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International audience; The functional preservation of the central nervous system (CNS) is based on the neuronal plasticity and survival. In this context, the neuroinflammatory state plays a key role and involves the microglial cells, the CNS-resident macrophages. In order to better understand the microglial contribution to the neuroprotection, microglia-derived extracellular vesicles (EVs) were isolated and molecularly characterized to be then studied in neurite outgrowth assays. The EVs, mainly composed of exosomes and microparticles, are an important cell-to-cell communication process as they exhibit different types of mediators (proteins, lipids, nucleic acids) to recipient cells. The medicinal leech CNS was initially used as an interesting model of microglia/neuron crosstalk due to their easy collection for primary cultures. After the microglia-derived EV isolation following successive methods, we developed their large-scale and non-targeted proteomic analysis to (i) detect as many EV protein markers as possible, (ii) better understand the biologically active proteins in EVs and (iii) evaluate the resulting protein signatures in EV-activated neurons. The EV functional properties were also evaluated in neurite outgrowth assays on rat primary neurons and the RNAseq analysis of the microglia-derived EVs was performed to propose the most representative miRNAs in microglia-derived EVs. This strategy allowed validating the EV isolation, identify major biological pathways in EVs and corroborate the regenerative process in EV-activated neurons. In parallel, six different miRNAs were originally identified in microglia-derived EVs including 3 which were only known in plants until now. The analysis of the neuronal proteins under the microglial EV activation suggested possible miRNA-dependent regulation mechanisms. Taken together, this combination of methodologies showed the leech microglial EVs as neuroprotective cargos across species and contributed to propose original EV-associated miRNAs whose functions will have to be evaluated in the EV-dependent dialog between microglia and neurons.

El rol neuromodulador de la serotonina ha sido conservado a lo largo de laevolución y se ha observado en invertebrados y en vertebrados. Los invertebradoshan sido utilizados como modelos de estudio de la modulación por serotonina, yaque la simpleza de sus sistemas nerviosos, permite correlacionar alteracionescomportamentales con modificaciones en los circuitos neuronales subyacentes adichos comportamientos. La posibilidad de identificar inequívocamente a lasneuronas serotoninérgicas, permite correlacionar su actividad con los efectosatribuidos a la serotonina. La serotonina es un importante neuromodulador en la sanguijuela: intervieneen la modulación de varios comportamientos tales como la alimentación, elacortamiento defensivo y la natación. Las neuronas serotoninérgicas Retziusconstituyen su fuente principal de liberación. La estimulación mecánica de la pieldel animal produce la activación de las células mecanosensoriales P. Dichaactivación es suficiente para evocar diversas respuestas motoras y a su vez, enparalelo, activar a las neuronas Retzius. Este trabajo de tesis tuvo como objetivo elestudio de la modulación serotoninérgica de un circuito sensoro-motor en el sistemanervioso central de la sanguijuela Hirudo medicinalis, basándose en la hipótesis deque la salida motora final estará determinada por la interacción de la via sensoromotoray la vía sensoro-moduladora. En la primera parte del trabajo se caracterizó la vía sensoro-motora entre lasneuronas mecanosensoriales sensibles a presión (P) y las motoneuronas annuluserectus (AE), que inervan los músculos responsables de las erección de los ánulos enque se divide la pared corporal del animal. Losresultados muestran que: - La motoneurona AE recibe dos señales simultáneas ante la activación de laneurona mecanosensorial P: una excitatoria y otra inhibitoria. La fase excitatoriaestá mediada por una sinápsis directa y la inhibitoria por una interacciónpolisináptica. - La interneurona pasiva 151 ejerce un rol modulador sobre la sinápsis P-AE,interactuando con la motoneurona AE y con la(s) interneurona(s) que media(n) lafase inhibitoria de la conexión P-AE. En la segunda parte del trabajo se analizó el efecto de la serotonina exógenay de antagonistas serotoninérgicos sobre las propiedades intrínsecas de las neuronas P y AEy sobre la interacción P-AE. - La aplicación de serotonina exógena produjo la hiperpolarización de las neuronas AE, por activación de una conductancia a cloro sensible a los antagonistasmetiotepina y mianserina. - La serotonina exógena causó una disminución de la amplitud de la faseinhibitoria de la interacción P-AE,y los antagonistas metiotepina y mianserinabloquearon este efecto. - La serotonina endógena ejercería una inhibición sobre la interacción P-AE. The role of serotonin as neuromodulator has been conserved along evolution,being observed in vertebrates as well as in invertebrates. Invertebrates have beenextensively used as models to study serotonin modulation because their simplenervous systems allow correlating behavioral changes with changes in the pattern ofactivity of the neural circuits that underlie specific behaviors. The preciseidentification of serotonin-containing neurons allows correlating their activity withthe effects attributed to this amine. Serotonin is an important neuromodulator in the leech: it is responsible forthe expression of feeding behavior and modulates shortening and swimming. Retziusneurons contain approximately half of the total ganglionic serotonin. Mechanicalstimulation of the skin activates mechanosensory cells sensitive to pressure, P cells. This activation is capable to evoke several motor responses and in parallel toactivate the serotonin-containing Retzius cells. The goal of this thesis work has beento evaluate the serotonin modulation of a sensory-motor circuit in the centralnervous system of the leech Hirudo medicinalis, based on the hypothesis that thefinal motor output results of the interaction between a sensory-modulatory and asensory-motor pathways. The aim of the first part of this work was to characterize the interactionbetween a pair of specific sensory and motor neurons: the P mechanosensoryneurons and the AE(annulus erector) cells, a pair of motor neurons that innervatethe muscles responsible for erecting the annuli into sharp ridges. The results showthat - The response of the AE neurons was composed of excitatory postsynapticpotentials (EPSPs) and of inhibitory postsynaptic potentials (IPSPs). The EPSPswere transmitted via a monosynaptic pathway, and the IPSPs via a polysynapticone. - The non-spiking neuron 151 is able to modulate the P-AE interaction, by meansof rectifying electrical synapses with AE motoneuron and with the interneuronallayer that mediates the inhibitory response. In the second part of this work we studied the effects of serotonin andserotonin receptor antagonists on the P-AE interaction and on the intrinsicproperties of the neurons involved. - Exogenously applied serotonin produced the hyperpolarization of AEmotorneurons, due to the activation of a chloride conductance that was sensitiveto methiothepine and mianserin. - Exogenously applied serotonin specifically diminished the amplitude of theinhibitory response to P cell stimulation, and this effect was also sensitive tomethiothepine and mianserin. - Endogenous serotonin would exert an inhbitory modulation on this sensory-motorcircuit. Fil: Iscla, Irene. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.