RESUMEN
BACKGROUND: Recent evidence suggests that exposure to intrauterine inflammation causes acute fetal brain injury and is linked to a spectrum of neurobehavioral disorders. In a rodent model of intrauterine inflammation induced by lipopolysaccharide (LPS) exposure in utero, activated microglia can be detected in the hippocampus of offspring survivors, as late as 60 days postnatal (DPN). Given that the hippocampus is important for learning and memory, these results suggest that in utero inflammation underlies long-term cognitive deficits observed in children/survivors. METHODS: An established mouse model of LPS-induced intrauterine inflammation was used to study hippocampal function from offspring at 44-59 DPN. Microgliosis was examined at 45 DPN. Extracellular field recordings of synaptic transmission were performed on acute hippocampal slices. RESULTS: LPS offspring mice displayed persistent microglial activation and increased CA3-CA1 excitatory synaptic strength, which can be explained in part by an increase in the probability of glutamate release, and reduced long-term synaptic potentiation compared to control mice. CONCLUSIONS: These results offer a mechanistic explanation for the cognitive and behavioral deficits observed in survivors of preterm birth caused by intrauterine inflammation.
Asunto(s)
Modelos Animales de Enfermedad , Hipocampo/fisiología , Efectos Tardíos de la Exposición Prenatal/metabolismo , Transmisión Sináptica/fisiología , Útero/metabolismo , Animales , Femenino , Hipocampo/efectos de los fármacos , Inflamación/inducido químicamente , Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Masculino , Ratones , Técnicas de Cultivo de Órganos , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Sobrevivientes , Transmisión Sináptica/efectos de los fármacos , Útero/efectos de los fármacos , Útero/patologíaRESUMEN
Cerebellar Purkinje cells (PCs) are particularly sensitive to cerebral ischemia, and decreased GABA(A) receptor function following injury is thought to contribute to PC sensitivity to ischemia-induced excitotoxicity. Here we examined the functional properties of the GABA(A) receptors that are spared following ischemia in cultured Purkinje cells from rat and in vivo ischemia in mouse. Using subunit-specific positive modulators of GABA(A) receptors, we observed that oxygen and glucose deprivation (OGD) and cardiac arrest-induced cerebral ischemia cause a decrease in sensitivity to the ß(2/3) -subunit-preferring compound, etomidate. However, sensitivity to propofol, a ß-subunit-acting compound that modulates ß(1-3) -subunits, was not affected by OGD. The α/γ-subunit-acting compounds, diazepam and zolpidem, were also unaffected by OGD. We performed single-cell reverse transcription-polymerase chain reaction on isolated PCs from acutely dissociated cerebellar tissue and observed that PCs expressed the ß(1) -subunit, contrary to previous reports examining GABA(A) receptor subunit expression in PCs. GABA(A) receptor ß(1) -subunit protein was also detected in cultured PCs by western blot and by immunohistochemistry in the adult mouse cerebellum and levels remained unaffected by ischemia. High concentrations of loreclezole (30 µm) inhibited PC GABA-mediated currents, as previously demonstrated with ß(1) -subunit-containing GABA(A) receptors expressed in heterologous systems. From our data we conclude that PCs express the ß(1) -subunit and that there is a greater contribution of ß(1) -subunit-containing GABA(A) receptors following OGD.
Asunto(s)
Isquemia Encefálica/metabolismo , Oxígeno/metabolismo , Células de Purkinje/metabolismo , Receptores de GABA-A/metabolismo , Animales , Western Blotting , Modelos Animales de Enfermedad , Glucosa/deficiencia , Inmunohistoquímica , Masculino , Ratones , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-DawleyRESUMEN
Cerebellar Purkinje cells (PC) are particularly vulnerable to ischemic injury and excitotoxicity, although the molecular basis of this sensitivity remains unclear. We tested the hypothesis that ischemia causes rapid down-regulation of GABA(A) receptors in cerebellar PC, thereby increasing susceptibility to excitotoxicity. Oxygen-glucose deprivation (OGD) caused a decline in functional GABA(A) receptors, within the first hour of re-oxygenation. Decreased amplitude of miniature inhibitory post-synaptic potentials confirmed that OGD caused a significant decrease in functional synaptic GABA(A) receptors and quantitative Western blot analysis demonstrated the loss of GABA(A) receptor current was associated with a decline in total receptor protein. Interestingly, the potent neuroprotectant allopregnanolone (ALLO) prevented the decline in GABA(A) receptor current and protein. Consistent with our in vitro data, global ischemia in mice caused a significant decline in total cerebellar GABA(A) receptor protein and PC specific immunoreactivity. Moreover, ALLO provided strong protection of PC and prevented ischemia-induced decline in GABA(A) receptor protein. Our findings indicate that ischemia causes a rapid and sustained loss of GABA(A) receptors in PC, whereas ALLO prevents the decline in GABA(A) receptors and protects against ischemia-induced damage. Thus, interventions which prevent ischemia-induced decline in GABA(A) receptors may represent a novel neuroprotective strategy.
Asunto(s)
Isquemia Encefálica/metabolismo , Fármacos Neuroprotectores/farmacología , Pregnanolona/farmacología , Células de Purkinje/metabolismo , Receptores de GABA-A/metabolismo , Animales , Western Blotting , Inmunohistoquímica , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Ratones , Técnicas de Placa-Clamp , Células de Purkinje/efectos de los fármacos , Células de Purkinje/patología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca(2+) influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.
Asunto(s)
Región CA1 Hipocampal/fisiología , Guanilato-Quinasas/metabolismo , Proteínas de la Membrana/metabolismo , Células Piramidales/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Animales , Línea Celular , Guanilato-Quinasas/aislamiento & purificación , Humanos , Inmunoprecipitación , Proteínas de la Membrana/aislamiento & purificación , Ratones , Unión Proteica , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/aislamiento & purificaciónRESUMEN
SK2- and KV4.2-containing K+ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca2+ source that provides Ca2+-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca2+ entry through NMDARs, whereas KV4.2-containing channel availability is increased by Ca2+ entry through SNX-482 (SNX) sensitive CaV2.3 R-type Ca2+ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca2+ entry through R-type Ca2+ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca2+ channels and KV4.2-containing K+ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca2+ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from CaV2.3 null mice. The results show that in the absence of CaV2.3 channels, apamin application still boosted EPSPs. The boosting effect of CaV2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from CaV2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and KV4.2-containing channels to CaV2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.
Asunto(s)
Apamina/farmacología , Canales de Calcio Tipo R/fisiología , Proteínas de Transporte de Catión/fisiología , Células Piramidales/efectos de los fármacos , Potenciales Sinápticos/efectos de los fármacos , Animales , Región CA1 Hipocampal/citología , Calcio/metabolismo , Canales de Calcio Tipo R/deficiencia , Canales de Calcio Tipo R/genética , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/genética , Potenciales Evocados/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Potenciales Postsinápticos Excitadores/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Placa-Clamp , Células Piramidales/metabolismo , Células Piramidales/fisiología , Venenos de Araña/farmacología , Potenciales Sinápticos/genética , Potenciales Sinápticos/fisiologíaRESUMEN
Allopregnanolone (ALLO) is a neurosteroid that has many functions in the brain, most notably neuroprotection and modulation of gamma-amino butyric acid (GABA) neurotransmission. Using a mouse model of cardiac arrest and cardiopulmonary resuscitation, we have previously demonstrated that ALLO protects cerebellar Purkinje cells (PCs) from ischemia in a GABA(A) receptor-dependent manner. In this study we examined the effect of sex on ALLO neuroprotection, observing that low dose ALLO (2 mg/kg) provided greater neuroprotection in females compared to males. At a higher dose of ALLO (8 mg/kg), both sexes were significantly protected from ischemic damage. Using an acute cerebellar slice preparation, whole cell voltage clamp recordings were made from PCs. Spontaneous inhibitory post synaptic currents (IPSCs) were analyzed and the response to physiological ALLO (10 nM) was significantly greater in female PCs compared to male. In contrast, recordings of miniature IPSCs, did not exhibit a sex difference in response to ALLO, suggesting that ALLO affects males and females differentially through a mechanism other than binding postsynaptic GABA(A) receptors. We conclude that the female brain has greater sensitivity to ALLO mediated potentiation of GABAergic neurotransmission, contributing to increased neuroprotection.