Neurons are MHC Class I-Dependent Targets for CD8 T Cells upon Neurotropic Viral Infection

Following infection of the central nervous system (CNS), the immune system is faced with the challenge of eliminating the pathogen without causing significant damage to neurons, which have limited capacities of renewal. In particular, it was thought that neurons were protected from direct attack by cytotoxic T lymphocytes (CTL) because they do not express major histocompatibility class I (MHC I) molecules, at least at steady state. To date, most of our current knowledge on the specifics of neuron-CTL interaction is based on studies artificially inducing MHC I expression on neurons, loading them with exogenous peptide and applying CTL clones or lines often differentiated in culture. Thus, much remains to be uncovered regarding the modalities of the interaction between infected neurons and antiviral CD8 T cells in the course of a natural disease. Here, we used the model of neuroinflammation caused by neurotropic Borna disease virus (BDV), in which virus-specific CTL have been demonstrated as the main immune effectors triggering disease. We tested the pathogenic properties of brain-isolated CD8 T cells against pure neuronal cultures infected with BDV. We observed that BDV infection of cortical neurons triggered a significant up regulation of MHC I molecules, rendering them susceptible to recognition by antiviral CTL, freshly isolated from the brains of acutely infected rats. Using real-time imaging, we analyzed the spatio-temporal relationships between neurons and CTL. Brain-isolated CTL exhibited a reduced mobility and established stable contacts with BDV-infected neurons, in an antigen- and MHC-dependent manner. This interaction induced rapid morphological changes of the neurons, without immediate killing or impairment of electrical activity. Early signs of neuronal apoptosis were detected only hours after this initial contact. Thus, our results show that infected neurons can be recognized efficiently by brain-isolated antiviral CD8 T cells and uncover the unusual modalities of CTL-induced neuronal damage

Nanowire based bioprobes for electrical monitoring of electrogenic cells

International audienceThe continuous miniaturization of electronic components and the emergence of nano-biotechnology has opened new perspectives to monitor electrical activities at the single cell level. Here, we describe the creation of very high surface-to-volume ratio passive devices (vertical nanowire probes) using large-scale fabrication process, allowing to follow the electrical activity of mammalian neurons. Based on conventional silicon processing, the silicon nanowires were silicided in platinum in order to improve their electrochemical performances and to guarantee their biocompatibility. Very high signal to noise ratio was achieved (up to 2000) when measuring spontaneous action potentials. Moreover, this bio-platform was used to record the impact of various bio-chemical and electrical stimulations on neuronal activity. To conclude, this study proposes a thorough comparison of the characteristics and performances of these new nanowire-based nanoprobes with the main alternative systems published up to now

Self-Aligned Functionalization Approach to Order Neuronal Networks at the Single-Cell Level

International audienceDespite significant progress, our knowledge of the functioning of the central nervous system still remains scarce to date. A better understanding of its behavior, in either normal or diseased conditions, goes through an increased knowledge of basic mechanisms involved in neuronal function, including at the single-cell level. This has motivated significant efforts for the development of miniaturized sensing devices to monitor neuronal activity with high spatial and signal resolution. One of the main challenges remaining to be addressed in this domain is, however, the ability to create in vitro spatially ordered neuronal networks at low density with a precise control of the cell location to ensure proper monitoring of the activity of a defined set of neurons. Here, we present a novel self-aligned chemical functionalization method, based on a repellant surface with patterned attractive areas, which permits the elaboration of low-density neuronal network down to individual cells with a high control of the soma location and axonal growth. This approach is compatible with complementary metal-oxide–semiconductor line technology at a wafer scale and allows performing the cell culture on packaged chip outside microelectronics facilities. Rat cortical neurons were cultured on such patterned surfaces for over one month and displayed a very high degree of organization in large networks. Indeed, more than 90% of the network nodes were settled by a soma and 100% of the connecting lines were occupied by a neurite, with a very good selectivity (low parasitic cell connections). After optimization, networks composed of 75% of unicellular nodes were obtained, together with a control at the micron scale of the location of the somas. Finally, we demonstrated that the dendritic neuronal growth was guided by the surface functionalization, even when micrometer scale topologies were encountered and we succeeded to control the extension growth along one-dimensional-aligned nanostructures with sub-micrometrical scale precision. This novel approach now opens the way for precise monitoring of neuronal network activity at the single-cell level