Real-time multisite observation of glutamate release in rat hippocampal slices.

A multichannel glutamate sensor was fabricated that consists of enzyme modified electrodes and has a high sensitivity and selectivity to glutamate. We placed a rat hippocampal slice on the sensor and monitored the current at four electrodes resulting from the stimulation with muscimol, a gamma-aminobutyric acid(A) (GABA(A)) receptor agonist. We obtained different glutamate concentration increases at the different positions, suppressed by bicuculline, a GABA(A) receptor antagonist. This demonstrated that the sensor can monitor the glutamate released via GABA(A) receptors pathways, and the difference in the concentrations may indicate differences in the distribution of GABA(A) receptor as well as diverse receptor functions. This multichannel sensor may be useful for non-invasive, real-time monitoring of glutamate distribution, which would make it a valuable tool for pharmacological analysis.

Nearly pure blue photoluminescent poly(2,7-[9-)3,5-bis[3,5- bis(benzyloxy)benzyloxy]benzyl)-9-(3,6-dioxaoctyl)]fluorene) in film.

The first unsymmetrically substituted polyfluorene bearing a bulky poly(benzyl ether) dendron and less bulky 3,6-dioxaoctyl groups in the 9-position was designed and synthesized, which gives almost a pure bluish photoluminescence with negligible weak greenish excimer emission around 520 nm even in a thermally annealed thin solid film.

Investigation of the molecular extraction process in single subpicoliter droplets using a near-infrared laser Raman trapping system

The near-infrared (NIR) laser Raman trapping system was applied to study liquid-liquid extraction in a single droplet in a subpicoliter range. The system trapped a single subpicoliter toluene droplet in water using the NIR laser beam and provided time-dependent optical images of the droplet during liquid-liquid extraction. The size of the trapped droplet gradually increased afterp-nonylphenol solution was added in water. The Raman spectra of the droplet prove that the increase of the droplet size was caused by the absorption of p-nonylphenol from the water. The distribution coefficient of p-nonylphenol in the sub-picoliter droplet was much higher than that in bulk solution.

Subnanoliter volume wall-jet cells combined with interdigitated microarray electrode and enzyme modified planar microelectrode

Miniaturized wall-jet type flow cells with an active volume of 0.042-15 nL were fabricated for use as highly sensitive electrochemical detectors for capillary electrophoresis/electrochemical detection and small on-line enzyme sensors. The cells consisted of three glass plates and a fused-silica capillary. Two of the plates had microfabricated flow channels and guide trenches for the capillary and working, reference, and counter electrodes. The other plate had a film electrode. When an interdigitated microarray electrode (total area, 66 microm x 64 microm; bandwidth and gap, 2 microm) was installed in the flow cell, the redox cycling enhanced the current at flow rates of less than 100 nL/min even though there were only eight pairs of microbands. A sharp dopamine peak enhanced by the redox cycling was observed when the cell was used for capillary electrophoresis. A square film electrode modified with glutamate oxidase and Os-poly(vinylpyridine) containing HRP was also installed in the flow cell and used to measure neurotransmitter release from cultured nerve cells. When the flow rate was relatively high, the response time of the modified electrode was comparable to that of a cylindrical carbon fiber electrode (33 microm o.d.) modified with the same enzyme and mediator. We observed a transient cathodic current response assigned to the glutamate release with the electrode in the flow cell in a suction mode measurement when we stimulated cultured nerve cells electrically with a dual microelectrode.

Real-time electrochemical imaging using an individually addressable multi-channel electrode.

We developed a real-time electrochemical imaging method that uses a multiple enzyme-modified microelectrode. The method will enable the investigation of the functions of biological materials and cells. To test its effectiveness, we imaged the two-dimensional concentration distribution for hydrogen peroxide and L-glutamate in a standard solution. The multiple electrode consists of an 8 x 8 array of 30 x 30 microm2 carbon micro electrode. Each electrode was connected to a 64-channel potentiostat that could apply a potential to all electrodes at the same time. The multiple electrode was coated with an Os-polyvinylpyridine based polymer (Os-gel) containing horse radish peroxidase (HRP) to detect hydrogen peroxide, which is a very common product of oxidase enzyme. When measuring glutamate, which is a well-known neurotransmitter in the mammalian central nerve system, we modified the electrode with a bilayer of Os-gel-HRP and GluOx. The detection limit of our method was 1 microM and images of the glutamate concentration-distribution changes induced by local injection of glutamate through microcapillary were obtained in real time.

Direct detection of uncaged glutamate and the laser photostimulation of cultured rat cortex.

The photostimulation of nerve cells using a caged compound is very useful because it is non-invasive and non-destructive compared with standard electrophysiological techniques. There are no methods, however, for continuously measuring the photo-uncaged 'free' compound concentration at high temporal and spatial resolutions which can detect how much uncaged compound has been applied to cells. Here, we used an electrochemical detection method for the real-time measurement of photo-uncaged glutamate. In this way, we were able to determine the amount of uncaged glutamate and investigate neural activities by tracing [Ca]i while simultaneously employing photostimulation and on-line glutamate measurement. The combination of an on-line sensor and laser-photostimulation with [Ca]i measurement could be a powerful tool with which to investigate synaptic connections and activities.

On-line electrochemical sensor for selective continuous measurement of acetylcholine in cultured brain tissue.

An on-line acetylcholine (ACh) sensor was developed in order to determine extracellular ACh concentrations without interference from choline (Ch). The sensor is composed of a small-volume enzymatic prereactor (22-microL inner volume) in which choline oxidase and catalase are immobilized in series. Carbon electrodes were modified with an acetylcholine esterase (AChE), choline oxidase (ChOx), and osmium poly(vinylpyridine)-based redox polymer containing horseradish peroxidase (Os-gel-HRP). The sensor sensitivity was 43.7 nA/microM (+/- 0.15, n = 3) for ACh under optimized conditions. Almost no response was seen when 100 microM Ch was continuously injected. The detection limit for ACh with the sensor was comparable to that obtained using liquid chromatography with electrochemical detection combined with an enzymatic reactor. The electrical stimulation of cultured rat hippocampal tissue resulted in an extracellular ACh increase of 20 nM (+/- 11 nM, n = 3). This increase was observed continuously with our online sensor combined with a microcapillary sampling probe located very close to the tissue. The continuous measurement of ACh and Ch using a split disk carbon film dual electrode in which one electrode surface was modified with ChOx/Os-gel-HRP and the other with AChE-ChOx/Os-gel-HRP bilayer film was also demonstrated to improve the response time by eliminating the prereactor.

Small-volume on-line sensor for continuous measurement of gamma-aminobutyric acid.

We report the first on-line electrochemical sensor for the continuous measurement of gamma-aminobutyric acid (GABA), which is a well-known inhibitory neurotransmitter in the nervous system. The sensor is composed of a glutamate oxidase (GluOx) and catalase immobilized small-volume enzymatic reactor and a glassy carbon (GC) electrode modified with a top layer film consisting of gabase and GluOx coimmobilized bovine serum albumin and an Ospoly(vinylpyrridine) bottom layer film containing horseradish peroxidase. The response of the sensor depends on the alpha-ketoglutarate concentration and is almost saturated when its concentration is 100 times higher than GABA. The sensor exhibits a sensitivity of 1.56 nA/microM for GABA under optimized conditions and shows almost no response when 10 microM glutamate is continuously injected. A detection limit of 0.1 microM is obtained with a linear range of 0.1-10 microM. GABA can be measured in the absence of alpha-ketoglutarate when there is L-glutamate in the sample solution, which is a typical condition for the extracellular measurement of cultured nerve cells.

Real-time detection of GABA-induced synaptic glutamate release in cultured rat cortex.

Glutamate is an important neurotransmitter in synaptic transmission. There are no methods, however, for continuous measurement of glutamate concentration at high temporal and spatial resolutions. We have developed a novel electrochemical detection method for the on-line measurement of glutamate release with nanomolar resolution in real time. Using this method, GABA was found to have a modulatory action on the synaptic glutamate release in cultured rat cortical cells. This synaptic modulation largely depends on the GABAA receptor and could be a key not only in neural development, but also in signal transduction in the brain. Our detection method is ideal for investigating such synaptic glutamate responses because of its higher sensitivity and real-time measurement capability.

Continuous monitoring of L-glutamate released from cultured nerve cells by an online sensor coupled with micro-capillary sampling.

A small volume L-glutamate online sensor was developed in order to monitor changes in the local concentration of L-glutamate released from cultured nerve cells. Syringe pump in the suction mode is used to sample extracellular fluid continuously from a glass micro-capillary and the concentration of L-glutamate can be determined by using a glassy carbon (GC) electrode modified with an Os-polyvinylpyridine mediator bottom film containing horseradish peroxidase and a bovine serum albumin top layer containing L-glutamate oxidase. The overall efficiency of L-glutamate detection with a sensor is 71% under optimum conditions due to an efficient enzymatic reaction at the modified electrode in the thin layer radial flow cell. As a result, we achieved a detection limit of 7-15 nM and a linear range of 50 nM to 10 microM. In an in vitro experiment, the extracellular fluid near a particular nerve cell can be sampled with this micro-pipet and continuously introduced into the modified GC electrode in the radial flow cell via suction provided by a syringe pump. The nerve cells are stimulated by the KCl in a glass capillary and the L-glutamate concentration change can be monitored by changing the distance between the sampling pipet and the nerve cells.

Concentration of extracellular L-glutamate released from cultured nerve cells measured with a small-volume online sensor.

An online sensor with a low detection limit for L-glutamate was developed in order to monitor the change in the extracellular L-glutamate concentration as a result of stimulated release from cultured nerve cells. The sensor consisted of a microdialysis (MD) probe fixed at the manipulator, a small-volume L-glutamate oxidase enzymatic reactor (0.75 mm i.d. and 2.5 cm long), and an electrochemical detector in a thin-layer radial flow cell with an active volume of 70-340 nL. Glassy carbon bulk or carbon film ring-disk electrodes were used as detectors by modifying them with Os poly(vinylpyridine) mediator containing horseradish peroxidase. The overall efficiency of L-glutamate detection with the sensor is 94% under optimum conditions, due to an efficient enzymatic reaction in the reactor and a high conversion efficiency in the radial flow cell. As a result, we achieved a sensitivity of 24.3 nA/muM and a detection limit of 7.2 nM (S/N = 3). The effect of interferents such as L-ascorbic acid can be minimized effectively by applying a low potential to the electrode for hydrogen peroxide detection (O mV) and via the ring-disk electrode geometry by using the disk for preoxidation. In the in vitro experiment, an MD probe for sampling was connected to a manipulator that controls distance between the probe and the stimulated cells. The cells were stimulated by KCl in a glass capillary or electrically with microarray film electrodes fabricated on a substrate. By using the sensor, we can monitor L-glutamate concentration changes at the submicromolar level caused by KCl stimulation of a single nerve cell and micromolar L-glutamate concentration increases caused by electrical stimulation of a brain slice. An increase in L-glutamate concentration can also be measured by positioning the probe near the cell that is connected synaptically to the stimulated cell.

Periodic synchronized bursting and intracellular calcium transients elicited by low magnesium in cultured cortical neurons.

1. In Mg(2+)-free external solution, rat cortical neurons in cultured networks entered a stable firing mode, consisting of regular bursts of action potentials superimposed on long-lasting depolarizations. The average separation between bursts varied from culture to culture, but was usually between 5 and 20 s. The distribution of burst intervals followed a Gaussian or normal distribution, with a standard deviation of typically 10% of the average burst period. 2. A gradually depolarizing pacemaker potential was never observed between bursts, but the threshold for action potentials during the quiescent phase was > or = 10 mV above the resting potential. No progressive change in conductance or excitability was observed during the quiescent period. Intracellular stimulation of action potentials did not reproduce the long-lasting depolarization. 3. Switching from current clamp to voltage clamp at the resting potential revealed large postsynaptic currents, mainly excitatory but with a small inhibitory component, at the same phase and frequency as the spike bursts, showing that periodic synaptic input is responsible for the burst-depolarizations. The current could be eliminated by local application of 2-amino-5-phosphonovaleric acid (APV) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the postsynaptic cell. In the presence of tetrodotoxin, irregular miniature excitatory postsynaptic currents were observed. 4. A fluorescent calcium indicator (fluo-3, 100 microM) was included in the whole-cell pipette solution, to allow simultaneous electrical and calcium measurements in the same cell. In current clamp, transient intracellular calcium increases were found, which were synchronized to the spike bursts. The Ca2+ rise lasted as long as the action potential burst, and was followed by an exponential decay considerably slower than that of the membrane potential. Calcium transients disappeared during voltage clamp at the resting potential, suggesting that calcium influx through voltage-dependent calcium channels greatly exceeds that through synaptic channels. 5. Multisite Ca2+ recording, after loading with fluo-3 acetoxymethyl (AM) ester, revealed that the onsets of burst-related calcium transients were synchronized in all active cells of each view-field, to within approximately 20 ms. Occasionally, secondary rhythms were observed in which only a subset of cells participated. The times to peak and the decay times of calcium transients varied among synchronized cells. 6. The pharmacology of the burst-related calcium transients was investigated by bath application of a variety of compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

Periodic bursting of cultured cortical neurons in low magnesium: cellular and network mechanisms.

The origin of the 0.1 Hz bursting of cultured rat cortical neurons in zero Mg2+ was studied by whole-cell recording, fluo-3 Ca2+ imaging, and multi-unit electrode array recording. A model is proposed in which spontaneous synaptic currents act as a random pacemaker.

Selective growth of sensory nerve fibers on metal oxide pattern in culture.

Metal oxides were used to study how the sensory nerve fibers recognize surface properties. Neurites selectively grow on the metal oxides deposited on silica glass, being guided along the axial direction of the patterns. The guiding ability depends on the electronegativity of the metal in metal oxide. Aluminum oxide or indium oxide patterns showed a remarkable ability to guide the growth direction. Neurites recognize the differences in surface properties (which are reflected by electronegativity) between metal oxides when the metal oxide substrata are only 1 micron in width.

Recognition of artificial microstructures by sensory nerve fibers in culture.

Dissociated culture of adult mouse dorsal root ganglion cells on glass plates, on which grating-associated microstructures (a repetition of microgrooves [mGRV] and microsteps [mSTP] of 0.1-10 micron) are fabricated by the conventional lithographic techniques, represents a remarkable bi-directional growth of their nerve fibers in the axial direction of the grating. Microscopical observation shows that the nerve fibers prefer to grow in the mGRV (70%), while their growth cones exhibit an even distribution onto the mGRV and mSTP. The efficiency of the nerve fibers to grow along the grating-axis are highly sensitive to a fine alteration of the width and depth of the mGRV. The preferential growth of the nerve fibers is thus due to a mechanical recognition of the microstructures by the growth cones and neurites.