Electric Field Modulation of Semiconductor Quantum Dot Photoluminescence: Insights Into the Design of Robust Voltage-Sensitive Cellular Imaging Probes.

The intrinsic properties of quantum dots (QDs) and the growing ability to interface them controllably with living cells has far-reaching potential applications in probing cellular processes such as membrane action potential. We demonstrate that an electric field typical of those found in neuronal membranes results in suppression of the QD photoluminescence (PL) and, for the first time, that QD PL is able to track the action potential profile of a firing neuron with millisecond time resolution. This effect is shown to be connected with electric-field-driven QD ionization and consequent QD PL quenching, in contradiction with conventional wisdom that suppression of the QD PL is attributable to the quantum confined Stark effect.

Material characterization and simulation for soft gels subjected to impulsive loading.

For impact and blast experiments of traumatic brain injury (TBI), soft gel materials are used as surrogates to imitate the mechanical responses of brain tissue. To properly model a viscoelastic gel brain in a surrogate head using a finite element (FE) model, material parameters such as the shear moduli and relaxation time at high strain rates are required. However, such information is scarce in the literature and its applicability for a range of dynamic conditions is unclear. We used an integrated experiment and simulation approach to efficiently determine mechanical properties of soft gels at finite strains, as well as over a wide range of strain rates. A novel impact experiment using a gel block was developed to capture the high strain rate behavior by maximizing the inherent shear wave motion at different impact conditions. A corresponding computational model was used to simulate the gel dynamics of the impact. Parametric simulations utilizing optimization and correlation analyses were used to calibrate multiple material parameters in the nonlinear viscoelastic model to the experimental data. The optimal parameters for gels, including Sylgards 184, 3-6636, and 527, were found. We ascertained the initial shear stiffening effect in gels at high strain rate loadings experimentally and incorporated this effect in the simulation. We have verified the integrated approach by comparing the material properties of the gels with analytical results based on shear wave propagation. This study provides a new approach to calibrate the material behavior of soft gels under high strain rate loading conditions.

Role of Interfacial Conditions on Blast Overpressure Propagation Into the Brain.

The complex interfacial condition between the human brain and the skull has been difficult to emulate in a surrogate system. Surrogate head models have typically been built using a homogeneous viscoelastic material to represent the brain, but the effect of different interfacial conditions between the brain and the skull on pressure transduction into the brain during blast has not been studied. In the present work, three interfacial conditions were generated in physical surrogate human head models. The first surrogate consisted of a gel brain separated from the skull by a layer of saline solution similar in thickness to the cerebrospinal fluid (CSF) layer in the human head: the fluid interface head model. The second surrogate head had the entire cranial cavity filled with the gel: the fixed interface head model. The third surrogate head contained a space-filling gel brain wrapped in a thin plastic film: the stick-slip interface head model. The human head surrogates were evaluated in a series of frontal blast tests to characterize the effect of skull-brain interfacial conditions on overpressure propagation into the gel brains. The fixed and the stick-slip interface head models showed nearly equal peak brain overpressures. In contrast, the fluid interface head model had much higher in-brain peak overpressures than the other two models, thus representing the largest transmission of forces into the gel brain. Given that the elevated peak overpressures occurred only in the fluid interface head model, the presence of the saline layer is likely responsible for this increase. This phenomenon is hypothesized to be attributed to the incompressibility of the saline and/or the impedance differences between the materials. The fixed interface head model showed pronounced high frequency energy content relative to the other two models, implying that the fluid and the stick-slip conditions provided better dampening. The cumulative impulse energy entering the three brain models were similar, suggesting that the interface conditions do not affect the total energy transmission over the positive phase duration of a blast event. This study shows that the fidelity of the surrogate human head models would improve with a CSF-emulating liquid layer.

Chemistries for Making Additive Nanolithography in OrmoComp Permissive for Cell Adhesion and Growth.

Two-photon lithography allows writing of arbitrary nanoarchitectures in photopolymers. This design flexibility opens almost limitless possibilities for biological studies, but the acrylate-based polymers frequently used do not allow for adhesion and growth of some types of cells. Indeed, we found that lithographically defined structures made from OrmoComp do not support E18 murine cortical neurons. We reacted OrmoComp structures with several diamines, thereby rendering the surfaces directly permissive for neuron attachment and growth by presenting a surface coating similar to the traditional cell biology coating achieved with poly-d-lysine (PDL) and laminin. However, in contrast to PDL-laminin coatings that cover the entire surface, the amine-terminated OrmoComp structures are orthogonally modified in deference to the surrounding glass or plastic substrate, adding yet another design element for advanced biological studies.

Methods for characterizing interspike intervals and identifying bursts in neuronal activity.

Neurons produce complex patterns of electrical spikes, which are often clustered in bursts. The patterns of spikes and bursts can change substantially when neurons are exposed to toxins and chemical agents. For that reason, characterization of these patterns is important for the development of neuron-based biosensors for environmental threat exposure. Here, we develop a quantitative approach to describe the distribution of interspike intervals, based on plotting histograms of the logarithm of the interspike interval. This approach provides a method for automatically classifying spikes into bursts, which does not depend on assumptions about the burst parameters. Furthermore, the approach provides a sensitive technique for detecting changes in spike and burst patterns induced by pharmacological exposure. Hence, it is suitable for use both as a research tool and for deployment in a neuron-based biosensor.

Characterization and detection of acceleration-induced cavitation in soft materials using a drop-tower-based integrated system.

The material response of biologically relevant soft materials, e.g., extracellular matrix or cell cytoplasm, at high rate loading conditions is becoming increasingly important for emerging medical implications including the potential of cavitation-induced brain injury or cavitation created by medical devices, whether intentional or not. However, accurately probing soft samples remains challenging due to their delicate nature, which often excludes the use of conventional techniques requiring direct contact with a sample-loading frame. We present a drop-tower-based method, integrated with a unique sample holder and a series of effective springs and dampers, for testing soft samples with an emphasis on high-rate loading conditions. Our theoretical studies on the transient dynamics of the system show that well-controlled impacts between a movable mass and sample holder can be used as a means to rapidly load soft samples. For demonstrating the integrated system, we experimentally quantify the critical acceleration that corresponds to the onset of cavitation nucleation for pure water and 7.5% gelatin samples. This study reveals that 7.5% gelatin has a significantly higher, approximately double, critical acceleration as compared to pure water. Finally, we have also demonstrated a non-optical method of detecting cavitation in soft materials by correlating cavitation collapse with structural resonance of the sample container.

Quantum Dot-Peptide-Fullerene Bioconjugates for Visualization of in Vitro and in Vivo Cellular Membrane Potential.

We report the development of a quantum dot (QD)-peptide-fullerene (C60) electron transfer (ET)-based nanobioconjugate for the visualization of membrane potential in living cells. The bioconjugate is composed of (1) a central QD electron donor, (2) a membrane-inserting peptidyl linker, and (3) a C60 electron acceptor. The photoexcited QD donor engages in ET with the C60 acceptor, resulting in quenching of QD photoluminescence (PL) that tracks positively with the number of C60 moieties arrayed around the QD. The nature of the QD-capping ligand also modulates the quenching efficiency; a neutral ligand coating facilitates greater QD quenching than a negatively charged carboxylated ligand. Steady-state photophysical characterization confirms an ET-driven process between the donor-acceptor pair. When introduced to cells, the amphiphilic QD-peptide-C60 bioconjugate labels the plasma membrane by insertion of the peptide-C60 portion into the hydrophobic bilayer, while the hydrophilic QD sits on the exofacial side of the membrane. Depolarization of cellular membrane potential augments the ET process, which is manifested as further quenching of QD PL. We demonstrate in HeLa cells, PC12 cells, and primary cortical neurons significant QD PL quenching (ΔF/F0 of 2-20% depending on the QD-C60 separation distance) in response to membrane depolarization with KCl. Further, we show the ability to use the QD-peptide-C60 probe in combination with conventional voltage-sensitive dyes (VSDs) for simultaneous two-channel imaging of membrane potential. In in vivo imaging of cortical electrical stimulation, the optical response of the optimal QD-peptide-C60 configuration exhibits temporal responsivity to electrical stimulation similar to that of VSDs. Notably, however, the QD-peptide-C60 construct displays 20- to 40-fold greater ΔF/F0 than VSDs. The tractable nature of the QD-peptide-C60 system offers the advantages of ease of assembly, large ΔF/F0, enhanced photostability, and high throughput without the need for complicated organic synthesis or genetic engineering, respectively, that is required of traditional VSDs and fluorescent protein constructs.

Azaspiracid-1 inhibits bioelectrical activity of spinal cord neuronal networks.

Azaspiracid-1 (AZA-1) is a recently identified phycotoxin that accumulates in molluscs and can cause severe human intoxications. For this study, we utilized murine spinal cord and frontal cortex neuronal networks grown over 64 channel microelectrode arrays (MEAs) to gain insights into the mechanism of action of AZA-1 on neuronal cells. Extracellular recordings of spontaneous action potentials were performed by monitoring mean spike rate as an assay of the efficacy of AZA-1 to alter the bioelectrical activity of neurons in the networks. Via slow onset, AZA-1 decreased the mean spike rate of the spinal cord neurons with an IC(50) of ca. 2.1nM, followed by partial recovery of original activity when toxin was removed. Pre-treatment with the GABA(A) receptor antagonist bicuculline led to an increased response of the neuronal networks to AZA-1 exposure and resulted in an irreversible inhibition of spike rate. AZA-1 did not cause any changes in frontal cortex networks upon drug exposure. In addition, whole-cell patch clamp recordings from spinal cord neurons showed that AZA-1 had no significant effect on the voltage-gated sodium (Na(+)) or calcium (Ca(2+)) currents, suggesting that the toxin affected synaptic transmission in the neuronal networks through a mechanism independent of these voltage-gated channels.

A portable microelectrode array recording system incorporating cultured neuronal networks for neurotoxin detection.

Cultured neuronal networks, which have the capacity to respond to a wide range of neuroactive compounds, have been suggested to be useful for both screening known analytes and unknown compounds for acute neuropharmacologic effects. Extracellular recording from cultured neuronal networks provides a means for extracting physiologically relevant activity, i.e. action potential firing, in a noninvasive manner conducive for long-term measurements. Previous work from our laboratory described prototype portable systems capable of high signal-to-noise extracellular recordings from cardiac myocytes. The present work describes a portable system tailored to monitoring neuronal extracellular potentials that readily incorporates standardized microelectrode arrays developed by and in use at the University of North Texas. This system utilizes low noise amplifier and filter boards, a two-stage thermal control system with integrated fluidics and a graphical user interface for data acquisition and control implemented on a personal computer. Wherever possible, off-the-shelf components have been utilized for system design and fabrication. During use with cultured neuronal networks, the system typically exhibits input referred noise levels of only 4-6 microVRMS, such that extracellular potentials exceeding 40 microV can be readily resolved. A flow rate of up to 1 ml/min was achieved while the cell recording chamber temperature was maintained within a range of 36-37 degrees C. To demonstrate the capability of this system to resolve small extracellular potentials, pharmacological experiments with cultured neuronal networks have been performed using ion channel blockers, tetrodotoxin and tityustoxin. The implications of the experiments for neurotoxin detection are discussed.

Acute neuropharmacologic action of chloroquine on cortical neurons in vitro.

Chloroquine, a common quinolone derivative used in the treatment of malaria, has been associated with neurologic side-effects including depression, psychosis and delirium. The neuropharmacologic effects of chloroquine were examined on cultured cortical neurons using microelectrode array (MEA) recording and the whole-cell patch clamp technique. Whole-cell patch clamp records under current-clamp mode also showed a chloroquine-induced depression of the firing rate of spontaneous action potentials by approximately 40%, consistent with the observations with the MEA recording, although no changes in either the baseline membrane potential or input resistance were observed. Voltage clamp recordings of spontaneous post-synaptic currents, recorded in the presence of tetrodotoxin, revealed no obvious changes in either the amplitude or rate of occurrence of inward currents with application of chloroquine at 10 microM, suggesting that the fundamental molecular mechanisms underlying spontaneous synaptic transmission may not be affected by acute application of the drug. In contrast, a concentration-dependent inhibition of whole-cell calcium current was observed in the presence of chloroquine. These acute neuropharmacologic changes were not accompanied by cytotoxic actions of the compound, even after exposure of up to 500 microM chloroquine for 7 h. These data suggest that chloroquine can depress in vitro neuronal activity, perhaps through inhibition of membrane calcium channels.

Neural stem cell differentiation in a cell-collagen-bioreactor culture system.

Neural stem cells and neural progenitors (NSCs/NPs) are capable of self-renewal and can give rise to both neurons and glia. Such cells have been isolated from the embryonic brain and immobilized in three dimensional collagen gels. The collagen-entrapped NSCs/NPs recapitulate CNS stem cell development and form functional synapses and neuronal circuits. However, the cell-collagen constructs from static conditions contain hypoxic, necrotic cores and the cells are short-lived. In the present study, NSCs/NPs isolated from embryonic day 13 rat cortical neuroepithelium are immobilized in type I collagen gels and cultured in NASA-designed rotating wall vessel (RWV) bioreactors for up to 9 weeks. Initially, during the first 2 weeks of culture, a lag phase of cellular growth and differentiation is observed in the RWV bioreactors. Accelerated growth and differentiation, with the cells beginning to form large aggregates (approximately 1 mm in diameter) without death cores, begins during the third week. The collagen-entrapped NSCs/NPs cultured in RWV show active neuronal generation followed by astrocyte production. After 6 weeks in rotary culture, the cell-collagen constructs contain over 10 fold greater nestin+ and GFAP+ cells and two-fold more TuJ1 gene expression than those found in static cultures. In addition, TuJ1+ neurons in RWV culture give rise to extensive neurite outgrowth and considerably more synapsin I+ pre-synaptic puncta surrounding MAP2+ cell bodies and dendrites. These results strongly suggest that the cell-collagen-bioreactor culture system supports long-term NSC/NP growth and differentiation, and RWV bioreactors can be useful in generating neural tissue like constructs, which may have the potential for cell replacement therapy.

Functional synapse formation among rat cortical neurons grown on three-dimensional collagen gels.

To investigate synaptic formation of neurons grown on three-dimensional (3D) collagen gels, neurons dissociated from embryonic rat cerebral cortices were seeded onto type-I collagen gels and cultured in serum-free medium for up to 2 weeks. Double-immunostaining for mitogen-activated protein-2 (MAP-2), a neuronal cell body and dendritic marker, and synapsin I, a synaptic vesicle antigen, was carried out to identify pre- and postsynaptic structural specializations, respectively. MAP-2(+) neuronal soma and dendrites were found to be surrounded by numerous puncta of synapsin I in a 1 week-old culture. Whole-cell patch clamp experiments demonstrated that the neurons grown on 3D gels exhibit sodium and potassium currents similar to those seen in 2D culture. Spontaneous action potentials were found in neurons that had been in culture for 8-12 days. In addition, spontaneous, bicuculline-sensitive gamma-aminobutyric acidergic postsynaptic currents were also present. This is the first demonstration of functional synapse formation among neurons grown on 3D collagen gels, suggesting that type-I collagen can be a promising material for neuronal regeneration.

Pharmacological effects of the marine toxins, brevetoxin and saxitoxin, on murine frontal cortex neuronal networks.

Brevetoxins and saxitoxins (STXs), which are produced by marine dinoflagellates, are very potent neurotoxins targeting separate sites of the alpha subunit of voltage-dependent sodium channels (VDSCs). An attractive approach for marine toxin detection relies on pharmacological modulation of VDSCs expressed in cells or tissues. While these function-based cellular assays exhibit the required sensitivity, they are typically slow and have limited potential use for field applications. Cultured neuronal networks grown on substrate integrated microelectrode arrays (MEAs) have emerged as a robust and sensitive approach for environmental threat detection. The present work describes the rapid effects of brevetoxin-2 (PbTx-2) and STX on embryonic murine frontal cortex neuronal networks on MEAs. Network recording parameters such as mean spike rate, burst rate, burst duration, number of spikes per burst and spike amplitude were analyzed before and after exposure to the toxins. STX produced fast and reversible inhibition of all electrophysiological parameters with IC(50)s ranging between 1.2 and 2.2nM. Although PbTx-2 also caused inhibition of most of the network electrophysiological parameters, it produced an increase in burst duration at lower concentrations (EC(50)=15+/-2 nM, n=4) followed by inhibition at higher ones (IC(50)=63+/-4 nM, n=4). Exposure of frontal cortex networks to PbTx-2 and STX also caused differential effects on spike amplitude. This work demonstrates that cultured neuronal networks not only could be used for pharmacological characterization of marine toxins but they also provide a tool with unique properties for their detection.

Toluene inhibits muscarinic receptor-mediated cytosolic Ca2+ responses in neural precursor cells.

Toluene is widely used as a component in industrial solvents and many toluene-containing products are abused via inhalation. While many studies have demonstrated its inhibitory effects on neuronal activity, the effects of toluene on receptor signaling in proliferating and differentiating neural precursor cells are presently unclear. Here, using digital video microscopy and Ca2+ imaging, we investigated the effects of acute exposure to toluene on the function of muscarinic acetylcholine receptors (mAChRs) expressed in neural precursor cells. The neural precursor cells were isolatedfrom embryonic day 13 (E13) rat cortex and expanded in serum-free medium containing basic fibroblast growth factor (bFGF). We found that the acetylcholine (ACh) analog carbachol (CCh) induced a dose-dependent increase in cytosolic Ca2+, which was blocked by the muscarinic receptor antagonist atropine in a reversible manner. Toluene was added to the perfusion medium and concentrations of toluene in the medium were determined by gas chromatographic analysis. Following imaging, the cells were fixed and processed for 5-bromo-2'-deoxyuridine (BrdU, cell proliferation marker) and beta-tubulin (TuJ1, neuronal marker) immunostaining. In the 5 day culture, most cells continued to divide (BrdU+), while afew cells differentiated into young neurons (TuJ1-). The CCh-induced Ca2+ elevations in proliferating (BrdU+TuJ1-) neural precursor cells were significantly reduced by acute exposure to 0.15 mM toluene and completely blocked by 10 mM toluene. Toluene's inhibition of muscarinic receptor-mediated Ca2+ signaling was rapid, reversible and dose-dependent with an IC50 value 0.5 mM. Since muscarinic receptors mediate cell proliferation and differentiation during neural precursor cell development, these results suggest that depression of muscarinic signaling may play a role in toluene's teratogenic effect on the developing nervous system.

Sensitivity of the neuronal network biosensor to environmental threats.

It is widely acknowledged that there is a critical need for broad-spectrum environmental threat detection. While cells/tissue-based biosensors have been discussed for many years as a means of meeting this critical need, these kinds of systems have met with logistic concerns, in particular with regard to stability. Our group has been working with cultured neuronal networks, which have the capacity to respond to a wide range of neuroactive compounds and are sufficiently robust to be shipped to end users. The basis of operation involves extracellular recording using thin-film microelectrode arrays where spontaneous bioelectrical activity, that is, spike firing, can be monitored in a noninvasive manner conducive for potentially long-term measurements. This work describes the current status of our efforts for the fabrication of a portable biosensor that incorporates cultured neuronal networks grown over standardized microelectrode arrays. Based on our protocol for aqueous phase sample introduction under constant flow conditions, minimal variation in mean spike rate is observed, consistent with temporal stability, such that changes of > 10% are readily distinguished. To demonstrate the capability of this system, changes are reported in mean spike rate and network synchronization resulting from exposure to different model environmental threats, cadmium and strychnine. The sensitivity of this assay approach and implications of the experimental findings for environmental threat detection are discussed.

Differential responses to ω-agatoxin IVA in murine frontal cortex and spinal cord derived neuronal networks.

ω-Agatoxin-IVA is a well known P/Q-type Ca(2+) channel blocker and has been shown to affect presynaptic Ca(2+) currents as well postsynaptic potentials. P/Q-type voltage gated Ca(2+) channels play a vital role in presynaptic neurotransmitter release and thus play a role in action potential generation. Monitoring spontaneous activity of neuronal networks on microelectrode arrays (MEAs) provides an important tool for examining this neurotoxin. Changes in extracellular action potentials are readily observed and are dependent on synaptic function. Given the efficacy of murine frontal cortex and spinal cord networks to detect neuroactive substances, we investigated the effects of ω-agatoxin on spontaneous action potential firing within these networks. We found that networks derived from spinal cord are more sensitive to the toxin than those from frontal cortex; a concentration of only 10nM produced statistically significant effects on activity from spinal cord networks whereas 50 nM was required to alter activity in frontal cortex networks. Furthermore, the effects of the toxin on frontal cortex are more complex as unit specific responses were observed. These manifested as either a decrease or increase in action potential firing rate which could be statistically separated as unique clusters. Administration of bicuculline, a GABAA inhibitor, isolated a single response to ω-agatoxin, which was characterized by a reduction in network activity. These data support the notion that the two clusters detected with ω-agatoxin exposure represent differential responses from excitatory and inhibitory neuronal populations.

Function and regulation of Vibrio campbellii proteorhodopsin: acquired phototrophy in a classical organoheterotroph.

Proteorhodopsins (PRs) are retinal-binding photoproteins that mediate light-driven proton translocation across prokaryotic cell membranes. Despite their abundance, wide distribution and contribution to the bioenergy budget of the marine photic zone, an understanding of PR function and physiological significance in situ has been hampered as the vast majority of PRs studied to date are from unculturable bacteria or culturable species that lack the tools for genetic manipulation. In this study, we describe the presence and function of a horizontally acquired PR and retinal biosynthesis gene cluster in the culturable and genetically tractable bioluminescent marine bacterium Vibrio campbellii. Pigmentation analysis, absorption spectroscopy and photoinduction assays using a heterologous over-expression system established the V. campbellii PR as a functional green light absorbing proton pump. In situ analyses comparing PR expression and function in wild type (WT) V. campbellii with an isogenic ΔpR deletion mutant revealed a marked absence of PR membrane localization, pigmentation and light-induced proton pumping in the ΔpR mutant. Comparative photoinduction assays demonstrated the distinct upregulation of pR expression in the presence of light and PR-mediated photophosphorylation in WT cells that resulted in the enhancement of cellular survival during respiratory stress. In addition, we demonstrate that the master regulator of adaptive stress response and stationary phase, RpoS1, positively regulates pR expression and PR holoprotein pigmentation. Taken together, the results demonstrate facultative phototrophy in a classical marine organoheterotrophic Vibrio species and provide a salient example of how this organism has exploited lateral gene transfer to further its adaptation to the photic zone.

Passaged neural stem cell-derived neuronal networks for a portable biosensor.

We have previously demonstrated a portable biosensor that utilizes networks of mammalian neurons on microelectrode arrays (MEAs) as the sensing element. These neuronal cultures on MEAs are derived from primary neuronal tissues and are short-lived. In order to extend the shelf life of neuronal networks for use in a fieldable sensor technology, a renewable source of networks is needed. Neural stem and progenitor cells are capable of self-renewal and differentiation into functional neuronal networks. The purpose of this study was to develop a strategy for growing passaged neural stem and progenitor cells on MEAs under controlled conditions to produce differentiated neurons and glia comprising functional neuronal networks. Primary and passaged neuroepithelial stem and progenitor cells dissociated from embryonic day 13 rat cortex were seeded on MEAs and maintained with serum-free medium containing basic fibroblast growth factor (bFGF) combined with brain-derived neurotrophic factor (BDNF). These culture conditions lead to abundant neurons, with astrocytes as supportive cells, forming synaptically linked networks of neurons. Spontaneous action potentials were best recorded from networks derived from primary or passaged progenitor cells 4-5 weeks after initial culture. The passaged progenitor cell-derived networks on MEAs responded to the GABA(A) antagonist bicuculline, the NMDA glutamate inhibitor APV, and the non-NMDA glutamate antagonist CNQX indicating active synapses were present. Passaged neural stem and progenitor cell-derived networks on MEAs have properties similar to networks derived from primary neuronal cultures and can serve as a renewable supply of sensor elements for detection of environmental threats.

Reconstruction of functional cortical-like tissues from neural stem and progenitor cells.

Neural stem and progenitor cells isolated from embryonic day 13 rat cerebral cortex were immobilized in three-dimensional type I collagen gels, and then the cell-collagen constructs were transferred to rotary wall vessel bioreactors and cultured in serum-free medium containing basic fibroblast growth factor (bFGF) combined with brain-derived neurotrophic factor for up to 10 weeks. Remarkably, the collagen-entrapped cells formed a complex two-layered structure that emulated to a certain extent the cerebral cortex of the embryonic brain in architecture and functionality. The surface layer (layer I) composed primarily of proliferating neural progenitor cells (nestin(+), vimentin(+), and PCNA(+)) predominantly expressed functional neurotransmitter receptors for cholinergic and purinergic agonists while differentiating cells (TuJ1(+) and GFAP(+)) in the deeper layer (layer II) contained differentiated neurons and astrocytes and mainly responded to GABAergic and glutamatergic agonists and to veratridine, which activates voltage-dependent Na(+) channels. An active synaptic vesicle recycling was demonstrated by neuronal networks in the deeper layer using the endocytotic marker FM1-43. Cell polarization forming the characteristic two-layered structure was found to associate with the bFGF and FGF receptor signaling. These engineered functional tissue constructs have a potential use as tissue surrogates for drug screening and detection of environmental toxins, and in neural cell replacement therapy.

Laser ablation of micropores for formation of artificial planar lipid bilayers.

Artificial lipid bilayers are a powerful tool for studying synthetic or reconstituted ion channels. Key to forming these lipid bilayers is having a small aperture in a septum separating two solution chambers. Traditional methods of aperture generation involve manually punching the aperture into the septum. While these techniques work, they are difficult to implement reliably and do not produce consistently sized apertures. Presented here is a method of using a UV excimer laser with a nanosecond scale pulse width to laser ablate apertures from 4 to 105 microm in 20 microm thick polycarbonate films for use in artificial lipid bilayer experiments. The data demonstrate that the apertures produced by laser ablation are highly reproducible and can support both the formation of stable, long-lasting lipid bilayers as well as the recording of ion channels incorporated into the bilayers.