Dries Braeken

Neurotoxicity of Alzheimer's disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio

The amyloid peptides Aβ40 and Aβ42 of Alzheimers disease are thought to contribute differentially to the disease process. Although Aβ42 seems more pathogenic than Aβ40, the reason for this is not well understood. We show here that small alterations in the Aβ42:Aβ40 ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ42:Aβ40 ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti‐amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non‐toxic intermediates.

Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to excitotoxicity

Influx of Ca2+ ions through α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors contributes to neuronal damage in stroke, epilepsy, and neurodegenerative disorders such as ALS. The Ca2+ permeability of AMPA receptors is largely determined by the glutamate receptor 2 (GluR2) subunit, receptors lacking GluR2 being permeable to Ca2+ ions. We identified a difference in GluR2 expression in motor neurons from two rat strains, resulting in a difference in vulnerability to AMPA receptor-mediated excitotoxicity both in vitro and in vivo. Astrocytes from the ventral spinal cord were found to mediate this difference in GluR2 expression in motor neurons. The presence of ALS-causing mutant superoxide dismutase 1 in astrocytes abolished their GluR2-regulating capacity and thus affected motor neuron vulnerability to AMPA receptor-mediated excitotoxicity. These results reveal a mechanism through which astrocytes influence neuronal functioning in health and disease.

GluR2 deficiency accelerates motor neuron degeneration in a mouse model of amyotrophic lateral sclerosis

AMPA receptor-mediated excitotoxicity has been implicated in the selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Motor neurons in vitro are particularly vulnerable to excessive AMPA receptor stimulation and one of the factors underlying this selective vulnerability is the presence of a large proportion of Ca2+-permeable (i.e. GluR2-lacking) AMPA receptors. However, the precise role of GluR2-lacking AMPA receptors in motor neuron degeneration remains to be defined. We therefore studied the impact of GluR2 deficiency on motor neuron death in vitro and in vivo. Cultured motor neurons from GluR2-deficient embryos displayed an increased Ca2+ influx through AMPA receptors and an increased vulnerability to AMPA receptor-mediated excitotoxicity. We deleted the GluR2 gene in mutant SOD1G93A mice by crossbreeding them with GluR2 knockout mice. GluR2 deficiency clearly accelerated the motor neuron degeneration and shortened the life span of mutant SOD1G93A mice. These findings indicate that GluR2 plays a pivotal role in the vulnerability of motor neurons in vitro and in vivo, and that therapies that limit Ca2+ entry through AMPA receptors might be beneficial in ALS patients.

A Multichannel Integrated Circuit for Electrical Recording of Neural Activity, With Independent Channel Programmability

Since a few decades, micro-fabricated neural probes are being used, together with microelectronic interfaces, to get more insight in the activity of neuronal networks. The need for higher temporal and spatial recording resolutions imposes new challenges on the design of integrated neural interfaces with respect to power consumption, data handling and versatility. In this paper, we present an integrated acquisition system for in vitro and in vivo recording of neural activity. The ASIC consists of 16 low-noise, fully-differential input channels with independent programmability of its amplification (from 100 to 6000 V/V) and filtering (1-6000 Hz range) capabilities. Each channel is AC-coupled and implements a fourth-order band-pass filter in order to steeply attenuate out-of-band noise and DC input offsets. The system achieves an input-referred noise density of 37 nV/√Hz, a NEF of 5.1, a CMRR >; 60 dB, a THD <; 1% and a sampling rate of 30 kS/s per channel, while consuming a maximum of 70 μA per channel from a single 3.3 V. The ASIC was implemented in a 0.35 μm CMOS technology and has a total area of 5.6 × 4.5 mm2. The recording system was successfully validated in in vitro and in vivo experiments, achieving simultaneous multichannel recordings of cell activity with satisfactory signal-to-noise ratios.

Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 μm CMOS chip

To cope with the growing needs in research towards the understanding of cellular function and network dynamics, advanced micro-electrode arrays (MEAs) based on integrated complementary metal oxide semiconductor (CMOS) circuits have been increasingly reported. Although such arrays contain a large number of sensors for recording and/or stimulation, the size of the electrodes on these chips are often larger than a typical mammalian cell. Therefore, true single-cell recording and stimulation remains challenging. Single-cell resolution can be obtained by decreasing the size of the electrodes, which inherently increases the characteristic impedance and noise. Here, we present an array of 16,384 active sensors monolithically integrated on chip, realized in 0.18 μm CMOS technology for recording and stimulation of individual cells. Successful recording of electrical activity of cardiac cells with the chip, validated with intracellular whole-cell patch clamp recordings are presented, illustrating single-cell readout capability. Further, by applying a single-electrode stimulation protocol, we could pace individual cardiac cells, demonstrating single-cell addressability. This novel electrode array could help pave the way towards solving complex interactions of mammalian cellular networks.

Substrate Topography Determines Neuronal Polarization and Growth In Vitro

The establishment of neuronal connectivity depends on the correct initial polarization of the young neurons. In vivo, developing neurons sense a multitude of inputs and a great number of molecules are described that affect their outgrowth. In vitro, many studies have shown the possibility to influence neuronal morphology and growth by biophysical, i.e. topographic, signaling. In this work we have taken this approach one step further and investigated the impact of substrate topography in the very early differentiation stages of developing neurons, i.e. when the cell is still at the round stage and when the first neurite is forming. For this purpose we fabricated micron sized pillar structures with highly reproducible feature sizes, and analyzed neurons on the interface of flat and topographic surfaces. We found that topographic signaling was able to attract the polarization markers of mouse embryonic neurons -N-cadherin, Golgi-centrosome complex and the first bud were oriented towards topographic stimuli. Consecutively, the axon was also preferentially extending along the pillars. These events seemed to occur regardless of pillar dimensions in the range we examined. However, we found differences in neurite length that depended on pillar dimensions. This study is one of the first to describe in detail the very early response of hippocampal neurons to topographic stimuli.

Superimposed topographic and chemical cues synergistically guide neurite outgrowth

Guidance of neuronal extensions is a complex process essential for linking neurons into complex functional networks underlying the workings of the neural system. Decades of research have suggested the ability of neuronal growth cones to integrate multiple types of cues during the extension process, but also have raised numerous still unanswered questions about synergy or antagonism between the superimposed chemical and mechanical signaling inputs. In this study, using a novel microfabricated analysis platform, we investigate the response of primary mouse embryonic hippocampal neurons to superimposed topographic and soluble chemical cues. We find that an optimal spatial frequency of topographic cues exists, maximizing the precision of the neurite extension. This optimal frequency can help the extending neurites navigate a topographically complex environment, providing pronounced directional selectivity. We also demonstrate that this cue can synergistically enhance attractive and suppress repulsive guidance by the bi-functional soluble cue Netrin-1, and eliminate the repulsive guidance by a chemorepellent Semaphorin3A (Sema3A). These results suggest that topographic cues can provide optimal periodic input into the guidance signaling processes involved in growth cone chemoattraction and can synergistically interact with chemical gradients of soluble guidance cues, shedding light on complex events accompanying the development of the functional nervous system.

Glutamate sensing with enzyme-modified floating-gate field effect transistors

Neurotransmitter release is the key factor of chemical messaging in the brain. Fast, sensitive and in situ detection of single cell neurotransmitter release is essential for the investigation of synaptic transmission under physiological or pathophysiological conditions. Although various techniques have been developed for detecting neurotransmitter release both in vitro and in vivo, the sensing of such events still remains challenging. First of all, the amount of neurotransmitter released during synaptic transmission is unknown because of the limited number of molecules released and the fast diffusion and reuptake of these molecules after release. On the other hand, advances in microelectronic biosensor devices have made possible the fast detection of various analytes with high sensitivity and selectivity. Specifically, enzyme-modified field-effect (ENFET) devices are attractive for such applications due to their fast response, small dimensions and the possibility to integrate a large number of sensors on the same chip. In this paper, we present a floating-gate FET device coated with glutamate oxidase (GLOD) layer. The surface chemistry was optimized for maximal enzyme loading and long-term stability, and characterized by quartz crystal microbalance and colorimetric assays. Enzyme loading was largest on poly-L-lysin-based surfaces combined with glutaraldehyde. The surface chemistry showed excellent stability for at least one month in Tris buffers stored at 4 degrees C. A glutamate detection limit of 10(-7) M has been obtained with the GLOD-coated FET and our sensor proved to be selective to glutamate only. We show that this biosensor is a promising tool for the in vitro detection of glutamate and can be extended to other neurotransmitters.

Localized electrical stimulation of in vitro neurons using an array of sub-cellular sized electrodes

The investigation of single-neuron parameters is of great interest because many aspects in the behavior and communication of neuronal networks still remain unidentified. However, the present available techniques for single-cell measurements are slow and do not allow for a high-throughput approach. We present here a CMOS compatible microelectrode array with 84 electrodes (with diameters ranging from 1.2 to 4.2 μm) that are smaller than the size of cell, thereby supporting single-cell addressability. We show controllable electroporation of a single cell by an underlying electrode while monitoring changes in the intracellular membrane potential. Further, by applying a localized electrical field between two electrodes close to a neuron while recording changes in the intracellular calcium concentration, we demonstrate activation of a single cell (∼270%, DF/F(0)), followed by a network response of the neighboring cells. The technology can be easily scaled up to larger electrode arrays (theoretically up to 137,000 electrodes/mm(2)) with active CMOS electronics integration able to perform high-throughput measurements on single cells.

An integrated multi-electrode-optrode array for in vitro optogenetics.

Modulation of a group of cells or tissue needs to be very precise in order to exercise effective control over the cell population under investigation. Optogenetic tools have already demonstrated to be of great value in the study of neuronal circuits and in neuromodulation. Ideally, they should permit very accurate resolution, preferably down to the single cell level. Further, to address a spatially distributed sample, independently addressable multiple optical outputs should be present. In current techniques, at least one of these requirements is not fulfilled. In addition to this, it is interesting to directly monitor feedback of the modulation by electrical registration of the activity of the stimulated cells. Here, we present the fabrication and characterization of a fully integrated silicon-based multi-electrode-optrode array (MEOA) for in vitro optogenetics. We demonstrate that this device allows for artifact-free electrical recording. Moreover, the MEOA was used to reliably elicit spiking activity from ChR2-transduced neurons. Thanks to the single cell resolution stimulation capability, we could determine spatial and temporal activation patterns and spike latencies of the neuronal network. This integrated approach to multi-site combined optical stimulation and electrical recording significantly advances today’s tool set for neuroscientists in their search to unravel neuronal network dynamics.