Brain-computer interface devices for patients with paralysis and amputation: a meeting report.

OBJECTIVE: The Food and Drug Administration's (FDA) Center for Devices and Radiological Health (CDRH) believes it is important to help stakeholders (e.g., manufacturers, health-care professionals, patients, patient advocates, academia, and other government agencies) navigate the regulatory landscape for medical devices. For innovative devices involving brain-computer interfaces, this is particularly important. APPROACH: Towards this goal, on 21 November, 2014, CDRH held an open public workshop on its White Oak, MD campus with the aim of fostering an open discussion on the scientific and clinical considerations associated with the development of brain-computer interface (BCI) devices, defined for the purposes of this workshop as neuroprostheses that interface with the central or peripheral nervous system to restore lost motor or sensory capabilities. MAIN RESULTS: This paper summarizes the presentations and discussions from that workshop. SIGNIFICANCE: CDRH plans to use this information to develop regulatory considerations that will promote innovation while maintaining appropriate patient protections. FDA plans to build on advances in regulatory science and input provided in this workshop to develop guidance that provides recommendations for premarket submissions for BCI devices. These proceedings will be a resource for the BCI community during the development of medical devices for consumers.

Microfluidic based contactless dielectrophoretic device: Modeling and analysis.

While there have been many attempts at patterning cells onto substrates, a reliable method for trapping cell clusters and forming cell arrays in a predefined geometry remains to be demonstrated. We intend to develop a multielectrode array platform to initially trap cells via dielectrophoresis (DEP) and to later measure their electrical activity. As a first step toward that objective, here we present an interdigitated microfabricated comb structure. We designed an optimal insulation layer via finite element modeling for maximum dielectrophoretic field strength in solution and minimal cell damage. The microfabricated structure was combined with a microfluidic channel to vertically constrain cell position. With the objective of capturing cells onto the substrate, we here show that there is an optimal thickness of dielectric which limits electrolysis in solution and still allows for sufficient dielectrophoretic force on the cells to pull them onto the surface.

Acute exposure of toluene transiently potentiates p42/44 mitogen-activated protein kinase (MAPK) activity in cultured rat cortical astrocytes.

It has been shown that the inhalation of toluene in rats can cause neuronal apoptosis in the central nervous system. However, the cellular and molecular effects of toluene directly on astrocytes are relatively unknown. We used primary cultures of astrocytes isolated from the neonatal rat cortex as a model to study the toluene effects on cell outcome and associated signal transduction pathways using immunostaining and Western blotting. We observed that acute toluene exposure significantly induced caspase-dependent cell apoptosis and transiently stimulated the activation of p42/44 mitogen-activated protein kinase (MAPK) in the primary astrocytes. Interestingly, the inhibition of the p42/44 MAPK signaling cascade by PD98059 in conjunction with the toluene treatment evoked more cellular apoptosis than toluene alone, suggesting that the toluene-induced transient MAPK activation may play a role in promoting cell survival during the toluene exposure.

Gene modulation in total brain induced by exposure to the bicyclic phosphorus ester trimethylolpropane phosphate (TMPP).

The effect of a single subconvulsive dose of the GABAergic convulsant trimethylolpropane phosphate (TMPP) on gene expression in total rat brain was examined using cDNA array analysis. Using threshold criteria that reduce the number of false positives to <1 gene per 3551 actively transcribed genes on the cDNA array, 41 genes/EST sequences were reproducibly modulated in response to 0.25 mg/kg TMPP. Several genes that were consistent with epileptogenesis and/or neuronal damage and repair mechanisms, such as trkB, alphaB-crystallin, and decorin, were modulated by TMPP exposure in the absence of clinical convulsions. Previous research indicates that rats exposed to subconvulsive doses of TMPP exhibit both "absence-like" EEG paroxysms and persisting central nervous system (CNS) sensitization, as evidenced by increased susceptibility to audiogenic seizures (AGS). Results of this study suggest that cDNA arrays can be used to identify gene modulation events induced by low-level exposure to a chemical convulsant in a reproducible manner.

Quantitative assessment of filter-based cDNA microarrays: gene expression profiles of human T-lymphoma cell lines.

MOTIVATION: While the use of cDNA microarrays for functional genomic analysis has become commonplace, relatively little attention has been placed on false positives, i.e. the likelihood that a change in measured radioactive or fluorescence intensity may reflect a change in gene expression when, in fact, there is none. Since cDNA arrays are being increasingly used to rapidly distinguish biomarkers for disease detection and subsequent assay development (Wellman et al., Blood, 96, 398-404, 2000), the impact of false positives can be significant. For the use of this technology, it is necessary to develop quantitative criteria for reduction of false positives with radioactively-labeled cDNA arrays. RESULTS: We used a single source of RNA (HuT78 T lymphoma cells) to eliminate sample variation and quantitatively examined intensity ratios using radioactively labeled cDNA microarrays. Variation in intensity ratios was reduced by processing microarrays in side-by-side (parallel mode) rather than by using the same microarray for two hybridizations (sequential mode). Based on statistical independence, calculation of the expected number of false positives as a function of threshold showed that a detection limit of [log(2)R] >0.65 with agreement from three replicates could be used to identify up- or down-modulated genes. Using this quantitative criteria, gene expression differences between two related T lymphoma cell lines, HuT78 and H9, were identified. The relevance of these findings to the known functional differences between these cell types is discussed.

The use of GABA(A) receptors expressed in neural precursor cells for cell-based assays.

GABA(A) receptors are known targets for certain classes of environmental neurotoxins and pharmaceutical compounds. Since few neural cell lines express functional GABA(A) receptors, the capacity to rapidly screen for compounds that affect GABA(A) receptor function is presently limited. Previous work has demonstrated that rat neural precursor cells express functional GABA(A) receptors that can be monitored via Ca(2+) imaging. This study examined GABA(A) receptor subunit expression to determine whether GABA(A) receptor function and its interactions with neurotoxins is preserved after passaging. Neural precursor cells isolated from embryonic day 13 rat brain were expanded in serum-free medium containing basic fibroblast growth factor and passaged three times. Reverse transcription-polymerase chain reaction analysis demonstrated early expression of abundant mRNAs encoding various GABA(A) receptor subunits. Ca(2+) imaging showed that the highly proliferating precursor cells in passaged cultures maintained expression of functional GABA(A) receptors. In addition, we showed that trimethylolpropane phosphate, a neurotoxin generated during partial pyrolysis of a synthetic ester turbine engine lubricant, potently inhibited muscimol (GABA(A) receptor agonist) but not depolarization-induced cytosolic Ca(2+) increase. The findings of this study suggest that neural precursor cells may be well suited for the evaluation of certain environmental neurotoxins with convulsant activity. The potential use of neural precursor cells in high-throughput screens for compounds acting on GABA(A) receptors is discussed.

Methods for short time series analysis of cell-based biosensor data.

This paper describes two approaches for sensing changes in spiking cells when only a limited amount of spike data is available, i.e., dynamically constructed local expansion rates and spike area distributions. The two methods were tested on time series from cultured neuron cells that exhibit spiking both autonomously and in the presence of periodic stimulation. Our tested hypothesis was that minute concentrations of toxins could affect the local statistics of the dynamics. Short data sets having relatively few spikes were generated from experiments on cells before and after being treated with a small concentration of channel blocker. In spontaneous spiking cells, local expansion rates show a sensitivity that correlates with channel concentration level, while stimulated cells show no such correlation. Spike area distributions on the other hand showed measurable differences between control and treated conditions for both types of spiking, and a much higher degree of sensitivity. Because these methods are based on analysis of short time series analysis, they might provide novel means for cell drug and toxin detection.

Characterization of acute neurotoxic effects of trimethylolpropane phosphate via neuronal network biosensors.

We have utilized cultured neuronal networks grown on microelectrode arrays to demonstrate rapid, reliable detection of a toxic compound, trimethylolpropane phosphate (TMPP). Initial experiments, which were performed blind, demonstrated rapid classification of the compound as a convulsant, a finding consistent with previous whole animal neurobehavioral studies. TMPP (2-200 microM) reorganized network spike activity into synchronous, quasi-periodic burst episodes. Integrated burst amplitudes invariably increased, reflecting higher spike frequencies within each burst. The variability of network burst parameters, quantified as coefficients of variation (CVs), was decreased. Mean CVs for burst duration, interburst interval, and burst rate were lowered by 42+/-13, 58+/-5.5, and 62+/-1.8%, respectively (mean+/-SEM, n=8 cultures, 197 channels). These changes in network activity paralleled the effects induced by bicuculline, a known disinhibitory and seizure-inducing drug, and confirmed classification of TMPP as a potential epileptogenic compound. Simple pharmacological tests permit exploration of mechanisms underlying observed activity shifts. The EC(50) for GABA inhibition of network activity was increased from 2.8 to 7.0 microM by 20 microM TMPP and to 20.5 microM by 200 microM TMPP. Parallel dose-response curves suggest that TMPP acts by a competitive antagonism of GABA inhibition, and are consistent with reported patch-clamp analysis of TMPP-induced reduction of inhibitory postsynaptic current amplitudes. The potency of TMPP in generating epileptiform activity in vitro was comparable to concentrations reported for in vivo studies. TMPP and bicuculline produced both increases and decreases in burst rate depending on native spontaneous bursting levels. These results demonstrate a need for multivariate analysis of network activity changes to yield accurate predictions of compound effects.

Design and demonstration of an automated cell-based biosensor.

Cell-based biosensors have the capacity to respond to a wide range of analytes in a physiologically relevant manner and appear well-suited for toxicity monitoring of both known and unknown analytes. One means of acquiring cellular functional information for biosensor applications involves extracellular recording from excitable cells, which can generate noninvasive and long-term measurements. Previous work from our laboratory described a prototype portable system capable of high signal-to-noise extracellular recordings, in spite of deficiencies in thermal control, fluidics handling, and absence of data acquisition (DAQ) capability. The present work describes a cell-based biosensor system that incorporates low noise amplifier and filter boards, a two-stage thermal control system with integrated fluidics and a flexible graphical user interface for DAQ and control implemented on a personal computer. Wherever possible, commercial off-the-shelf components have been utilized for system design and fabrication. The system exhibits input-referred noise levels of 5-10 microV(RMS), such that extracellular potentials exceeding 50-60 microV can be readily resolved. In addition, the biosensor system is capable of automated temperature and fluidics control. Flow rates can range from 0-2.5 ml/min, while the cell recording chamber temperature is maintained within a range of 36-37 degrees C. To demonstrate the capability of this system to resolve small extracellular potentials, recordings from embryonic chick cardiac myocytes have been performed.

Identification of differential gene expression profiles in rat cortical cells exposed to the neuroactive agents trimethylolpropane phosphate and bicuculline.

Recent technological advancements in microfabrication combined with the rapid acquisition of full genome sequence data have led to the development of DNA arrays that have the capacity to monitor the expression levels of thousands of genes simultaneously. The development of this technology enables the use of functional genomics approaches to identify molecular markers associated with cellular responsiveness to cytotoxic exposures. Databases containing unique cell-response profiles associated with specific toxicants or classes of toxicants can then be used in conjunction with cell-based biosensor platforms for environmental surveillance and toxicological assessment. An important issue that must be addressed, however, is whether DNA arrays can be used to identify transient gene modulation events in a reproducible manner. To address this issue, we utilized a primary embryonic rat (day 18) cortical cell model system and examined the RNA of both chemically treated and untreated cells using radioisotope-labeled cDNA probes and commercially available nylon membrane arrays. Using this approach, we examined experimental variability, basal gene expression variability, the occurrence of false positives, and the reproducibility of gene expression profiles obtained after chemical exposure. Minimal differences in gene modulation were observed between RNA samples from independently cultured cortical cells when array experiments were conducted in parallel (Pearson correlation coefficient for gene intensities =0.98). In contrast, significant differences in gene expression were observed between array experiments conducted at different times with an identical RNA source (Pearson correlation coefficient for gene intensities=0.91). Our results suggest the effect of basal gene activity differences in independently isolated cell cultures is negligible and that experimental variability possibly associated with the handling of RNA samples, differences in reverse transcription efficiency, hybridization, and/or signal acquisition are the primary contributors to variability in measurements. Using cDNA array analysis of unexposed cells from three independent cell culture preparations, we calculated false positive gene modulation events as a function of the threshold absolute value of log(2) >1.0. The number of false positives using this criteria was 1-10 gene/ESTs/5109 actively transcribed gene/ESTs represented on the array. Using three independent replicate experiments of untreated cortical cell cultures, we determined that a threshold criterion of absolute value of log(2) >0.63 for triplicate experiments would reduce the expected number of false positives in our experiments to less than one. Using this criterion, reproducible gene expression profiles were identified in cortical cells exposed to the neuroactive agents trimethylolpropane phosphate and bicuculline.

Ethanol blocks cytosolic Ca2+ responses triggered by activation of GABA(A) receptor/Cl- channels in cultured proliferating rat neuroepithelial cells.

GABA(A) receptor/Cl- channels and voltage-gated Ca2+ channels are believed to be important sites of ethanol action in the CNS. Acute exposure of ethanol potentiates GABA(A) receptor/Cl- channel activity and inhibits voltage-gated Ca2+ channels in a number of preparations, mostly post-mitotic neurons. The effects of ethanol on these channels in primary cultures of undifferentiated neural precursor cells remain unknown. To address this issue, we examined the effects of ethanol on GABA(A) agonist-activated elevation of cytosolic Ca2+ in an in vitro model of the cortical neuroepithelium derived from rat basic fibroblast growth factor-expanded neural precursor cells. We found a potent inhibition of GABA(A)-activated elevation of cytosolic Ca2+ by ethanol in actively proliferating cells. Since we had recently demonstrated that GABA(A) receptor activation depolarizes these cells and elevates their cytosolic Ca2+, we tested whether the effects of ethanol involved both GABA(A) receptors and voltage-gated Ca2+ channels. Both extracellular K+- and muscimol-induced cytosolic Ca2+ elevations were abolished by nitrendipine, indicating that both depolarizing stimuli triggered Ca2+ influx through L-type voltage-gated Ca2+ channels. Exposure of proliferating cells to different concentrations of ethanol revealed that the drug was more potent in blocking muscimol-induced compared to K+-evoked cytosolic Ca2+ elevations. These results raise the possibility that ethanol blocks GABAergic stimulation of cytosolic Ca2+ levels in proliferating precursors primarily by interacting with GABA(A) receptor/Cl- channels and secondarily with voltage-gated Ca2+ channels.

Detection of physiologically active compounds using cell-based biosensors.

Cell-based biosensors are portable devices that contain living biological cells that monitor physiological changes induced by exposure to environmental perturbations such as toxicants, pathogens or other agents. Methods of detecting physiological changes include extracellular electrical recordings, optical measurements, and, in the future, functional genomics and proteomics. Several technical developments are occurring that will increase the feasibility of cell-based biosensors for field applications; these developments include stem cell and 3D culture technologies. Possible scenarios for the use of cell-based biosensors include broad-range detectors of unknown threat agents and functional assessment of identified agents.

Neurophysiologic effects of chemical agent hydrolysis products on cortical neurons in vitro.

The neurophysiologic effects of chemical agent hydrolysis products were examined on cultured cortical neurons using multielectrode array (MEA) recording and the whole-cell patch clamp technique. Measurement of neuronal network extracellular potentials showed that the primary hydrolysis product of soman, pinacolyl methylphosphonic acid (PMPA), inhibited network mean burst and spike rates with an EC50 of approximately 2 mM. In contrast, the degradation product of sarin, isopropyl methylphosphonic acid (IMPA), and the final common hydrolysis product of both soman and sarin, methylphosphonic acid (MPA), failed to affect neuronal network behavior at concentrations reaching 5 mM. Closer examination of the effects of PMPA (2 mM) on discriminated extracellular units revealed that mean spike amplitude was slightly diminished to 95 +/- 1% (mean +/- S.E.M., n = 6, P < 0.01) of control. Whole-cell patch clamp records under current clamp mode also showed a PMPA-induced depression of the firing rate of spontaneous action potentials (APs) to 36 +/- 6% (n = 5, P < 0.001) of control. In addition, a minor depression with exposure to PMPA was observed in spontaneous and evoked AP amplitude to 93 +/- 3% (n = 5, P < 0.05) of control with no change in either the baseline membrane potential or input resistance. Preliminary voltage clamp recordings indicated a reduction in the occurrence of spontaneous inward currents with application of PMPA. These findings suggest that PMPA, unlike MPA or IMPA, may more readily interfere with one or more aspects of excitatory synaptic transmission. Furthermore, the data demonstrate that the combination of extracellular microelectrode array and patch clamp recording techniques facilitates analysis of compounds with neuropharmacologic effects.

Trimethylolpropane phosphate induces epileptiform discharges in the CA1 region of the rat hippocampus.

The actions of trimethylolpropane phosphate (TMPP), an ethyl bicyclophosphate convulsant produced during the partial pyrolysis of some phosphate ester-based lubricants, were tested on CA1 neurons of rat hippocampal slices using intracellular recording techniques. Bath application of TMPP (0.1-100 microM) induced spontaneous paroxysmal depolarizing shifts and the associated spontaneous epileptiform bursts followed by after-hyperpolarizations in 63% of neurons tested. The TMPP-induced epileptiform bursts were blocked by muscimol, a gamma-aminobutyric acid A (GABA(A)) receptor agonist, diazepam (DZP), a GABA(A)-benzodiazepine ionophore complex agonist, or baclofen, a GABA(B) receptor agonist. While bath application of muscimol, DZP, or baclofen suppressed spontaneous activity in CA1 neurons not previously exposed to TMPP, subsequent application of TMPP (10 microM) reversed the actions of muscimol and diazepam, but not baclofen. TMPP (0.1-100 microM) also induced membrane hyperpolarization associated with an increase in peak input resistance and inward rectification in 33% of neurons tested or membrane depolarization associated with an increase in input resistance in 17% of neurons tested. In summary, TMPP induced epileptiform activities in hippocampal CA1 neurons. The epileptogenic effects of TMPP are consistent with its interaction with GABA(A)-benzodiazepine receptors.

Identification of target genes responsive to JP-8 exposure in the rat central nervous system.

Concern for the health risk associated with occupational exposure to jet fuel has emerged in the Department of Defense. Jet propulsion fuel-8 (JP-8) is the fuel used in most US and North Atlantic Treaty Organization (NATO) jet aircraft, and will be the predominant fuel both for military land vehicles and aircraft into the twenty-first century. JP-8 exhibits reduced volatility and lower benzene content as compared to JP-4, the predominant military aircraft fuel before 1992, possibly suggesting greater occupational exposure safety. However, the higher rates of occupational exposure through fueling and maintenance of increasingly larger numbers of aircraft/vehicles raise concerns with respect to toxicity. Clinical studies of workers experiencing long-term exposure to certain jet fuels demonstrated deficits in CNS function, including fatigue, neurobehavioral changes, psychiatric disorders, and abnormal electroencephalogram (EEG). In the present study, cDNA nylon arrays (Atlas Rat 1.2 Array, Clontech Laboratories, Palo Alto, CA) were utilized to measure changes in gene expression in whole brain tissue of rats exposed repeatedly to JP-8, under conditions that simulated possible real-world occupational exposure (6 h/day for 91 days) to JP-8 vapor at 1,000 mg/m3. Gene expression analysis of the exposure group compared to the control group revealed a modulation of several genes, including glutathione S-transferase Yb2 subunit (GST Yb2); cytochrome P450 IIIAl (CYP3A1); glucose-dependent insulinotropic peptide (GIP); alpha1-proteinase inhibitor (alpha1-AT); polyubiquitin; GABA transporter 3 (GAT-3); and plasma membrane Ca2+-transporting ATPase (brain isoform 2) (PMCA2). The implications of these vapor-induced changes in gene expression are discussed.

Stimulation of unitary T-type Ca(2+) channel currents by calmodulin-dependent protein kinase II.

The effect of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) stimulation on unitary low voltage-activated (LVA) T-type Ca(2+) channel currents in isolated bovine adrenal glomerulosa (AG) cells was measured using the patch-clamp technique. In cell-attached and inside-out patches, LVA channel activity was identified by voltage-dependent inactivation and a single-channel conductance of approximately 9 pS in 110 mM BaCl(2) or CaCl(2). In the cell-attached patch, elevation of bath Ca(2+) from 150 nM to 1 microM raised intracellular Ca(2+) in K(+)-depolarized (140 mM) cells and evoked an increase in the LVA Ca(2+) channel probability of opening (NP(o)) by two- to sixfold. This augmentation was associated with an increase in the number of nonblank sweeps, a rise in the frequency of channel opening in nonblank sweeps, and a 30% reduction in first latency. No apparent changes in the single-channel open-time distribution, burst lengths, or openings/burst were apparent. Preincubation of AG cells with lipophilic or peptide inhibitors of CaMKII in the cell-attached or excised (inside-out) configurations prevented the rise in NP(o) elicited by elevated Ca(2+) concentration. Furthermore, administration of a mutant recombinant CaMKIIalpha exhibiting cofactor-independent activity in the absence of elevated Ca(2+) produced a threefold elevation in LVA channel NP(o). These data indicate that CaMKII activity is both necessary and sufficient for LVA channel activation by Ca(2+).

Immobilization of neural cells in three-dimensional matrices for biosensor applications.

To overcome logistical difficulties with current designs of cell- or tissue-based biosensors which have individual cells or tissue slices immobilized on membranes or microelectrode arrays, we have proposed a system that uses three-dimensional cultures of neural cells immobilized in hydrogel matrices. In this design, immobilized cells would be maintained in a reservoir and then transferred to a detector platform when needed for analysis. The development of such a system relies upon a renewable supply of cells and the ability to culture cells for long periods of time in three-dimensions while maintaining their physiological function. To investigate the ability to culture neural cells in 3D matrices, embryonic rat cortical neurons and astrocytes were immobilized by matrix entrapment in a novel sugar poly(acrylate) hydrogel and collagen gels. The sugar poly(acrylate) hydrogel does not appear to support neural cell growth as a result of a lack of cell adherence, small pore size and, possibly, harshness of synthesis conditions. In contrast, collagen gels support the growth of cortical neurons, astrocytes, as well as neural progenitor cells. Evidence is also presented from immunocytochemistry and patch-clamp measurements which shows that neural progenitor cells proliferate in culture and can be induced to differentiate into neural cell types. Thus, they potentially represent a renewable cell source.

Synaptic connectivity in hippocampal neuronal networks cultured on micropatterned surfaces.

Embryonic rat hippocampal neurons were grown on patterned silane surface in order to organize synapse formations in a controlled manner. The surface patterns were composed of trimethoxysilylpropyl-diethylenetriamine (DETA) lines separated by tridecafluoro-1,1,2,2-tetrahydrooctyl-1-dimethylchlorosilane (13F) spaces. Pre- and post-synaptic specializations were identified by immunostaining for synapsin I and microtubule-associated protein-2 (MAP-2). Functional synaptic connections were examined by recording simultaneously from pairs of neurons using the whole-cell configuration of the patch-clamp technique. Spontaneous and evoked synaptic currents were recorded in neurons cultured for 2-14 days. The formation of functional connections was accompanied by the appearance of spontaneous synaptic currents (SSCs), which could be detected after approximately 3 days in culture in the absence of evoked synaptic currents (ESCs). ESCs were detected only after approximately 7 days in culture, mostly in the form of unidirectional synaptic connections. Other forms of synaptic connectivity, such as bidirectional and autaptic connections, were also identified. Both transient GABAergic and glutamatergic signals mediated the transmissions between communicating cells. These results demonstrate the combination of various types of synaptic connections forming simple and complex networks in neurons cultured on line (DETA)-space (13F) patterns. Finally, precisely synchronized SSCs were recorded in neuron pairs cultured on pattern indicating the existence of a fast-acting feedback mechanism mediated by pre-synaptic GABA(A) receptors.

Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

Investigation of in vitro toxicity of jet fuels JP-8 and Jet A.

The in vitro cytotoxicity and electrophysiological toxicity of Jet Propulsion-8 (JP-8 jet fuel) on four cell types: H4IIE liver cell line, NIH Swiss 3T3 cell line, neuroblastoma x glioma NG108-15 cells, and embryonic hippocampal neurons were investigated. H4IIE cells exposed to Jet A (a commercial fuel) and JP-8 demonstrated identical toxicity with an IC50 of 12.6 +/- 0.4 micrograms/ml for the two fuels. Comparison of H4IIE and NIH/3T3 toxicity to JP-8 revealed that NIH/3T3 cells were more sensitive to JP-8 than H4IIE cells, with an IC50 8.5 +/- 0.1 micrograms/ml. JP-8 exposure for the hippocampal neurons proved to be highly toxic (IC50 of < 2 micrograms/ml), while in contrast, the NG108-15 cells were much less sensitive. Electrophysiological examination of NG108-15 cells showed that administration of JP-8 at 1 microgram/ml did not alter significantly any of the electrophysiological properties. However, exposure to JP-8 at 10 micrograms/ml during a current stimulus of +46 pA decreased the amplitude of the action potential to 83 +/- 7% (n = 4), the rate of rise, dV/dtMAX to 50 +/- 8% (n = 4), and the spiking rate to 25 +/- 11% (n = 4) of the corresponding control levels. These results demonstrate JP-8 induced cytotoxic varies among cell types. The possible mechanisms underlying these observations are presented.