Constant pressure fluid infusion into rat neocortex from implantable microfluidic devices.

Implantable electrode arrays capable of recording and stimulating neural activity with high spatial and temporal resolution will provide a foundation for future brain computer interface technology. Currently, their clinical impact has been curtailed by a general lack of functional stability, which can be attributed to the acute and chronic reactive tissue responses to devices implanted in the brain. Control of the tissue environment surrounding implanted devices through local drug delivery could significantly alter both the acute and chronic reactive responses, and thus enhance device stability. Here, we characterize pressure-mediated release of test compounds into rat cortex using an implantable microfluidic platform. A fixed volume of fluorescent cell marker cocktail was delivered using constant pressure infusion at reservoir backpressures of 0, 5 and 10 psi. Affected tissue volumes were imaged and analyzed using epifluorescence and confocal microscropies and quantitative image analysis techniques. The addressable tissue volume for the 5 and 10 psi infusions, defined by fluorescent staining with Hoescht 33342 dye, was significantly larger than the tissue volume addressed by simple diffusion (0 psi) and the tissue volume exhibiting insertion-related cell damage (stained by propidium iodide). The results demonstrate the potential for using constant pressure infusion to address relevant tissue volumes with appropriate pharmacologies to alleviate reactive biological responses around inserted neuroprosthetic devices.

A micromachined silicon multielectrode for multiunit recording.

A 16-channel multielectrode was used to record propagating action potentials from multiple units in the ventral nerve cord of the cricket Gryllus bimaculatus. The multielectrode was fabricated using photolithographic and bulk silicon etching techniques. The fabrication differs from standard methods in its use of deep reactive ion etching (DRIE) to form the bulk electrode structure. This technique enables the fabrication of relatively thick (>100 microm), rigid structures whose top surface can have any form of thin film electronics. The multielectrode tested in this paper consists of 16 narrow silicon bridges, 150 microm wide and 350 microm tall, spaced evenly over a centimeter, with passive rectangular gold recording sites on the top surface. The nerve cord was placed perpendicularly across the bridges. In this geometry, the nerve spans a 350 microm deep, 450 microm wide trench between each recording site, permitting adequate isolation of recording sites from each other and a platinum ground plane. Spike templates for eight neurons were formed using principle component analysis and clustering of the concatenated multichannel waveforms. Clean templates were generated from a 40 s recording of stimulus evoked activity. Conduction velocities ranged from 2.59+/-0.05 to 4.99+/-0.12 m/s. Two limitations of extracellular electrode arrays are the resolution of overlapping spikes and relation of discriminated units to known anatomy. The high density, precise positioning, and controlled impedance of recording sites achievable in microfabricated devices such as this one will aid in overcoming these limitations. The rigid devices fabricated using this process offer stable positioning of recording sites over relatively large distances (several millimeters) and are suitable for clamping or squeezing of nerve cords.

Dark field imaging of biological macromolecules with the scanning transmission electron microscope.

A scanning transmission electron microscope (STEM) equipped with a field emission gun has been employed for the examination of biological macromolecules at high resolution. The quality of micrographs obtained with the STEM is dependent upon the quality of the substrate used to support biological objects because the image contrast in dark field is proportional to the mass density of the specimen. In order to reduce deleterious effects of the substrates on the image quality, we have developed a method of fabricating substrates consisting of very thin, very clean carbon films supported on very clean fenestrated plastic films. These films are approximately 15 A thick. Well-known biological macromolecules such as glutamine synthetase and tobacco mosaic virus (both stained) and low-density lipoprotein and ferritin (both unstained were placed on these substrates and examined with the STEM by using various modes of contrast. The micrographs obtained by using the dark field mode of contrast employing an annular detector were free from phase contrast, as expected. Using this contrast mode, we have been able to directly observe (in-focus) 2.5- to 4.4-A lattice spacings in the ferritin core. The effect of electron radiation damage on the helical structure of tobacco mosaic virus was also examined. Micrographs as well as corresponding optical diffraction patterns obtained with moderately low doses showed very clear helical structure from both sides of the virus. In addition, the (11.5 A)(-1) layer lines indicated the effective resolution attained on these particles.