death. Attempts to interfere with the colchicine-induced apoptosis in 1-week-old cultures showed that colchicine-induced PI uptake and formation of apoptotic nuclei were temporarily prevented by coapplication of the protein synthesis inhibitor cycloheximide. Application of the pancaspase inhibitor z-VAD-fmk almost completely abolished the formation of active caspase 3 protein and apoptotic nuclei induced by colchicine, but the formation of necrotic nuclei increased correspondingly and the PI uptake was unaffected. We conclude that colchicine induces caspase 3-dependent apoptotic cell death of dentate granule cells in hippocampal brain slice cultures, but the apoptotic cell death is highly dependent on the developmental stage of the cultures.
Asunto(s)
Apoptosis/efectos de los fármacos , Colchicina/toxicidad , Giro Dentado/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotoxinas/toxicidad , Clorometilcetonas de Aminoácidos/farmacología , Animales , Bencimidazoles , Caspasa 3 , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Cicloheximida/farmacología , Giro Dentado/metabolismo , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes , Inmunohistoquímica , Necrosis , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Propidio/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Organotypic hippocampal slice cultures are used increasingly in experimental models of neurodegeneration, together with various histological, biochemical and electrophysiological markers of cell death. While functional electrophysiological changes typically occur early, histological changes appear later, with loss of dendritic immunoreactivity for microtubule-associated protein 2 (MAP2) among the earliest. In this study we compared the temporal changes of four different histological markers for neurodegeneration after oxygen-glucose deprivation (OGD) of rat hippocampal slice cultures. Within an observation period of 24 h after OGD, shrinkage of Hoechst 33342 stained neuronal nuclei both occurred before, and was completed faster, than loss of MAP2 staining, which again started earlier and progressed faster towards complete loss than the increase in cellular uptake of propidium iodide and Fluoro-Jade B staining of degenerating neurons. We conclude that shrinkage of Hoechst 33342 stained neuronal nuclei detected by image analysis is an early and easily quantifiable indicator of neuronal degeneration in hippocampal slice cultures.
Asunto(s)
Muerte Celular/fisiología , Núcleo Celular/patología , Hipocampo/patología , Degeneración Nerviosa/patología , Células Piramidales/patología , Animales , Bencimidazoles , Hipoxia de la Célula/fisiología , Colorantes , Colorantes Fluorescentes , Glucosa/farmacología , Proteínas Asociadas a Microtúbulos/análisis , Técnicas de Cultivo de Órganos , Oxígeno/farmacología , Propidio , Células Piramidales/química , Ratas , Ratas WistarRESUMEN
This paper reviews the current state of the use of organotypic brain slice cultures for neurotoxicological and neuropharmacological screening and mechanistic studies, as exemplified by excitotoxin application. At present, no in vitro systems have been approved by the regulatory authorities for neurotoxicity testing. For the evaluation of the slice culture method, organotypic hippocampal slice cultures were exposed to toxic doses of the excitotoxins, glutamate, N-methyl-D-aspartate (NMDA), kainic acid and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and the glial toxin, DL-alpha-aminoadipic acid (DLAAA). Neuronal cell death was quantified by propidium iodide (PI) uptake, and visualised by Fluoro-Jade (FJ) staining. General cell death was monitored by lactate dehydrogenase (LDH) release into the culture medium. EC50 values for the different compounds, based on PI uptake after exposure for 48 hours in entire cultures, were: glutamate, 3.5 mM; DL-AAA, 2.3 mM; kainic acid, 13 microM; NMDA, 11 microM; and AMPA, 3.7 microM. In the slice cultures, the hippocampal subfields displayed the same differences in vulnerability as those observed in vivo. When subfield analysis was performed on the cultures, the CA1 subfield was most susceptible to glutamate, NMDA and AMPA, while CA3 was most susceptible to kainic acid. The amount of LDH release for DL-AAA was about four times that of L-glutamate, in accordance with the additional toxic effect on glial cells, which was also found by confocal microscopy to stain for FJ. In conclusion, it was found that organotypic brain slice culture, combined with standardised protocols and quantifiable markers, such as PI and FJ staining, is a relevant and feasible in vitro system for neurotoxicity testing. Considering the amount and quality of the available published data, it is recommended that the brain slice culture method could be subjected to pre-validation and formal validation for inclusion in a tiered in vitro neurotoxicity testing scheme to supplement and replace conventional animal tests.
Asunto(s)
Encéfalo/efectos de los fármacos , Neurotoxinas/toxicidad , Técnicas de Cultivo de Órganos/métodos , Pruebas de Toxicidad/métodos , Alternativas a las Pruebas en Animales , Animales , Muerte Celular/efectos de los fármacos , Ácido Glutámico/farmacología , Hipocampo/efectos de los fármacos , Ácido Kaínico/farmacología , Microscopía Fluorescente , Degeneración Nerviosa , Neuronas/efectos de los fármacos , Propidio/metabolismo , Ratas , Ratas WistarRESUMEN
Despite extensive research, the current theories on serotonergic dysfunctions and cortisol hypersecretion do not provide sufficient explanations for the nature of depression. Rational treatments aimed at causal factors of depression are not available yet. With the currently available antidepressant drugs, which mainly target serotonin, less than two thirds of depressed patients achieve remission. There is now evidence that inflammatory and neurodegenerative (I&ND) processes play an important role in depression and that enhanced neurodegeneration in depression may-at least partly-be caused by inflammatory processes. Multiple inflammatory-cytokines, oxygen radical damage, tryptophan catabolites-and neurodegenerative biomarkers have been established in patients with depression and these findings are corroborated by animal models of depression. A number of vulnerability factors may predispose towards depression by enhancing inflammatory reactions, e.g. lower peptidase activities (dipeptidyl-peptidase IV, DPP IV), lower omega-3 polyunsaturated levels and an increased gut permeability (leaky gut). The cytokine hypothesis considers that external, e.g. psychosocial stressors, and internal stressors, e.g. organic inflammatory disorders or conditions, such as the postpartum period, may trigger depression via inflammatory processes. Most if not all antidepressants have specific anti-inflammatory effects, while restoration of decreased neurogenesis, which may be induced by inflammatory processes, may be related to the therapeutic efficacy of antidepressant treatments. Future research to disentangle the complex etiology of depression calls for a powerful paradigm shift, i.e. by means of a high throughput-high quality screening, including functional genetics and genotyping microarrays; established and novel animal and ex vivo-in vitro models for depression, such as new transgenic mouse models and endophenotype-based animal models, specific cell lines, in vivo and ex vivo electroporation, and organotypic brain slice culture models. This screening will allow to: 1) discover new I&ND biomarkers, both at the level of gene expression and the phenotype; and elucidate the underlying molecular I&ND pathways causing depression; and 2) identify new therapeutic targets in the I&ND pathways; develop new anti-I&ND drugs for these targets; select existing anti-I&ND drugs or substances that could augment the efficacy of antidepressants; and predict therapeutic response by genetic I&ND profiles.
Asunto(s)
Trastorno Depresivo/etiología , Trastorno Depresivo/fisiopatología , Encefalitis/complicaciones , Encefalitis/fisiopatología , Degeneración Nerviosa/etiología , Degeneración Nerviosa/fisiopatología , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Causalidad , Citocinas/metabolismo , Trastorno Depresivo/tratamiento farmacológico , Modelos Animales de Enfermedad , Encefalitis/metabolismo , Humanos , Degeneración Nerviosa/metabolismo , Estrés Oxidativo/fisiología , Estrés Psicológico/complicaciones , Estrés Psicológico/metabolismo , Estrés Psicológico/fisiopatología , Triptófano/metabolismoRESUMEN
Hippocampus-associated genes that orchestrate the formation of the compact stratum pyramidale are largely unknown. The BTB (broad complex, tramtrack, bric-a-brac)-zinc finger gene Zbtb20 (also known as HOF, Znf288, Zfp288) encodes two protein isoforms, designated Zbtb20(S) and Zbtb20(L), which are expressed in newborn pyramidal neurons of the presumptive hippocampus in mice. Here, we have generated transgenic mice with ectopic expression of Zbtb20(S) and Zbtb20(L) in immature pyramidal neurons differentiated from multipotent non-hippocampal precursors. The subiculum and posterior retrosplenial areas in these mice were transformed into a three-layered hippocampus-like cortex with a compact homogenous pyramidal cell layer. Severe malformations of lamination occur in neocortical areas, which coincide with a deficiency in expression of cortical lamination markers. The alterations in cortical cytoarchitecture result in behavioral abnormalities suggestive of a deficient processing of visual and spatial memory cues in the cerebral cortex of adult Zbtb20 transgenic mice. Overall, our in vivo data suggest that Zbtb20 functions as a molecular switch for a pathway that induces invariant pyramidal neuron morphogenesis and suppression of cell fate transitions in newborn neurons.
Asunto(s)
Corteza Cerebral/crecimiento & desarrollo , Células Piramidales/crecimiento & desarrollo , Factores de Transcripción/fisiología , Animales , Movimiento Celular/genética , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Ratones , Ratones Transgénicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Células Piramidales/citología , Células Piramidales/metabolismo , Factores de Transcripción/genética , Dedos de Zinc/genéticaRESUMEN
In addition to its well-known hematopoietic effects, erythropoietin (EPO) also has neuroprotective properties. However, hematopoietic side effects are unwanted for neuroprotection, underlining the need for EPO-like compounds with selective neuroprotective actions. One such compound, devoid of hematopoietic bioactivity, is the chemically modified, EPO-derivative carbamylerythropoietin (CEPO). For comparison of the neuroprotective effects of CEPO and EPO, we subjected organotypic hippocampal slice cultures to oxygen-glucose deprivation (OGD) or N-methyl-d-aspartate (NMDA) excitotoxicity. Hippocampal slice cultures were pretreated for 24 h with 100 IU/ml EPO (=26 nM) or 26 nM CEPO before OGD or NMDA lesioning. Exposure to EPO and CEPO continued during OGD and for the next 24 h until histology, as well as during the 24 h exposure to NMDA. Neuronal cell death was quantified by cellular uptake of propidium iodide (PI), recorded before the start of OGD and NMDA exposure and 24 h after. In cultures exposed to OGD or NMDA, CEPO reduced PI uptake by 49+/-3 or 35+/-8%, respectively, compared to lesion-only controls. EPO reduced PI uptake by 33+/-5 and 15+/-8%, respectively, in the OGD and NMDA exposed cultures. To elucidate a possible mechanism involved in EPO and CEPO neuroprotection against OGD, the integrity of alpha-II-spectrin cytoskeletal protein was studied. Both EPO and CEPO significantly reduced formation of spectrin cleavage products in the OGD model. We conclude that CEPO is at least as efficient neuroprotectant as EPO when excitotoxicity is modeled in mouse hippocampal slice cultures.
Asunto(s)
Eritropoyetina/análogos & derivados , Eritropoyetina/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Glucosa/deficiencia , Hipoxia Encefálica/metabolismo , N-Metilaspartato/farmacología , Fármacos Neuroprotectores/farmacología , Animales , Animales Recién Nacidos , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Calpaína/antagonistas & inhibidores , Colorantes , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Hipoxia Encefálica/patología , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Propidio/farmacocinética , Cloruro de TolonioRESUMEN
We investigated whether neurotoxic effects of the mitochondrial toxin 3-nitropropionic acid (3-NP) in hippocampal slice cultures are dependent on glucose levels in the culture medium and whether such effects occur via apoptosis or necrosis. In addition, 3-NP toxicity was investigated at two developmental stages of the cultures, prepared from rat brain at postnatal day 5-7 and grown in Neurobasal medium for 1 or 3 weeks. Cultures were exposed to 3-NP in the presence of high (25 mM), normal (5 mM), or low (3 mM) glucose for 48 h, followed by 48 h incubation in medium without 3-NP. Cellular propidium iodide (PI) uptake and lactate dehydrogenase (LDH) efflux into the medium revealed time- and dose-dependent cell death by 3-NP, with EC(50) values of about 60 microM in high or normal glucose. Regional vulnerability, as assessed by PI uptake and MAP2 immunostaining, in 3-week-old cultures was as follows: CA1 > CA3 > fascia dentata. In low glucose much lower concentrations of 3-NP (25 microM) triggered neurotoxicity. One-week-old cultures were less susceptible to 3-NP toxicity than 3-week-old cultures, but the dentate granule cells were relatively more affected in the immature cultures. We found no evidence for apoptotic cell death by 3-NP in 3-week-old cultures, but in 1-week-old cultures the putative apoptotic marker c-JUN/AP1 and nuclear fragmentation (Hoechst) were significantly increased in the dentate granule cells.
Asunto(s)
Glucosa/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Propionatos/toxicidad , Animales , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glucosa/farmacología , Hipocampo/citología , Técnicas In Vitro , L-Lactato Deshidrogenasa/metabolismo , Nitrocompuestos , Propidio/farmacocinética , Ratas , Ratas Wistar , Factores de Tiempo , Factor de Transcripción AP-1/metabolismoRESUMEN
Quantitative glucose and lactate metabolism was assessed in continuously perfused organotypic hippocampal slices under control conditions and during exposure to glutamate and drugs that interfere with aerobic and anaerobic metabolism. On-line detection was possible with a system based on slow perfusion rates, a half-open (medium/air interface) tissue chamber and a flow injection analytic system equipped with biosensors for glucose and lactate. Under basal conditions about 50% of consumed glucose was converted to lactate in hippocampal slice cultures. Using medium containing lactate (5 mm) instead of glucose (5 mm) significant lactate uptake was observed, but this uptake was less than the net uptake of lactate equivalents in glucose-containing medium. Glucose deprivation experiments suggested lactate efflux from glycogen stores. The effects of drugs compromising or stimulating energy metabolism, i.e. 2-deoxyglucose, 3-nitropropionic acid, alpha-cyano-4-hydroxycinnamate, l-glutamate, d-asparate, ouabain and monensin, were tested in this flow system. The data show that maintaining Na+ and K+ gradients consumed much of the energy but do not support the hypothesis that l-glutamate stimulates glycolysis in hippocampal slice cultures.