RESUMEN
Neurotransmitter spillover is a form of communication not readily predicted by anatomic structure. In the cerebellum, glutamate spillover from climbing fibers recruits molecular layer interneurons in the absence of conventional synaptic connections. Spillover-mediated signaling is typically limited by transporters that bind and reuptake glutamate. Here, we show that patterned expression of the excitatory amino acid transporter 4 (EAAT4) in Purkinje cells regulates glutamate spillover to molecular layer interneurons. Using male and female Aldolase C-Venus knock-in mice to visualize zebrin microzones, we find larger climbing fiber-evoked spillover EPSCs in regions with low levels of EAAT4 compared with regions with high EAAT4. This difference is not explained by presynaptic glutamate release properties or postsynaptic receptor density but rather by differences in the glutamate concentration reaching receptors on interneurons. Inhibiting glutamate transport normalizes the differences between microzones, suggesting that heterogeneity in EAAT4 expression is a primary determinant of differential spillover. These results show that neuronal glutamate transporters limit extrasynaptic transmission in a non-cell-autonomous manner and provide new insight into the functional specialization of cerebellar microzones.SIGNIFICANCE STATEMENT Excitatory amino acid transporters (EAATs) help maintain the fidelity and independence of point-to-point synaptic transmission. Whereas glial transporters are critical to maintain low ambient levels of extracellular glutamate to prevent excitotoxicity, neuronal transporters have more subtle roles in shaping excitatory synaptic transmission. Here we show that the patterned expression of neuronal EAAT4 in cerebellar microzones controls glutamate spillover from cerebellar climbing fibers to nearby interneurons. These results contribute to fundamental understanding of neuronal transporter functions and specialization of cerebellar microzones.
Asunto(s)
Cerebelo/metabolismo , Transportador 4 de Aminoácidos Excitadores/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Ácido Glutámico/metabolismo , Interneuronas/metabolismo , Transmisión Sináptica/fisiología , Animales , Transportador 4 de Aminoácidos Excitadores/genética , Ratones , Células de Purkinje/metabolismo , Sinapsis/metabolismoRESUMEN
The ability to process complex spatiotemporal information is a fundamental process underlying the behavior of all higher organisms. However, how the brain processes information in the temporal domain remains incompletely understood. We have explored the spatiotemporal information-processing capability of networks formed from dissociated rat E18 cortical neurons growing in culture. By combining optogenetics with microelectrode array recording, we show that these randomly organized cortical microcircuits are able to process complex spatiotemporal information, allowing the identification of a large number of temporal sequences and classification of musical styles. These experiments uncovered spatiotemporal memory processes lasting several seconds. Neural network simulations indicated that both short-term synaptic plasticity and recurrent connections are required for the emergence of this capability. Interestingly, NMDA receptor function is not a requisite for these short-term spatiotemporal memory processes. Indeed, blocking the NMDA receptor with the antagonist APV significantly improved the temporal processing ability of the networks, by reducing spontaneously occurring network bursts. These highly synchronized events have disastrous effects on spatiotemporal information processing, by transiently erasing short-term memory. These results show that the ability to process and integrate complex spatiotemporal information is an intrinsic property of generic cortical networks that does not require specifically designed circuits.
Asunto(s)
Corteza Cerebral/citología , Corteza Cerebral/fisiología , Red Nerviosa/fisiología , Neuronas/fisiología , Percepción Espacial/fisiología , Percepción del Tiempo/fisiología , Estimulación Acústica , Células Cultivadas , Humanos , Microelectrodos , Música/psicología , Red Nerviosa/citología , Optogenética , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
The number of neurotransmitter-filled vesicles released into the synaptic cleft with each action potential dictates the reliability of synaptic transmission. Variability of this fundamental property provides diversity of synaptic function across brain regions, but the source of this variability is unclear. The prevailing view is that release of a single (univesicular release, UVR) or multiple vesicles (multivesicular release, MVR) reflects variability in vesicle release probability, a notion that is well-supported by the calcium-dependence of release mode. However, using mouse brain slices, we now demonstrate that the number of vesicles released is regulated by the size of the readily-releasable pool, upstream of vesicle release probability. Our results point to a model wherein protein kinase A and its vesicle-associated target, synapsin, dynamically control release site occupancy to dictate the number of vesicles released without altering release probability. Together these findings define molecular mechanisms that control MVR and functional diversity of synaptic signaling.
Asunto(s)
Transmisión Sináptica , Vesículas Sinápticas/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ratones , Modelos Biológicos , Sinapsinas/metabolismoRESUMEN
Neuronal inclusions composed of α-synuclein (α-syn) characterize Parkinson's Disease (PD) and Dementia with Lewy bodies (DLB). Cognitive dysfunction defines DLB, and up to 80% of PD patients develop dementia. α-Syn inclusions are abundant in the hippocampus, yet functional consequences are unclear. To determine if pathologic α-syn causes neuronal defects, we induced endogenous α-syn to form inclusions resembling those found in diseased brains by treating hippocampal neurons with α-syn fibrils. At seven days after adding fibrils, α-syn inclusions are abundant in axons, but there is no cell death at this time point, allowing us to assess for potential alterations in neuronal function that are not caused by neuron death. We found that exposure of neurons to fibrils caused a significant reduction in mushroom spine densities, adding to the growing body of literature showing that altered spine morphology is a major pathologic phenotype in synucleinopathies. The reduction in spine densities occurred only in wild type neurons and not in neurons from α-syn knockout mice, suggesting that the changes in spine morphology result from fibril-induced corruption of endogenously expressed α-syn. Paradoxically, reduced postsynaptic spine density was accompanied by increased frequency of miniature excitatory postsynaptic currents (EPSCs) and presynaptic docked vesicles, suggesting enhanced presynaptic function. Action-potential dependent activity was unchanged, suggesting compensatory mechanisms responding to synaptic defects. Although activity at the level of the synapse was unchanged, neurons exposed to α-syn fibrils, showed reduced frequency and amplitudes of spontaneous Ca2+ transients. These findings open areas of research to determine the mechanisms that alter neuronal function in brain regions critical for cognition at time points before neuron death.
Asunto(s)
Hipocampo/citología , Neuronas/efectos de los fármacos , alfa-Sinucleína/toxicidad , Animales , Calcio/metabolismo , Células Cultivadas , Endotoxinas/toxicidad , Antagonistas del GABA/farmacología , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiología , Técnicas de Placa-Clamp , Fosfopiruvato Hidratasa/metabolismo , Picrotoxina/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Potenciales Sinápticos/efectos de los fármacos , Tetrodotoxina/farmacología , Transducción Genética , alfa-Sinucleína/metabolismoRESUMEN
Bacopa monnieri is a plant used as a nootropic in Ayurveda, a 5000-year-old system of traditional Indian medicine. Although both animal and clinical studies supported its role as a memory enhancer, the molecular and cellular mechanism underlying Bacopa's nootropic action are not understood. In this study, we used deep sequencing (RNA-Seq) to identify the transcriptome changes upon Bacopa treatment on SH-SY5Y human neuroblastoma cells. We identified several genes whose expression levels were regulated by Bacopa. Biostatistical analysis of the RNA-Seq data identified biological pathways and molecular functions that were regulated by Bacopa, including regulation of mRNA translation and transmembrane transport, responses to oxidative stress and protein misfolding. Pathway analysis using the Ingenuity platform suggested that Bacopa may protect against brain damage and improve brain development. These newly identified molecular and cellular determinants may contribute to the nootropic action of Bacopa and open up a new direction of investigation into its mechanism of action.