Biophysics of microchannel-enabled neuron-electrode interfaces.

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.

Second Harmonic Generation for time-resolved monitoring of membrane pore dynamics subserving electroporation of neurons.

Electroporation of neurons, i.e. electric-field induced generation of membrane nanopores to facilitate internalization of molecules, is a classic technique used in basic neuroscience research and recently has been proposed as a promising therapeutic strategy in the area of neuro-oncology. To optimize electroporation parameters, optical techniques capable of delivering time and spatially-resolved information on electroporation pore formation at the nanometer scale would be advantageous. For this purpose we describe here a novel optical method based on second harmonic generation (SHG) microscopy. Due to the nonlinear and coherent nature of SHG, the 3D radiation lobes from stained neuronal membranes are sensitive to the spatial distribution of scatterers in the illuminated patch, and in particular to nanopore formation.We used phase-array analysis to computationally study the SHG signal as a function of nanopore size and nanopore population density and confirmed experimentally, in accordance with previous work, the dependence of nanopore properties on membrane location with respect to the electroporation electric field; higher nanopore densities, lasting < 5 milliseconds, are observed at membrane patches perpendicular to the field whereas lower density is observed at partly tangent locations. Differences between near-anode and near-cathode cell poles are also measured, showing higher pore densities at the anodic pole compared to cathodic pole. This technique is promising for the study of nanopore dynamics in neurons and for the optimization of novel electroporation-based therapeutic approaches.

Developmental expression of the oligodendrocyte myelin glycoprotein in the mouse telencephalon.

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.

Integrating multi-unit electrophysiology and plastic culture dishes for network neuroscience.

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.

Intersection of microwire electrodes with proximal CA1 stratum-pyramidale neurons at insertion for multiunit recordings predicted by a 3-D computer model.

It is of broad interest in the context of neuronal multiunit extracellular recordings to understand electrode-tissue interactions in order to maximize the number of recordable units and to minimize experimental artifacts due to mechanical tissue alteration. Toward this goal, a computer model of microwire electrode insertion in hippocampus CA1 area was developed, firstly to provide estimates of the number of electrode-neuron intersections affecting recordable (local) neurons and, secondly, to determine optimal insertion/electrode parameters that minimize the number of intersections. The model predicts that in hippocampus CA1 area, using an electrode 50 microm in diameter, only 10% of the recordable neurons (those within 50 microm of the electrode), would remain collision free. Moreover, the model also predicts that inhibitory neurons are less prone to be intersected by the electrode, resulting in a 2 to threefold higher percentage of collision-free interneurons than expected from the relative densities of pyramidal cells and interneurons. Furthermore, the model confirms, in agreement with experimental observations, that electrode tilting with respect to the main neuronal axis increases the number of intact neurons (fourfold for a 50-microm electrode at 45 degrees when compared to 0 degrees , i.e., an insertion normal to the cell body layer).

Extracellular potentials in low-density dissociated neuronal cultures.

The detection of extracellular potentials by means of multi-electrode arrays (MEA) is a useful technique for multi-site long-term monitoring of cultured neuronal activity with single-cell resolution. To optimize the geometry of the MEA it is advantageous to localize the cellular compartments that constitute the generators of these signals. For this purpose, an in vitro technique for the detection of extracellular signals with subcellular resolution has been developed. It makes use of easy-to-manufacture large-tip pipettes, monitoring of electrode-cell gap resistance for precise electrode positioning and low-density (100 cells/mm(2)) dissociated hippocampal cultures. Negative monophasic extracellular spikes, typically 60 microV, were measured over putative axonal processes and monophasic, biphasic and triphasic signals were recorded over the soma. A compartmental simulation suggests that different somatic conductance densities of Na(+) (1-10 mS/cm(2)) and K(+) (5-10 mS/cm(2)) channels can produce characteristic somatic extracellular potentials, with a variety of shapes similar to those observed experimentally.