Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular. / Los procesos de plasticidad sináptica homeostática (PSH) comprenden una serie de mecanismos compensatorios dependientes del grado de actividad de los circuitos neuronales, de manera que perturbaciones que aumenten o disminuyan crónicamente la actividad del circuito son compensadas mediante distintos mecanismos que causan una disminución, o aumento, respectivamente, de la actividad. Estos fenómenos podrían mantener la actividad del circuito dentro de un rango dinámico. Para detectar estos fenómenos de plasticidad, suele requerirse la incubación de las neuronas durante días en agentes farmacológicos que induzcan un aumento o disminución de la actividad, previo a realizar mediciones. No obstante, en nuestro laboratorio fue previamente descrito, en neuronas piramidales de hipocampo de rata, un fenómeno de PSH de rápido desarrollo (horas en lugar de días). Este fenómeno se caracteriza por un aumento en el tiempo de la frecuencia y amplitud de las corrientes excitatorias en miniatura, y se desarrolla espontáneamente durante las 8-12 horas de incubación, sin intervenciones farmacológicas. Para evaluar los mecanismos subyacentes a los cambios en la frecuencia y amplitud se deben realizar distintos experimentos de corrientes evocadas por estimulación presináptica. Sin embargo, los eventos evocados mediante protocolos de estimulación resultan de carácter polifásico e irregular (i.e. corrientes múltiples, seguidas una de otra, sin forma reproducible), lo cual no permite realizar los análisis. Otras evidencias previas indican que el componente regular y monofásico puede ser aislado al utilizar una solución de registro alta en cationes divalentes (Ca2+ y Mg2+). Dado que el uso de dicha solución podría afectar la observación del fenómeno homeostático mismo, en este trabajo se estudiaron, mediante la técnica de voltage-clamp, los efectos que podría tener el uso de esta solución sobre el aumento de la frecuencia y amplitud de los eventos en miniatura. Se determinó la existencia de un proceso homeostático en estas condiciones, observándose un aumento en el tiempo de la frecuencia de los eventos en miniatura, no pudiéndose determinar concluyentemente el aumento en la amplitud. Esta observación valida futuros estudios complementarios de corrientes evocadas en presencia de altos divalentes para investigar posibles mecanismos de expresión del fenómeno. / Homeostatic synaptic plasticity processes comprise a series of compensatory mechanisms that depend on the activity levels of the neuronal circuits, so that perturbations that upregulate or downregulate chronically the activity of the circuit are counterbalanced by different mechanisms that downregulate or upregulate the activity of the circuit, respectively. These processes may allow keeping the activity of neuronal networks in a dynamic range. To detect these plasticity phenomena, it is usually necessary to incubate neurons for a few days in presence of pharmacological agents that either upregulate or downregulate their activity prior to measurements. However, our laboratory has described a homeostatic plasticity process of rapid development (hours instead of days, which is what is usually observed). This phenomenon is observed in pyramidal neurons of rat hippocampus slices incubated for 8-12 hours and is characterized by an increase in time of the frequency and amplitude of miniature excitatory synaptic currents. To evaluate the underlying mechanisms of the changes in frequency and amplitude it is necessary to carry out recordings of evoked currents. However, and in concordance with previous preliminary data from this laboratory, the evoked events in these cells and conditions displayed polyphasic and irregular characteristics, which rendered the analysis unfeasible. However, other evidence suggests that the monophasic, more regular component of the evoked response can be isolated using a recording solution high in divalent cations (Ca2+ and Mg2+). However, the use of this solution could have secondary side effects that alter the measurement of the plasticity phenomenon. Here, the voltage-clamp technique was used to examine whether the increase in frequency and amplitude of the miniature events can be observed in these conditions. The frequency change was observed, but the detection of the amplitude change was not conclusive. These observations validate future studies of evoked currents in high divalent cations to investigate possible mechanisms of expression of the homeostatic phenomenon.

Programa de Doctorado: Biotecnología en ciencias de la salud (Biotecnología y Bíomedicina)

Doctor en Ciencias con Mención en Biología Molecular, Celular y Neurociencias. / Dans plusieurs neurones du SNC l'activité somato-dendritique infraliminaire peut se propager passivement dans l'axone et augmenter transitoirement la transmission synaptique spontanée et évoquée par le potentiel d'action de manière dépendante du Ca2+. Les mécanismes sousjacents à ce type de plasticité synaptique, appelée facilitation "analogique" ou "analogo-digitale", restent largement inconnus pour la plupart des synapses centrales, principalement en raison de la difficulté à réaliser des enregistrements directs des petits boutons présynaptiques. Ici, nous utilisons de la photolyse de Ca2+ et de l'imagerie aux niveau des terminaisons présynaptiques individuelles des interneurones de la couche moléculaire du cervelet (ICM), combinées à des enregistrements électrophysiologiques avec la technique du patch. Nous décrivons un nouveau mécanisme de facilitation analogo-digitale qui est dépendant du Ca2+ infraliminaire et dans lequel la fraction et la cinétique du pool de vésicules dites “prêtes à être libérées” (readily releasable pool ou RRP en anglais), est modulée par des modifications de la probabilité d'occupation des sites d'ancrage dans des contacts synaptiques individuels. Nos résultats ajoutent une nouvelle dimension à la compréhension de la manière dont l'activité infraliminaire module le flux d'information dans les circuits neuronaux / In several neurons of the CNS, subthreshold somatodendritic activity can spread passively into the axon and transiently enhance spontaneous and spike-evoked synaptic transmission in a Ca2+-dependent and graded manner. Available evidence about the underlying mechanism of this type of synaptic plasticity, called “analog” or “analog to digital” facilitation (ADF), remains largely incomplete for the majority of central synapses, mainly due to the experimental inaccessibility to the small presynaptic boutons. Here we use both Ca2+ photolysis and imaging at individual presynaptic terminals of the rat cerebellar molecular layer interneurons (MLIs), combined with whole-cell paired recordings from synaptically connected MLIs, to report a novel subthreshold Ca2+-dependent mechanism for ADF whereby the fraction and the kinetics of the pool of vesicles available for immediate release, the readily releasable pool (RRP), are modulated by changing the docking site occupancy probability in single synaptic contacts. Our results add a new dimension in the understanding of how subthreshold activity modulates information flow in neuronal circuits. / 31 de diciembre, 2019

No description available.

Lesioned axons do not regenerate in the adult mammalian central nervous system, owing to the overexpression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1-40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3 (GSK3beta) and ERK1/2 in axon regeneration failure in lesioned cortical neurons. We also investigated whether pharmacological blockage of GSK3beta and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: i) cerebellar granule cells and ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3beta inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Lastly these regenerative effects were corroborated in the lesioned EHP in NgR1 -/- mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections. On the other hand, and focused in the OMgp, by using recording electrophysiological nano-devices we found that, OMgp has a role in synaptic transmission, since it can induce excitatory postsynaptic potentials (EPSPs) in cultured hippocampal neurons.