by Keyword: Synapse

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Bosch, M., Castro, J., Sur, M., Hayashi, Y., (2017). Photomarking relocalization technique for correlated two-photon and electron microcopy imaging of single stimulated synapses Synapse Development - Methods and Protocols (Methods in Molecular Biology) (ed. Poulopoulos , A.), Humana Press (New York, USA) 1538, 185-214

Synapses learn and remember by persistent modifications of their internal structures and composition but, due to their small size, it is difficult to observe these changes at the ultrastructural level in real time. Two-photon fluorescence microscopy (2PM) allows time-course live imaging of individual synapses but lacks ultrastructural resolution. Electron microscopy (EM) allows the ultrastructural imaging of subcellular components but cannot detect fluorescence and lacks temporal resolution. Here, we describe a combination of procedures designed to achieve the correlated imaging of the same individual synapse under both 2PM and EM. This technique permits the selective stimulation and live imaging of a single dendritic spine and the subsequent localization of the same spine in EM ultrathin serial sections. Landmarks created through a photomarking method based on the 2-photon-induced precipitation of an electrodense compound are used to unequivocally localize the stimulated synapse. This technique was developed to image, for the first time, the ultrastructure of the postsynaptic density in which long-term potentiation was selectively induced just seconds or minutes before, but it can be applied for the study of any biological process that requires the precise relocalization of micron-wide structures for their correlated imaging with 2PM and EM.

Keywords: Correlated imaging, DAB, Dendritic spine, Photobranding, Photoetching, Photomarking, Postsynaptic density, Serial-section transmission electron microscopy, Synapse, Time-lapse live two-photon fluorescence microscopy

Llorens, F., Del Rio, J. A., (2012). Unraveling the neuroprotective mechanisms of PrPC in excitotoxicity Prion 6, (3), 245-251

Knowledge of the natural roles of cellular prion protein (PrPC) is essential to an understanding of the molecular basis of prion pathologies. This GPIanchored protein has been described in synaptic contacts, and loss of its synaptic function in complex systems may contribute to the synaptic loss and neuronal degeneration observed in prionopathy. In addition, Prnp knockout mice show enhanced susceptibility to several excitotoxic insults, GABAA receptor-mediated fast inhibition was weakened, LTP was modified and cellular stress increased. Although little is known about how PrPC exerts its function at the synapse or the downstream events leading to PrPCmediated neuroprotection against excitotoxic insults, PrPC has recently been reported to interact with two glutamate receptor subunits (NR2D and GluR6/7). In both cases the presence of PrPC blocks the neurotoxicity induced by NMDA and Kainate respectively. Furthermore, signals for seizure and neuronal cell death in response to Kainate in Prnp knockout mouse are associated with JNK3 activity, through enhancing the interaction of GluR6 with PSD-95. In combination with previous data, these results shed light on the molecular mechanisms behind the role of PrPC in excitotoxicity. Future experimental approaches are suggested and discussed.

Keywords: Prion protein, Excitotoxicity, Neuroprotection, Glutamate receptors, Synapse, prionopathy

Llorens, Franc, Gil, Vanessa, Antonio del Rio, Jose, (2011). Emerging functions of myelin-associated proteins during development, neuronal plasticity, and neurodegeneration FASEB Journal 25, (2), 463-475

Adult mammalian central nervous system (CNS) axons have a limited regrowth capacity following injury. Myelin-associated inhibitors (MAIs) limit axonal outgrowth, and their blockage improves the regeneration of damaged fiber tracts. Three of these proteins, Nogo-A, MAG, and OMgp, share two common neuronal receptors: NgR1, together with its coreceptors [p75(NTR), TROY, and Lingo-1]; and the recently described paired immunoglobulin-like receptor B (PirB). These proteins impair neuronal regeneration by limiting axonal sprouting. Some of the elements involved in the myelin inhibitory pathways may still be unknown, but the discovery that blocking both PirB and NgR1 activities leads to near-complete release from myelin inhibition, sheds light on one of the most competitive and intense fields of neuroregeneration study in recent decades. In parallel with the identification and characterization of the roles and functions of these inhibitory molecules in axonal regeneration, data gathered in the field strongly suggest that most of these proteins have roles other than axonal growth inhibition. The discovery of a new group of interacting partners for myelin-associated receptors and ligands, as well as functional studies within or outside the CNS environment, highlights the potential new physiological roles for these proteins in processes, such as development, neuronal homeostasis, plasticity, and neurodegeneration.-Llorens, F., Gil, V., del Rio, J. A. Emerging functions of myelin-associated proteins during development, neuronal plasticity, and neurodegeneration.

Keywords: MAIs, Neural stem cells, Synapse formation

Madronal, Noelia, Lopez-Aracil, Cristina, Rangel, Alejandra, del Rio, Jose A., Delgado-Garcia, Jose M., Gruart, Agnes, (2010). Effects of Enriched Physical and Social Environments on Motor Performance, Associative Learning, and Hippocampal Neurogenesis in Mice PLoS ONE 5, (6), e11130

We have studied the motor abilities and associative learning capabilities of adult mice placed in different enriched environments. Three-month-old animals were maintained for a month alone (AL), alone in a physically enriched environment (PHY), and, finally, in groups in the absence (SO) or presence (SOPHY) of an enriched environment. The animals' capabilities were subsequently checked in the rotarod test, and for classical and instrumental learning. The PHY and SOPHY groups presented better performances in the rotarod test and in the acquisition of the instrumental learning task. In contrast, no significant differences between groups were observed for classical eyeblink conditioning. The four groups presented similar increases in the strength of field EPSPs (fEPSPs) evoked at the hippocampal CA3-CA1 synapse across classical conditioning sessions, with no significant differences between groups. These trained animals were pulse-injected with bromodeoxyuridine (BrdU) to determine hippocampal neurogenesis. No significant differences were found in the number of NeuN/BrdU double-labeled neurons. We repeated the same BrdU study in one-month-old mice raised for an additional month in the above-mentioned four different environments. These animals were not submitted to rotarod or conditioned tests. Non-trained PHY and SOPHY groups presented more neurogenesis than the other two groups. Thus, neurogenesis seems to be related to physical enrichment at early ages, but not to learning acquisition in adult mice.

Keywords: Long-term potentiation, Adult neurogenesis, Synaptic transmission, Cell proliferation, CA3-CA1 synapse, Granule cells