Multielectrode array technology constitutes a promising approach for the characterization of the activity-dependent neuronal plasticity underlying information processing in the nervous system. For this purpose, long-term monitoring and stimulation of cultured neuronal networks with one-to-one neuron-sensor interfacing is advantageous. Existing neurochips that meet these specifications have made use of custom 3D structures requiring clean-room intensive microfabrication techniques. Low-cost fabrication procedures with potential for mass production would facilitate progress in the area. To this end, we have developed a sandwich structure comprising an elastomeric film, microstructured by replica moulding and microhole punching, for neuronal patterning, and a standard planar microelectrode array (MEA), for stimulation and recording. The elastomeric film includes microwells for cell body confinement, and microchannels capable of guiding neurites for network topology specification. The device is formed by overlaying the elastomeric structures on planar arrays. The combination of replica moulding, rapid prototyping and planar MEAs results in low-cost neurochips accessible to most neurophysiology labs. Single neuron patterning and recordings of extracellular potentials are demonstrated.
We have previously described the use of microchannels (μChannels) as substrate-integrated equivalents of micropipettes and advantageous neuron-electrode interface enhancers. The use of μChannels to establish stable recording and stimulation of threading axons results in a high signal-to-noise ratio (SNR), potentially high-throughput and low-cost alternative to conventional substrate-embedded microelectrodes. Here we confirm the consistent achievement of high SNRs with μChannels and systematically characterize the impact of μChannel geometry on the measured signals via numerical simulations and in vitro experiments. We demonstrate and rationalize how channels with a length of ≤300 μm and channel cross section of ≤12 μm(2) support spontaneous formation of seals and yield spike sizes in the millivolt range. Despite the low degree of complexity involved in their fabrication and use, μChannel devices provide a single-unit mean SNR of 101 ± 76, which compares favourably with the SNR obtained from typical microelectrode arrays.
Abstract-Towards establishing electrical interfaces with patterned in vitro neurons, we have previously described the fabrication of hybrid elastomer-glass devices polymer-on-multielectrode array technology and obtained single-electrode recordings of extracellular potentials from confined neurons (Claverol-Tinturé et al., 2005). Here, we demonstrate the feasibility of spatially localized multisite recordings from individual microchannel-guided neurites extending from microwell-confined somas with good signal-to-noise ratios (20 dB) and spike magnitudes of up to 300 V.Single-cell current source density (scCSD) analysis of the spatio-temporal patterns of membrane currents along individual processes is illustrated.
The oligodendrocyte myelin glycoprotein is a glycosylphosphatidylinositol-anchored protein expressed by neurons and oligodendrocytes in the central nervous system. Attempts have been made to identify the functions of the myelin-associated inhibitory proteins (MAIPs) after axonal lesion or in neurodegeneration. However, the developmental roles of some of these proteins and their receptors remain elusive. Recent studies indicate that NgR1 and the recently discovered receptor PirB restrict cortical synaptic plasticity. However, the putative factors that trigger these effects are unknown. Because Nogo-A is mostly associated with the endoplasmic reticulum and myelin associated glycoprotein appears late during development, the putative participation of OMgp should be considered. Here, we examine the pattern of development of OMgp immunoreactive elements during mouse telencephalic development. OMgp immunoreactivity in the developing cortex follows the establishment of the thalamo-cortical barrel field. At the cellular level, we located OMgp neuronal membranes in dendrites and axons as well as in brain synaptosome fractions and axon varicosities. Lastly, the analysis of the barrel field in OMgp-deficient mice revealed that although thalamo-cortical connections were formed, their targeting in layer IV was altered, and numerous axons ectopically invaded layers II-III. Our data support the idea that early expressed MAIPs play an active role during development and point to OMgp participating in thalamo-cortical connections.
The electrophysiological characterisation of cultured neurons is of paramount importance for drug discovery, safety pharmacology and basic research in the neurosciences. Technologies offering low cost, low technical complexity and potential for scalability towards high-throughput electrophysiology on in vitro neurons would be advantageous, in particular for screening purposes. Here we describe a plastic culture substrate supporting low-complexity multi-unit loose-patch recording and stimulation of developing networks while retaining manufacturability compatible with low-cost and large-scale production. Our hybrid polydimethylsilane (PDMS)-on-polystyrene structures include chambers (6 mm in diameter) and microchannels (25 microm x 3.7 microm x 1 mm) serving as substrate-embedded recording pipettes. Somas are plated and retained in the chambers due to geometrical constraints and their processes grow along the microchannels, effectively establishing a loose-patch configuration without human intervention. We demonstrate that off-the-shelf voltage-clamp, current-clamp and extracellular amplifiers can be used to record and stimulate multi-unit activity with the aid of our dishes. Spikes up to 50 pA in voltage-clamp and 300 microV in current-clamp modes are recorded in sparse and bursting activity patterns characteristic of 1 week-old hippocampal cultures. Moreover, spike sorting employing principal component analysis (PCA) confirms that single microchannels support the recording of multiple neurons. Overall, this work suggests a strategy to endow conventional culture plasticware with added functionality to enable cost-efficient network electrophysiology.
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