RESUMEN
In a model of homeostatic plasticity, hippocampal slice culture CA3 pyramidal neurons responded to excitatory synapse inactivity by enhancing glutamate release through an increased number of miniature excitatory post-synaptic currents, mEPSCs and excitatory pre-synaptic terminals. Also accompanying these changes was a specific reduction in the expression of a "fast" calcium transporter, the plasma membrane calcium ATPase, PMCA2a. This transporter normally influences glutamate release from excitatory terminals where it helps control calcium levels. The reduction in PMCA2a expression occurred within 2 days of synapse inactivity; it was specific and reversible in young and mature hippocampal slice cultures and required removal of NMDA receptor mediated activity. Furthermore, the enhanced mEPSCs in the model were resistant to pharmacological inhibition of PMCA transporter activity. Reduced expression of PMCA2a during homeostatic plasticity therefore provides a mechanism to remodel pre-synaptic Ca2+ dynamics as a flexible way to alter glutamate release.
Asunto(s)
Plasticidad Neuronal/efectos de los fármacos , ATPasas Transportadoras de Calcio de la Membrana Plasmática/biosíntesis , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Regulación hacia Abajo , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Ácido Glutámico/metabolismo , Homeostasis , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Quinoxalinas/farmacología , Ratas , Ratas Wistar , Receptores de Glutamato/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/metabolismoRESUMEN
Neuroplastin-65 is a brain-specific, synapse-enriched member of the immunoglobulin (Ig) superfamily of cell adhesion molecules. Previous studies highlighted the importance of neuroplastin-65 for long-term potentiation (LTP), but the mechanism was unclear. Here, we show how neuroplastin-65 activation of mitogen-activated protein kinase p38 (p38MAPK) modified synapse strength by altering surface glutamate receptor expression. Organotypic hippocampal slice cultures treated with the complete extracellular fragment of neuroplastin-65 (FcIg1-3) sustained an increase in the phosphorylation of p38MAPK and an inability to induce LTP at hippocampal synapses. The LTP block was reversed by application of the p38MAPK inhibitor SB202190, suggesting that p38MAPK activation occurred downstream of neuroplastin-65 binding and upstream of the loss of LTP. Further investigation revealed that the mechanism underlying neuroplastin-65-dependent prevention of LTP was a p38MAPK-dependent acceleration of the loss of surface-exposed glutamate receptor subunits that was reversed by pretreatment with the p38MAPK inhibitor SB202190. Our results indicate that neuroplastin-65 binding and associated stimulation of p38MAPK activity are upstream of a mechanism to control surface glutamate receptor expression and thereby influence plasticity at excitatory hippocampal synapses.
Asunto(s)
Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Receptores AMPA/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Biotina , Western Blotting , Estimulación Eléctrica , Electrofisiología , Hipocampo/citología , Imidazoles/farmacología , Fragmentos Fc de Inmunoglobulinas/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Fosforilación , Piridinas/farmacología , Ratas , Receptores AMPA/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Homeostatic plasticity is a phenomenon whereby synaptic strength is scaled in the context of the activity that the network receives. Here, we have analysed excitatory and inhibitory synapses in a model of homeostatic plasticity where rat organotypic hippocampal slice cultures were deprived of excitatory synaptic input by the NMDA and AMPA/KA glutamate receptor antagonists, AP5 and CNQX. We show that chronic excitatory synapse deprivation generates an excitable CA3 network where enhanced amplitude and frequency of spontaneous excitatory post-synaptic potentials were associated with increased glutamate receptor subunit expression and increased number and size of synapsin 1 and VGLUT1 positive puncta. Intact spontaneous inhibitory post-synaptic potentials coincided with persistent expression of the GABA-A receptor alpha subunit and GAD65 and an enhancement of parvalbumin-positive puncta. In this model of homeostatic plasticity, scaling up of synaptic excitation and maintenance of fast synaptic inhibition promote an excitable, but stable, CA3 network.
Asunto(s)
Hipocampo , Homeostasis , Red Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Sinapsis/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Aminopiridinas/farmacología , Animales , Biomarcadores/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/anatomía & histología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Red Nerviosa/anatomía & histología , Técnicas de Placa-Clamp , Ratas , Receptores AMPA/metabolismo , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Técnicas de Cultivo de Tejidos , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Plasma membrane Ca2+ ATPases (PMCAs) are critical regulators of intracellular Ca2+ concentration ([Ca2+]i). Specific isoforms have also been demonstrated to interact and co-localise with members of the synapse-associated protein (SAP) family in hippocampal dendritic spines. Presently, only indirect evidence of changes in PMCA protein expression during postnatal development exists, therefore we chose to examine the postnatal developmental protein expression patterns of PMCAs 1-4 and the SAP proteins SAP102 and PSD95. Using Western blotting analysis, we compared the postnatal expression in the in vivo hippocampus to the expression within in vitro organotypic hippocampal slice cultures; a valid model of the developing hippocampus. All PMCA and SAP family members studied showed a marked increase in protein expression levels throughout the postnatal time course both in vivo and in vitro. SAP102 and the ubiquitously expressed PMCAs 1 and 4 followed a similar time course of expression within the in vivo and in vitro preparations. In contrast, the neurone-specific PMCA isoforms 2 and 3 and PSD95 displayed slight differences in early postnatal development. However, and most importantly, their expression > or = 14 days in vitro (DIV) was similar to that in vivo. The results of this study demonstrate that postnatal expression of all PMCAs, SAP102 and PSD95 is similar in both the in vivo hippocampus and the in vitro organotypic hippocampal slice culture. Our results support the use of organotypic hippocampal slice cultures for future investigations of the importance of PMCAs for neuronal Ca2+ handling and SAP family member interactions.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Espinas Dendríticas/enzimología , Hipocampo/enzimología , Hipocampo/crecimiento & desarrollo , Proteínas del Tejido Nervioso/metabolismo , Membranas Sinápticas/enzimología , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Señalización del Calcio/fisiología , Proteínas de Transporte de Catión/metabolismo , Neuropéptidos/metabolismo , Técnicas de Cultivo de Órganos , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Ratas , Ratas Wistar , Transmisión Sináptica/fisiología , Factores de Tiempo , Regulación hacia Arriba/fisiologíaRESUMEN
Organotypic hippocampal slice cultures can be used to study hippocampal biochemistry and physiology over a chronic period on the days to weeks timescale. In order to validate the organotypic hippocampal slice culture for our ongoing studies of synaptic function, we have compared, using Western blotting, the levels of a number of synaptic proteins from in vitro organotypic hippocampal slice cultures with those from in vivo hippocampal slices prepared from age-matched controls. We chose to follow the developmental expression of the neuroplastin (np) family of immunoglobulin related cell adhesion molecules (CAMs), np65, a brain specific isoform highly expressed in hippocampal neurones and np55 a more widely expressed isoform and two synaptic marker proteins, synaptophysin, a pre-synaptic marker and post-synaptic density protein-95, PSD95, a post-synaptic marker. All showed increasing expression over the developmental time period, both in vivo and in vitro. The level of both neuroplastins was also consistent between the in vivo and in vitro preparations, whereas the level of PSD95 was markedly increased in the organotypic hippocampal slice cultures while the level of synaptophysin was slightly decreased. Whilst these findings may indicate some differences in the composition and organisation of synapses, the developmental expression profiles of these synaptic proteins within organotypic hippocampal slice cultures suggests they are a valid model for the study of synapse function and development in vitro.