HCN1 channels in cerebellar Purkinje cell promote late stages of learning and constrain synaptic inhibition

Abstract Neural computations rely on ion channels that modify neuronal responses to synaptic inputs. While single cell recordings suggest diverse and neurone type-specific computational functions for HCN1 channels, their behavioural roles in any single neurone type are not clear. Using a battery of behavioural assays, including analysis of motor learning in vestibulo-ocular reflex and rotarod tests, we find that deletion of HCN1 channels from cerebellar Purkinje cells selectively impairs late stages of motor learning. Because deletion of HCN1 modifies only a subset of behaviours involving Purkinje cells, we asked whether the channel also has functional specificity at a cellular level. We find that HCN1 channels in cerebellar Purkinje cells reduce the duration of inhibitory synaptic responses but, in the absence of membrane hyperpolarization, do not affect responses to excitatory inputs. Our results indicate that manipulation of subthreshold computation in a single neurone type causes specific modifications to behaviour. © 2013 The Physiological Society

Studying the neurogenic potential of local progenitors in the adult olfactory bulb and their biocompatibility with graphene

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 03-07-201

IGF-I: A Key Growth Factor that Regulates Neurogenesis and Synaptogenesis from Embryonic to Adult Stages of the Brain

The generation of neurons in the adult mammalian brain requires the activation of quiescent neural stem cells (NSCs). This activation and the sequential steps of neuron formation from NSCs are regulated by a number of stimuli, which include growth factors. Insulin-like growth factor-I (IGF-I) exert pleiotropic effects, regulating multiple cellular processes depending on their concentration, cell type, and the developmental stage of the animal. Although IGF-I expression is relatively high in the embryonic brain its levels drop sharply in the adult brain except in neurogenic regions, i.e., the hippocampus (HP) and the subventricular zone-olfactory bulb (SVZ-OB). By contrast, the expression of IGF-IR remains relatively high in the brain irrespective of the age of the animal. Evidence indicates that IGF-I influences NSC proliferation and differentiation into neurons and glia as well as neuronal maturation including synapse formation. Furthermore, recent studies have shown that IGF-I not only promote adult neurogenesis by regulating NSC number and differentiation but also by influencing neuronal positioning and migration as described during SVZ-OB neurogenesis. In this article we will revise and discuss the actions reported for IGF-I signaling in a variety of in vitro and in vivo models, focusing on the maintenance and proliferation of NSCs/progenitors, neurogenesis, and neuron integration in synaptic circuits.The work in Carlos Vicario laboratory was funded by grants from the Spanish Ministerio de Ciencia e Innovación and Ministerio de Economía y Competitividad (MICINN and MINECO; BFU2007-61230, BFU2010-1963 and SAF2013-4759R, the Instituto de Salud Carlos III (ISCIII; CIBERNED CB06/05/0065), and the Comunidad de Madrid (S2011/BMD-2336) to CV. VN and ÇD were supported by a FPI Fellowship from the MICINN and MINECO and grants from CIBERNED and the European Union in the 7th framework program.Peer reviewedPeer Reviewe

Distinct effects of BDNF and NT-3 on the dendrites and presynaptic boutons of developing olfactory bulb GABAergic interneurons in vitro

Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) are known to regulate neuronal morphology and the formation of neural circuits, yet the neuronal targets of each neurotrophin are still to be defined. To address how these neurotrophins regulate the morphological and synaptic differentiation of developing olfactory bulb (OB) GABAergic interneurons, we analyzed the effect of BDNF and NT-3 on GABA+-neurons and on different subtypes of these neurons: tyrosine hydroxylase (TH+); calretinin (Calr+); calbindin (Calb+); and parvalbumin (PVA+). These cells were generated from cultured embryonic mouse olfactory bulb neural stem cells (eOBNSCs) and after 14 days in vitro (DIV), when the neurons expressed TrkB and/or TrkC receptors, BDNF and NT-3 did not significantly change the number of neurons. However, long-term BDNF treatment did produce a longer total dendrite length and/or more dendritic branches in all the interneuron populations studied, except for PVA+-neurons. Similarly, BDNF caused an increase in the cell body perimeter in all the interneuron populations analyzed, except for PVA+-neurons. GABA+- and TH+-neurons were also studied at 21 DIV, when BDNF produced significantly longer neurites with no clear change in their number. Notably, these neurons developed synaptophysin+ boutons at 21 DIV, the size of which augmented significantly following exposure to either BDNF or NT-3. Our results show that in conditions that maintain neuronal survival, BDNF but not NT-3 promotes the morphological differentiation of developing OB interneurons in a cell-type-specific manner. In addition, our findings suggest that BDNF and NT-3 may promote synapse maturation by enhancing the size of synaptic boutons.This study was funded by grants from MINECO (Grant Numbers: SAF2013-47596-R, SAF2016-80419-R and CIBERNED CB06/05/0065), the Comunidad de Madrid (Grant Number S2011/ BMD-2336) and Fundación Ramón Areces (Grant Number CIVP18A3941) to C.V

In Vitro Evaluation of Biocompatibility of Uncoated Thermally Reduced Graphene and Carbon Nanotube-Loaded PVDF Membranes with Adult Neural Stem Cell-Derived Neurons and Glia

Graphene, graphene-based nanomaterials (GBNs), and carbon nanotubes (CNTs) are being investigated as potential substrates for the growth of neural cells. However, in most in vitro studies, the cells were seeded on these materials coated with various proteins implying that the observed effects on the cells could not solely be attributed to the GBN and CNT properties. Here, we studied the biocompatibility of uncoated thermally reduced graphene (TRG) and poly(vinylidene fluoride) (PVDF) membranes loaded with multi-walled CNTs (MWCNTs) using neural stem cells isolated from the adult mouse olfactory bulb (termed aOBSCs). When aOBSCs were induced to differentiate on coverslips treated with TRG or control materials (polyethyleneimine-PEI and polyornithine plus fibronectin-PLO/F) in a serum-free medium, neurons, astrocytes, and oligodendrocytes were generated in all conditions, indicating that TRG permits the multi-lineage differentiation of aOBSCs. However, the total number of cells was reduced on both PEI and TRG. In a serum-containing medium, aOBSC-derived neurons and oligodendrocytes grown on TRG were more numerous than in controls; the neurons developed synaptic boutons and oligodendrocytes were more branched. In contrast, neurons growing on PVDF membranes had reduced neurite branching, and on MWCNTs-loaded membranes oligodendrocytes were lower in numbers than in controls. Overall, these findings indicate that uncoated TRG may be biocompatible with the generation, differentiation, and maturation of aOBSC-derived neurons and glial cells, implying a potential use for TRG to study functional neuronal networks.This work was funded by grants from the European Union in the 7th Framework Program research project “HARCANA” (Grant Agreement No. NMP3-LA-2008-213277) to CV-A, ML-M, VA, and CJ, and the Spanish Ministerio de Economía y Competitividad (MINECO: CIBERNED CB06/05/0065, SAF2013-47596R, and CSIC 201220E098) to CV-A, and MINECO (MAT2013-48107-C3) to RV, and from Millipore Iberica (Ref. no: 050601140007) to RM-M. CERM is grateful to IAP (7-05 FS2) and FRS-FNRS for financial support.Peer reviewedPeer Reviewe

In vitro evaluation of biocompatibility of uncoated thermally reduced graphene and carbon nanotube-loaded PVDF membranes with adult neural stem cell-derived neurons and glia

Graphene, graphene-based nanomaterials (GBNs), and carbon nanotubes (CNTs) are being investigated as potential substrates for the growth of neural cells. However, in most in vitro studies, the cells were seeded on these materials coated with various proteins implying that the observed effects on the cells could not solely be attributed to the GBN and CNT properties. Here, we studied the biocompatibility of uncoated thermally reduced graphene (TRG) and poly(vinylidene fluoride) (PVDF) membranes loaded with multi-walled CNTs (MWCNTs) using neural stem cells isolated from the adult mouse olfactory bulb (termed aOBSCs). When aOBSCs were induced to differentiate on coverslips treated with TRG or control materials (polyethyleneimine-PEI and polyornithine plus fibronectin- PLO/F) in a serum-free medium, neurons, astrocytes, and oligodendrocytes were generated in all conditions, indicating that TRG permits the multi-lineage differentiation of aOBSCs. However, the total number of cells was reduced on both PEI and TRG. In a serum-containing medium, aOBSC-derived neurons and oligodendrocytes grown on TRG were more numerous than in controls; the neurons developed synaptic boutons and oligodendrocytes were more branched. In contrast, neurons growing on PVDF membranes had reduced neurite branching, and on MWCNTs-loaded membranes oligodendrocytes were lower in numbers than in controls. Overall, these findings indicate that uncoated TRG may be biocompatible with the generation, differentiation, and maturation of aOBSC-derived neurons and glial cells, implying a potential use for TRG to study functional neuronal networks

Morphological Diversity of Calretinin Interneurons Generated From Adult Mouse Olfactory Bulb Core Neural Stem Cells

Neural stem cells (NSCs) in the olfactory bulb (OB) core can generate mature interneurons in the adult mice brain. The vast majority of these adult generated cells express the calcium-binding protein Calretinin (CalR), and they migrate towards different OB layers. However, these cells have yet to be fully characterized and hence, to achieve this we injected retroviral particles expressing GFP into the OB core of adult animals and found that the CalR neurons generated from NSCs mainly migrate to the granule cell layer (GCL) and glomerular layer (GL) in similar proportions. In addition, since morphology and function are closely related, we used three-dimensional imaging techniques to analyze the morphology of these adult born cells, describing new subtypes of CalR interneurons based on their dendritic arborizations and projections, as well as their localization in the GCL or GL. We also show that the migration and morphology of these newly generated neurons can be altered by misexpressing the transcription factor Tbr1 in the OB core. Therefore, the morphology acquired by neurons located in a specific OB layer is the result of a combination of both extrinsic (e.g., layer allocation) and intrinsic mechanisms (e.g., transcription factors). Defining the cellular processes and molecular mechanisms that govern adult neurogenesis might help better understand brain circuit formation and plasticity, as well as eventually opening the way to develop strategies for brain repair.This work was funded by grants from the Spanish “Ministerio de Economía y Competitividad and Ministerio de Ciencia e Innovación” (MINECO and MICINN: SAF 2013-4759R, SAF 2016-80419-R, PID 2019-109059RB-100, and CIBERNED CB06/05/0065), from the “Comunidad de Madrid” (S2011/BMD-2336) and from the CSIC Intramural program (Refs. 201220E098 and 201320E054) to CV

Thermally reduced graphene is a permissive material for neurons and astrocytes and de novo neurogenesis in the adult olfactory bulb in vivo

Graphene and graphene-based nanomaterials (GBNs) are being investigated as potential substrates for the growth of neural stem cells (NSCs), neurons and glia in cell culture models. In contrast, reports testing the effects of graphene directly with adult neural cells in vivo are missing. Here we studied the biocompatibility of thermally reduced graphene (TRG) with neurons and glia, as well as with the generation of new neurons in the adult brain in vivo. TRG injected in the brain together with a retroviral vector expressing GFP to label dividing progenitor cells in the core of the adult olfactory bulb (OB) did not alter de novo neurogenesis, neuronal and astrocyte survival nor did it produce a microglial response. These findings indicate that TRG may be a biocompatible material with neuronal and glial cells in vivo and support its use in studies of brain repair and function.We are grateful to M.J. Roman for her technical support. This work was funded by grants from the European Union in the 7th Framework Program research project “HARCANA” (Grant Agreement No. NMP3-LA-2008-213277) to M.A.L.-M. and C.V.-A, the Spanish Ministerio de Economía y Competitividad (MINECO: CIBERNED CB06/05/0065, SAF2013-47596R, and CSIC 201220E098) to C.V.-A, from the Spanish Ministry of Science and Innovation (MiCInn: MAT 2010e18749) to R.V, from MINECO (SAF2010-15173) to R. M.-M, and from MINECO (BFU2011-26339) and INCRECyT project from European Social Fund, PCyTA and JCCM to E.D.M.Peer Reviewe