Optical monitoring of neural networks evoked by focal electrical stimulation on microelectrode arrays using FM dyes.

Patch-clamping or microelectrode arrays (MEA) are conventional methods to monitor the electrical activity in biological neural networks in vitro. Despite the effectiveness of these techniques, there are disadvantages including the limited number of electrodes and the predetermined location of electrodes in MEAs. In particular, these drawbacks raise a difficulty in monitoring a number of neurons outnumbering the electrodes. Here, we propose an optical technique to determine the effective range of focal electrical stimulation using FM dyes in neural networks grown on planar-type MEAs. After 3 weeks in culture, electrical stimulation was delivered to neural networks via an underlying electrode in the presence of FM dyes. The stimulation induced the internalization of the dye into the neurons around the stimulating electrodes. Fluorescent images of dye distribution successfully showed the effects of focal stimulation. A range of stimulus amplitudes and frequencies were examined to collect fluorescence images. FM-dye uptake after electrical stimulation resulted in the labeling of cells up to approximately 300 microm away from the stimulating electrode. Fluorescence intensity increased proportionally to stimulation amplitude. Tetrodotoxin was shown to inhibit the labeling of neurons except those located immediately adjacent (within 40 microm) from the stimulating electrode. In the presence of AMPA and NMDA receptors antagonists, the FM-dye labeling appeared within 80 microm from the electrode, indicating directly evoked neural networks via blocking of glutamatergic synaptic transmission. These results showed that FM dyes can be a useful tool for monitoring activity-dependent synaptic events and determining the effect of focal stimulation in cultured neural networks.

Modulation of cultured neural networks using neurotrophin release from hydrogel-coated microelectrode arrays.

Polyacrylamide and poly(ethylene glycol) diacrylate hydrogels were synthesized and characterized for use as drug release and substrates for neuron cell culture. Protein release kinetics was determined by incorporating bovine serum albumin (BSA) into hydrogels during polymerization. To determine if hydrogel incorporation and release affect bioactivity, alkaline phosphatase was incorporated into hydrogels and a released enzyme activity determined using the fluorescence-based ELF-97 assay. Hydrogels were then used to deliver a brain-derived neurotrophic factor (BDNF) from hydrogels polymerized over planar microelectrode arrays (MEAs). Primary hippocampal neurons were cultured on both control and neurotrophin-containing hydrogel-coated MEAs. The effect of released BDNF on neurite length and process arborization was investigated using automated image analysis. An increased spontaneous activity as a response to the released BDNF was recorded from the neurons cultured on the top of hydrogel layers. These results demonstrate that proteins of biological interest can be incorporated into hydrogels to modulate development and function of cultured neural networks. These results also set the stage for development of hydrogel-coated neural prosthetic devices for local delivery of various biologically active molecules.

Functionalized hydrogel surfaces for the patterning of multiple biomolecules.

Patterning of multiple proteins and enzymes onto biocompatible surfaces can provide multiple signals to control cell attachment and growth. Acrylamide-based hydrogels were photo-polymerized in the presence of streptavidin-acrylamide, resulting in planar gel surfaces functionalized with the streptavidin protein. This surface was capable of binding biotin-labeled biomolecules. The proteins fibronectin and laminin, the enzyme alkaline phosphatase, and the photo-protein R-phycoerythrin were patterned using soft lithographic techniques. Polydimethylsiloxane stamps were used to transfer biotinylated proteins onto streptavidin-conjugated hydrogel surfaces. Stamped biomolecules were spatially resolved to feature sizes of 10 mum. Fluorescence measurements were used to assess protein transfer and enzyme functionality on modified surfaces. Our results demonstrate that hydrogel surfaces can be patterned with multiple proteins and enzymes, with retention of biological and catalytic activity. These surfaces are biocompatible and provide cues for cell attachment and growth. (c) 2006 Wiley Periodicals, Inc. J Biomed Mater Res 2007.

Automatic selection of parameters for vessel/neurite segmentation algorithms.

An automated method is presented for selecting optimal parameter settings for vessel/neurite segmentation algorithms using the minimum description length principle and a recursive random search algorithm. It trades off a probabilistic measure of image-content coverage against its conciseness. It enables nonexpert users to select parameter settings objectively, without knowledge of underlying algorithms, broadening the applicability of the segmentation algorithm, and delivering higher morphometric accuracy. It enables adaptation of parameters across batches of images. It simplifies the user interface to just one optional parameter and reduces the cost of technical support. Finally, the method is modular, extensible, and amenable to parallel computation. The method is applied to 223 images of human retinas and cultured neurons, from four different sources, using a single segmentation algorithm with eight parameters. Improvements in segmentation quality compared to default settings using 1000 iterations ranged from 4.7%-21%. Paired t-tests showed that improvements are statistically significant (p < 0.0005). Most of the improvement occurred in the first 44 iterations. Improvements in description lengths and agreement with the ground truth were strongly correlated (p = 0.78).

Extracellular recordings from patterned neuronal networks using planar microelectrode arrays.

Neuronal cell networks have been reconstructed on planar microelectrode arrays (MEAs) from dissociated hippocampal pyramidal neurons. Microcontact printing (microCP) and a photoresist-liftoff method were used to selectively localize poly-L-lysine (PLL) on the surface of MEAs. Haptotaxis led to the organization of the neurons into networks localized adjacent to microelectrodes. Various grids of PLL with 2-25-microm-wide lines spaced by 50-200 microm with 15-25-microm nodes at intersection points were used to guide cell body attachment and neurite outgrowth. Bursting activity with spike amplitude attenuation was observed, and multichannel recordings detected instances of coincident firing activity. Finally, we present here an extracellular recording from a approximately 2 microm bundle of guided neurites.