Neuron network activity scales exponentially with synapse density.

Neuronal network output in the cortex as a function of synapse density during development has not been explicitly determined. Synaptic scaling in cortical brain networks seems to alter excitatory and inhibitory synaptic inputs to produce a representative rate of synaptic output. Here, we cultured rat hippocampal neurons over a three-week period to correlate synapse density with the increase in spontaneous spiking activity. We followed the network development as synapse formation and spike rate in two serum-free media optimized for either (a) neuron survival (Neurobasal/B27) or (b) spike rate (NbActiv4). We found that while synaptophysin synapse density increased linearly with development, spike rates increased exponentially in developing neuronal networks. Synaptic receptor components NR1, GluR1 and GABA-A also increase linearly but with more excitatory receptors than inhibitory. These results suggest that the brain's information processing capability gains more from increasing connectivity of the processing units than increasing processing units, much as Internet information flow increases much faster than the linear number of nodes and connections.

Multivariate FTIR analysis of substrates for protein, polysaccharide, lipid and microbe content: potential for solid-state fermentations.

Components of fermentation processes such as protein, polysaccharide and lipid, as well as microbes, such as fungi grown on solid substrates, are difficult to measure in situ. The potential of Fourier Transform Infrared (FTIR) analysis of solid-state fermentations from mid-infrared absorption spectra has been investigated. The problem under consideration was to build a calibration model containing no irrelevant information to enable a multivariate mathematical approach for prediction of component concentrations. Methods for solid sample preparation and preprocessing of FTIR data were developed to assure Beer-Lambert law compliance and produce a well-conditioned multivariate system. The model was tested using composite samples of zein protein, corn starch and azolectin lipid, and corn samples containing known levels of fungal contamination. Preliminary concentration estimates were remarkably close to the correct values, with less than 5% standard error of prediction for all components measured.

Long-term stability of grafted polyethylene glycol surfaces for use with microstamped substrates in neuronal cell culture.

Crucial to long-term stability of neuronal micropatterns is functional retention of the underlying substratum while exposed to cell culture conditions. We report on the ability of covalently bound PEG films in long-term cell culture to continually retard protein adhesion and cell growth. PDMS microstamps were used to create poly-d-lysine (PDL) substrates permissive to cell attachment and growth, and polyethylene glycol (PEG) substrates were used to minimize protein and cell adhesion. Film thickness was measured using null ellipsometry and atomic force microscopy (AFM). Organosilane film structure was examined using Fourier transform infrared (FT-IR) spectroscopy. Long-term film stability in cell culture conditions was tested by immersion in 0.1 M sodium phosphate buffer pH 7.4 for up to one month. Null ellipsometry and water contact measurements indicated that organosilane films were stable up to one month, whereas the PEG film thickness declined rapidly after day 25. Hippocampal cells plated at 200 cells/mm2 on uniform PEG substrates gave a steady increase in biofilm thickness on PEG films throughout the culture, possibly from proteins of neuronal origin. We found that all the layers in the cross-linking procedure were stable in cell culture conditions, with the exception of PEG, which degraded after day 25.

Early-pregnancy proteinuria in diabetes related to preeclampsia.

OBJECTIVE: To test the hypothesis that the risk of preeclampsia in diabetic mothers is increased with incipient diabetic nephropathy as well as with overt nephropathy. METHODS: Pregnancy outcome was studied in 311 women with class B-RF diabetes from two institutions. Using 104 women without chronic hypertension followed at the University of California, San Francisco, we constructed a receiver-operating characteristic curve relating 24-hour urinary total protein before 20 weeks' gestation to the subsequent development of preeclampsia. From the curve, a predictive cutoff level of proteinuria was selected and tested in two validation groups not used to construct the curve: 158 women without chronic hypertension followed at the University of Cincinnati and 49 women with chronic hypertension from both institutions. RESULTS: The receiver-operating characteristic curve showed an increased risk of preeclampsia with early-pregnancy proteinuria of 190 mg/day or more. In the Cincinnati validation group, the rate of preeclampsia was 7% in women with early-pregnancy proteinuria of less than 190 mg/day, 31% with proteinuria of 190-499 mg/day, and 38% with proteinuria of 500 mg/day or more. In the chronic-hypertension validation group, the rates were 0, 50, and 58%, respectively. By multiple logistic regression, the increased risk of preeclampsia with proteinuria above 190 mg/day persisted after controlling for the effects of parity, chronic hypertension, retinopathy, and glycemic control. CONCLUSIONS: Diabetic gravidas with early-pregnancy proteinuria of 190-499 mg/day are at increased risk for preeclampsia. The risk is comparable to that in women with overt diabetic nephropathy and is independent of chronic hypertension. We speculate that diabetic women with proteinuria in this range have incipient or subclinical diabetic nephropathy.

Multisite hippocampal slice recording and stimulation using a 32 element microelectrode array.

A technique has been developed in which a planar array of 32 microelectrodes, arranged in a 4 by 8 pattern with 200 micron separation, is used to record from and stimulate the hippocampal slice preparation at multiple sites. Control of media flow past the tissue is critical to observe signals and preserve viability. Active suppression circuitry is used to prevent device saturation due to large stimulation artifacts. The field potentials recorded are spatially unique and provide a 2-dimensional description of the underlying population activity in the various pyramidal strata and subpopulations. Multisite stimulation is also possible with the array, permitting the experimenter to quickly stimulate and record from brain slices in many spatial patterns.

A high-speed multichannel neural data acquisition system for IBM PC compatibles.

An inexpensive 32-channel data acquisition system has been constructed for use in acquiring neuroelectric data from a multiple element electrode array. Direct memory access (DMA) techniques allowed a maximum aggregate sampling rate of 652 ksamples/s, or 20.4 ksamples/s on 32 channels. The 8-bit analog-to-digital (A/D) conversion circuitry lies on a single plug-in card installed in an IBM PC-compatible AT & T 6300 Plus personal computer, although the card may be used in an IBM PC with no reduction in sampling performance. Data have been taken from 32 points on the surface of the rat hippocampal slice preparation. Assembly language graphics routines permitted rapid display of raw and processed data. Signal processing routines can be executed and results can be displayed to provide rapid feedback to an experimenter.

Differentiated B104 neuroblastoma cells are a high-resolution assay for micropatterned substrates.

The B104 neuroblastoma cell line was investigated for use as an assay for predicting the patterning of primary neurons. B104 cells were grown on four uniform substrates with the result that the cells preferred, in descending order, poly-D-lysine (PDL), phenyltrichlorosilane (PTCS), coverslip glass, and silicon dioxide coated coverslips. B104 cells were then grown on micropatterned PDL grids on silicon dioxide coated substrates with excellent patterning. Compliance of somata to the pattern, defined as the percentage of cell bodies in a grid field located on the grid pattern, was 86% after 8 h. Neurites were not as compliant, since only 10% of background areas were free of neurites and connected cells. Compliance at longer time periods was greatly reduced. With the addition of the differentiating agent dibutyrylcyclicAMP (DBcAMP), the compliance of somata was maintained at high levels for up to 72 h. Also, the compliance of neurites greatly increased (70%) and showed positive improvement with longer pattern path lengths, contrary to B104 cells without DBcAMP. At longer times neurite compliance was reduced (12% at 28 h and 44% at 72 h). Although there are differences in substrate preferences, the B104 system with DBcAMP appears to be a useful tool in the investigation of the technology of patterned substrates.

Modulation of neural network activity by patterning.

Using neuronal cultures on microelectrode arrays, researchers have shown that recordable electrical activity can be influenced by chemicals in the culture environment, thus demonstrating potential applicability to biosensors or drug screening. Since practical success requires the design of robust networks with repeatable, reliable responses understanding the sources of variation is important. In this report, we used lithographic technologies to confine neurons to highly defined patterns (40 microm wide stripes); in turn these patterns gave us a measure of control over the local density of neurons (100-500 cells/mm(2)). We found that the apparent electrical activity of the network, as measured by the fraction of electrodes from which signals were recordable, increases 8-10-fold with greater local density. Also, average-firing rates of the active neurons increased 3-5-fold. We conclude that patterned networks offer one means of controlling and enhancing the responsiveness of cultured neural networks.

Two-dimensional current source density analysis of propagation delays for components of epileptiform bursts in rat hippocampal slices.

Recordings of epileptiform burst activity in the CA1 region of the transverse rat hippocampal slices were made with a 32-channel surface electrode array. The 200 microns interelectrode resolution, the simultaneity of the data, and the use of a two-dimensional current source density analysis allowed accurate measurement of population spike peak times. Differences were found in the apparent propagation delays among 3 burst components: the compound action potential (CAP) along the Schaffer collaterals, the first population spike directly driven by the CAP, and the second (and succeeding) population spikes representing the bursting, epileptiform component. Delay measurements were applied to epileptiform bursts recorded in slices treated with picrotoxin (PTX), pentylenetetrazol (PTZ), and 0-Mg2+ medium. In 0-Mg2+ medium all components propagate at nearly the same velocity. In the PTZ and PTX media the second population spike propagated more slowly than the CAP. The first population spike propagated at the same velocity as the CAP for orthodromic Schaffer collateral stimulation. The first population spike propagated at the same, slower velocity as the second spike for antidromic Schaffer collateral stimulation.

Identification of Fourier transform infrared photoacoustic spectral features for detection of Aspergillus flavus infection in corn.

Aspergillus flavus and other pathogenic fungi display typical infrared spectra which differ significantly from spectra of substrate materials such as corn. On this basis, specific spectral features have been identified which permit detection of fungal infection on the surface of corn kernels by photoacoustic infrared spectroscopy. In a blind study, ten corn kernels showing bright greenish yellow fluorescence (BGYF) in the germ or endosperm and ten BGYF-negative kernels were correctly classified as infected or not infected by Fourier transform infrared photoacoustic spectroscopy. Earlier studies have shown that BGYF-positive kernels contain the bulk of the aflatoxin contaminating grain at harvest. Ten major spectral features, identified by visual inspection of the photoacoustic spectra of A. flavus mycelium grown in culture versus uninfected corn, were interpreted and assigned by theoretical comparisons of the relative chemical compositions of fungi and corn. The spectral features can be built into either empirical or knowledge-based computer models (expert systems) for automatic infrared detection and segregation of grains or kernels containing aflatoxin from the food and feed supply.

A flexible perforated microelectrode array for extended neural recordings.

A flexible and perforated 32-element planar microelectrode array has been fabricated and used to measure evoked potentials in brain slices. Electrodes are spaced 200 microns apart in a 4 x 8 array and are sandwiched between layers of insulating polyimide. The polyimide sandwich is lifted off its substrate, making it flexible so that it could shape to contoured tissues. Prior to lift off, holes are etched to expose recording sites 15 microns in diameter and to create perforations which allow increased circulation of artificial cerebrospinal fluid to the recording surface of the tissue and, hence, increased viability. Comparisons of evoked potentials measured over time showed an average increase of 10 h to the viability of the slice while using the perforated versus nonperforated arrays.

Micrometer resolution silane-based patterning of hippocampal neurons: critical variables in photoresist and laser ablation processes for substrate fabrication.

Toward the goal of creating patterns of primary hippocampal neurons in low density culture, we investigated techniques to fabricate microminiature grids of organofunctional silanes on glassy surfaces. A new photoresist (PR) process, Selective Silane Removal (SSR), was developed and compared to two previously developed techniques which use PR and laser patterning. The grid patterns consisted of 27 combinations of path width, length, and intersection (node diameter). The background consisted of squares bounded by the paths. The best neuron patterning was observed on substrates produced by the SSR process where cytophilic aminosilane is uniformly deposited and selectively removed from the background. Controlling water during aminosilane deposition was critical to good neuronal growth and patterning. Oxygen plasma etching of background regions prior to cytophobic phenylsilane binding significantly reduced off-pattern cell growth. Up to 90% of somata grown on these substrates complied to the pattern, and an average of 77% of background regions were free of neurites or cells connected to the pattern. The highest laser energy density, 120 mJ/cm2, produced the best compliance on lased substrates, with an average of 35% of background regions free of connected cells and neurites, but considerable variation across the surface. On substrates with excellent patterning, compliance to nodes was found to be dependent on pattern dimensions, with 20-micron node diameters and 80-micron internodal path lengths increasing compliance.

Long-term maintenance of patterns of hippocampal pyramidal cells on substrates of polyethylene glycol and microstamped polylysine.

For neurons to attach and remain in precise micropatterns for weeks in culture, background molecules that remain nonpermissive for extended culture durations need to be identified. Nonpermissive background molecules of either polyethylene glycol (PEG) or the amino acid serine (C3H7NO3) were evaluated. The foreground regions were microstamped with 3-, 5-, or 10-micron lines of poly-D-lysine (PDL), which promotes neural attachment and growth. After 29 days in culture the foreground compliance, or the fraction of all live somata which rested on the desired PDL surface, averaged 86% for serine and 90% for PEG, with only a small decline. The background compliance, or the fraction of square areas in the pattern background which were free of neurite extension, declined from highs of 40% and 55% (midculture) to 5.5% and 12% (29 days) for serine and PEG, respectively. Images of the cultures suggest that PEG is significantly more effective as a nonpermissive substrate. We conclude that these materials, especially PEG, are adequate for the maintenance of long-term patterned cultures of neurons. We believe that this is the first report of high-quality long-term patterning of cultured neurons.

Neural network pattern recognition of photoacoustic FTIR spectra and knowledge-based techniques for detection of mycotoxigenic fungi in food grains.

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS), a highly sensitive probe of the surfaces of solid substrates, is used to detect toxigenic fungal contamination in corn. Kernels of corn infected with mycotoxigenic fungi, such as Aspergillus flavus, display FTIR-PAS spectra that differ significantly form spectra of uninfected kernels. Photoacoustic infrared spectral features were identified, and an artificial neural network was trained to distinguish contaminated form uncontaminated corn by pattern recognition. Work is in progress to integrate epidemiological information about cereal crop fungal disease into the pattern recognition program to produce a more knowledge-based, and hence more reliable and specific, technique. A model of a hierarchically organized expert system is proposed, using epidemiological factors such as corn variety, plant stress and susceptibility to infection, geographic location, weather, insect vectors, and handling and storage conditions, in addition to the analytical data, to predict Al. flavus and other kinds of toxigenic fungal contamination that might be present in food grains.

Real-time multichannel neural spike recognition with DSPs.

Data acquisition technology that uses relatively inexpensive digital signal processors (DSPs) for the rapid acquisition of neuronal action potential (spike) data was investigated. Three spike sorting techniques were considered. The simplest classification routine tested was peak windowing. Spikes were considered to belong to the same class when their peak amplitude fell within a user-specified interval. Another approach required computing the RMS error between stored templates of each unit's spike waveform and the spike most recently detected. The new spike was classified according to the minimum of the RMS errors computed. In the third algorithm, two optimal coefficient vectors, called principal components, were computed from previously acquired template waveforms and stored. These principal components were used to compute two vector products for each newly detected spike waveform vector. The resulting two values were features that were used to classify the waveform. The test system that was constructed is described, and results obtained with each of the algorithms are compared and evaluated.

Microstamp patterns of biomolecules for high-resolution neuronal networks.

A microstamping technique has been developed for high-resolution patterning of proteins on glass substrates for the localisation of neurons and their axons and dendrites. The patterning process uses a microfabricated polydimethylsiloxane stamp with micrometer length features to transfer multiple types of biomolecules to silane-derivatised substrates, using glutaraldehyde as a homobifunctional linker. To test the efficacy of the procedure, substrates are compared in which poly-d-lysine (PDL) was physisorbed and patterned by photoresist with those stamped with PDL. Fluorescein isothiocyanate labelled poly-l-lysine was used to verify the presence and uniformity of the patterns on the glass substrates. As a biological assay, B104 neuroblastoma cells were plated on stamped and physisorbed glass coverslips. Pattern compliance was determined as the percentage of cells on the pattern 8 h after plating. Results indicate that the stamping and photoresist patterning procedure are equivalent. Substrates stamped with PDL had an average pattern compliance of 52.6 +/- 4.4%, compared to 54.6 +/- 8.1% for physisorbed substrates. Measures of background avoidance were also equivalent. As the procedure permits successive stamping of multiple proteins, each with its own micropattern, it should be very useful for defining complex substrates to assist in cell patterning and other cell guidance studies.

Chronic network stimulation enhances evoked action potentials.

Neurons cultured on multielectrode arrays almost always lack external stimulation except during the acute experimental phase. We have investigated the effects of chronic stimulation during the course of development in cultured hippocampal neural networks by applying paired pulses at half of the electrodes for 0, 1 or 3 r/day for 8 days. Spike latencies increased from 4 to 16 ms as the distance from the stimulus increased from 200 to 1700 microm, suggesting an average of four synapses over this distance. Compared to no chronic stimulation, our results indicate that chronic stimulation increased evoked spike counts per stimulus by 50% at recording sites near the stimulating electrode and increased the instantaneous firing rate. On trials where both pulses elicited responses, spike count was 40-80% higher than when only one of the pulses elicited a response. In attempts to identify spike amplitude plasticity, we found mainly amplitude variation with different latencies suggesting recordings from neurons with different identities. These data suggest plastic network changes induced by chronic stimulation that enhance the reliability of information transmission and the efficiency of multisynaptic network communication.