Μm Tip Diameter Research Articles

EVALUATION OF FABRICATED SOLID MICRONEEDLES AS SMART APPROACH FOR TRANSDERMAL DRUG DELIVERY SYSTEM OF ASTAXANTHIN

Objective: The lack of drugs to effectively cross the stratum corneum (SC), has recently been a significant barrier to transdermal administration. In order to increase the effectiveness of transdermal distribution, this issue has been solved through the development of micron-scale needles. The objective is to develop, formulate and evaluate biocompatible polymeric solid microneedles with a TDDS-loaded Astaxanthin patch involving the poke and patch method. Methods: The solid microneedle arrays were fabricated using an SLA printer with high-resolution potential and was examined using scanning electron microscopy (25 microns at the z-axis and 140 microns at the x-axis, respectively). Fabricated Astaxanthin transdermal film was evaluated by many characterization parameters. The developed microneedle was examined for skin insertion and a drug permeation study was carried out across the porcine skin. Results: Solid MN arrays of 1.85 µm tip-to-tip distance, 600 µm height, 300 µm width, and 30 µm tip diameter, were created using biocompatible Class I Dental SG resin. Microneedle crossed stratum corneum layer and penetrated porcine skin with 381.356 mm in depth, with no structural change. Transdermal patches loaded with astaxanthin drug was developed and using various polymer concentration consistent, good, and transparent films were created of thickness between 0.85±0.07 mm to 0.87±0.01 mm range, with average weights ranging from 168.02±1.05 to 172.22±1.25 10/cm2), Folding Endurance with 10-12 folds was reported for formulated transdermal films; also tensile strength was 0.414±0.002 kg/mm2 and drug content was 94.41±0.42% of the best formulation reported. According to studies on drug permeation, Astaxanthin transdermal patches did not significantly permeate porcine skin without being pierced by microneedles the medication exhibited good penetration characteristics. Conclusion: Upon evaluation, formulated transdermal film was reported to be best for solid microneedle-TDDS of astaxanthin for improved patient compliance, bioavailability, and biomedical applications.

A Miniaturized Ultrasonic Micro-Hole Perforator for Minimally Invasive Craniotomy.

Micro-hole perforation on skull is urgently desired for minimally invasive insertion of micro-tools in brain for diagnostic or treatment purpose. However, a micro drill bit would easily fracture, making it difficult to safely generate a micro-hole on the hard skull. In this study, we present a method for ultrasonic vibration assisted micro-hole perforation on skull in a manner similar to subcutaneous injection on soft tissue. For this purpose, a high amplitude miniaturized ultrasonic tool with a 500 μm tip diameter micro-hole perforator was developed with simulation and experimental characterization. In-depth investigation of micro-hole generation mechanism was performed with systematic experiments on animal skull with a bespoke test rig; effects of vibration amplitude and feed rate on hole forming characteristics were systematically studied. It was observed that by exploiting skull bone's unique structural and material properties, the ultrasonic micro-perforator could locally damage bone tissue with micro-porosities, induce sufficient plastic deformation to bone tissue around the micro-hole and refrain elastic recovery after tool withdraw, generating a micro-hole on skull without material. Under optimized conditions, high quality micro-holes could be formed on the hard skull with a force (<1 N) even smaller than that for subcutaneous injection on soft skin. This study would provide a safe and effective method and a miniaturized device for micro-hole perforation on skull for minimally invasive neural interventions.

Fabricating High-Resolution and High-Dimensional Microneedle Mold through the Resolution Improvement of Stereolithography 3D Printing

Microneedles are transdermal drug delivery tools that can be fabricated simply, economically, and rapidly using SLA 3D printing. However, SLA 3D printing has a limitation in that the resolution is slightly lowered when the microneedle is precisely printed. To solve this issue, we optimized the SLA 3D printing conditions such as printing angle, needle height, aspect ratio, and spacing between the microneedles for high-resolution microneedle fabrication. The sharpest microneedle tip was obtained when the printing angle was adjusted to 60° in both the x and y axes. The aspect ratio and the spacing between the microneedles did not affect the output of the sharp tip. Under optimal conditions, the microneedles with 1180 ± 20 µm height, 490 ± 20 µm base, and 30.2 ± 3.4 µm tip diameter were obtained. The dissolving microneedle patch, prepared using the 3D printed microneedle as a mold, penetrated the porcine skin ex vivo. When the printing angle was 60° in the x and y axes, the area of the single stacking layer, including the microneedle tip, increased, and thus the sharp tip could be printed. A high-dimensional, side-notched arrowhead (SNA) microneedle was fabricated by applying the SLA 3D printing condition. Moreover, a letter-type microneedle patch was fabricated using the customized characteristics of 3D printing. Consequently, high-resolution and high-dimensional microneedles were successfully fabricated by adjusting the printing angle using a general SLA 3D printer, and this technology will be applied to the manufacture of drug delivery tools and various microstructures.

Investigation on MRR and Machining Gap of Micro Reciprocated Wire-EDM for SKD11

In this paper, the investigational work is carried out on the micro reciprocated wire electrical discharge machining (wire-EDM) of SKD11 using circularly travelled wire with Φ30 μm as tool electrode, aiming to achieve the fabrication of micro parts with high cost-effect, efficiency and precision. The experiments are systematically performed using central composite design approach of response surface methodology to study the effect of open voltage, discharge capacitance, pulse duration and feed rate on material removal rate (MRR) and machining gap. The mathematical models of MRR and machining gap are developed by analysis of variance, and whilst verified by confirmation experiments with ≤ 8.38% and 6.70% prediction errors for MRR and machining gap, respectively. Besides, multi-objective optimization of parameter combinations is attempted by non-dominated sorting genetic algorithm-II. Finally, a typical experimental sample, micro gear is successfully machined with 1194.38 μm tip diameter under the condition of No. 14 optimal solution.

Optimization Design and Fabrication of Polymer Micro Needle by Hot Embossing Method

This paper presents the design, fabrication and testing of a solid polymer microneedle. Mechanical behavior of the microneedle was simulated by ANSYS via the results of suffered and strength calculations. The results shows, that the maximum stresses of polymer MNs is far less than 3.183 MPa, which is the allowable pressure needed. Taguchi method was used help in data analysis and prediction of optimum parameter settings, a series of experiments were conducted to verify the impact of embossing temperature, embossing pressure, and embossing time on the microneedle’s quality. According to the result from the Taguchi experiment, S/N ratio is calculated to find the best combination settings for microneedle’s height. The highest value of S/N ratio (54.403) for product height is determined as optimal initial parameter settings, achieved by 130 °C, 11 MPa and 150 s for PMMA microneedle with optimized size of 550 µm height, 250 µm base diameter and 50 µm tip diameter. Embossing temperature and embossing pressure are the significant parameters in the experiment. The proposed method was verified by a set of experiments, the force for insertion as well as the modes of mechanical failure were examined.

Suction electrode recording in locus coeruleus of newborn rat brain slices reveals network bursting comprising summated non-synchronous spiking

The brainstem locus coeruleus (LC) controling behaviors like arousal, sleep, breathing, pain or opioid withdrawal is an established model for spontaneous action potential synchronization. Such synchronous ‘spiking’ might produce an extracellular field potential (FP) which is a crucial tool for neural network analyses. We found using ≥10 μm tip diameter suction electrodes in newborn rat brainstem slices that the LC generates at ∼1 Hz a robust rhythmic FP (rFP). During distinct rFP phases, LC neurons discharge with a jitter of ±33 ms single spikes that summate to a ∼200 ms-lasting population burst. The rFP is abolished by blocking voltage-gated Na+ channels with tetrodotoxin (TTX, 50 nM) or gap junctions with mefloquine (100 μM) and activating μ-opioid receptors with [D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin (DAMGO, 1 μM). Raising superfusate K+ from 3 to 7 mM either increases rFP rate or transforms its pattern to slower and longer multipeak bursts similar to those during early recovery from DAMGO. The results show that electrical coupling of neonatal LC neurons does not synchronize their spiking as previously proposed. They also indicate that both increased excitability (by elevated K+) and recovery from inhibition (by opioids) can enhance spike desynchronization to transform the population burst pattern. Both observations show that this gap junction-coupled neural network has a more complex connectivity than currently assumed. These new findings along with the inhibitory drug effects that are in line with previous reports based on single neuron recording point out that field potential analysis is pivotal to further the understanding of this brain circuit.

In Situ Monitoring of Pb2+ Leaching from the Galvanic Joint Surface in a Prepared Chlorinated Drinking Water.

A novel method using a micro-ion-selective electrode (micro-ISE) technique was developed for in situ lead monitoring at the water-metal interface of a brass-leaded solder galvanic joint in a prepared chlorinated drinking water environment. The developed lead micro-ISE (100 μm tip diameter) showed excellent performance toward soluble lead (Pb2+) with sensitivity of 22.2 ± 0.5 mV decade-1 and limit of detection (LOD) of 1.22 × 10-6 M (0.25 mg L-1). The response time was less than 10 s with a working pH range of 2.0-7.0. Using the lead micro-ISE, lead concentration microprofiles were measured from the bulk to the metal surface (within 50 μm) over time. Combined with two-dimensional (2D) pH mapping, this work clearly demonstrated that Pb2+ ions build-up across the lead anode surface was substantial, nonuniform, and dependent on local surface pH. A large pH gradient (ΔpH = 6.0) developed across the brass and leaded-tin solder joint coupon. Local pH decreases were observed above the leaded solder to a pH as low as 4.0, indicating it was anodic relative to the brass. The low pH above the leaded solder supported elevated lead levels where even small local pH differences of 0.6 units (ΔpH = 0.6) resulted in about four times higher surface lead concentrations (42.9 vs 11.6 mg L-1) and 5 times higher fluxes (18.5 × 10-6 vs 3.5 × 10-6 mg cm-2 s-1). Continuous surface lead leaching monitoring was also conducted for 16 h.

Development of a Microbiosensor for Acetate Detection in Biofilms

We developed an amperometric acetate microbiosensor capable of measuring in situ sub-micromolar concentration of acetate inside biofilms. The operating principle of the microbiosensor utilized electrochemically active biofilms (EABs) to convert acetate to electrons on the microelectrode tip. The acetate microbiosensor consisted of a 30 µm glass-covered carbon wire working electrode with G.sulfurreducens biofilm on the tip recessed into a glass outer case approximately 200 μm and a Ag/AgCl reference electrode, as shown in Figure 1. The glass outer case terminated to a 30 µm tip diameter where acetate could flow into and diffuse up to the active tip. Acetate-free G.sulfurreducens growth medium served as electrolyte. The microbiosensor showed a linear response between 50 µM and 1.6 mM acetate concentration with a 79 ± 8 µM limit of detection (S/N=2). The operating potential of the microbiosensor was selected as +300 mVAg/AgCl satisfying the limiting current condition. The microbiosensor also demonstrated a higher specificity to acetate over other possible metabolites that could interfere with acetate measurements. To test the microbiosensor, we measured local acetate concentration inside G. sulfurreducens biofilms grown on a 1 in2 electrode in a three-electrode bioreactor operated in fed-batch mode. Measuring acetate profiles in G.sulfurreducens biofilms is critical to understand acetate limitations and acetate diffusion and to test the developed microbiosensor in G.sulfurreducens biofilms which utilizes acetate as the electron donor. The microbiosensor was used to measure acetate depth profiles within ~225 µm thick G.sulfurreducens biofilm under no current generation (open circuit potential) and current generation (at +300 mVAg/AgCl) conditions. For depth profile measurements, bulk acetate concentration in the bioreactor was set to 1.5 mM by considering the linear calibration range of the microbiosensor. The acetate concentration remained at bulk values throughout the biofilm at open circuit potential when the biofilm was not respiring. However, the acetate concentration decreased from the bulk values to below the detection limit within 200 μm when current was allowed to pass at +300 mVAg/AgCl. This showed that the biofilm was acetate-limited due to the zero acetate concentration at the bottom of the biofilm. Figure 1

Fabrication of Diamond-Like Carbon Microgears in Room-Temperature Curing Nanoimprint Lithography Using Ladder-Type Hydrogen Silsesquioxane

ABSTRACTWe investigated the fabrication of convex diamond-like carbon (DLC) based microgears in room-temperature curing nanoimprint lithography (RTC-NIL) using the ladder-type hydrogen silsesquioxane (HSQ), as an application for the medical micro electro mechanical system (MEMS). The HSQ which is an inorganic polymer of sol-gel system turns into a gel when exposed to air and has the siloxane bond. Therefore, the HSQ was used as RT-imprinting material, and also used as an oxide mask material in electron cyclotron resonance (ECR) oxygen (O2) ion shower etching. We fabricated the polydimethylsiloxane (PDMS) mold with concave microgear patterns which has 40, 50 and 60 μm-tip diameter and 300 nm-depth. We carried out the RTC-NIL process using the PDMS mold under the following optimum conditions of 0.10 MPa-imprinting pressure and 1.0 min-imprinting time. We found that the residual layer of imprinted HSQ microgear patterns was removed with ECR trifluoromethane (CHF3) ion shower under the following conditions of 300 eV-ion energy and 2.0 min-etching time, and then microgears of the HSQ on the DLC film were etched with ECR O2 ion shower under the following conditions of 400 eV-ion energy and 10 min-etching time. As a result, the convex DLC based microgears which have 40, 50 and 60 μm-tip diameter and 400 nm-height were fabricated with high accuracy in the new fabrication process of RTC-NIL.

Fiber‐Optic Probes for Small‐Scale Measurements of Scalar Irradiance

A new method for producing fiber-optic microprobes for scalar irradiance (=fluence rate) measurements is described. Such fine-scale measurements are important in many photobiological disciplines. With the new method, it is possible to cast spherical 30-600 μm wide light integrating sensor tips onto tapered or untapered optical fibers. The sensor tip is constructed by first casting a clear polymethyl methacrylate (PMMA) sphere (~80% of the size of the final probe tip diameter) onto the optical fiber via dip-coating. Subsequently, the clear sphere is covered with light diffusing layers of PMMA mixed with TiO2 until the fiber probe exhibits a satisfactory isotropic response (typically ±5-10%). We also present an experimental setup for measuring the isotropic response of fiber-optic scalar irradiance probes in air and water. The fiber probes can be mounted in a syringe equipped with a needle, facilitating retraction of the spherical fiber tip. This makes it, e.g. possible to cut a hole in cohesive tissue with the needle before inserting the probe. The light-collecting properties of differently sized scalar irradiance probes (30, 40, 100, 300 and 470 μm) produced by this new method were compared to probes produced with previously published methods. The new scalar irradiance probes showed both higher throughput of light, especially for blue light, as well as a better isotropic light collection over a wide spectral range. The new method also allowed manufacturing of significantly smaller scalar irradiance microprobes (down to 30 μm tip diameter) than hitherto possible, and such sensors allow minimally invasive high-resolution scalar irradiance measurements in thin biofilms, leaves and animal tissues.

Fabrication of DLC-Based Micro-Gear Patterns by Room-Temperature Curing Nanoimprint Lithography Using Glass-Like Carbon Molds

ABSTRACTThe fabrication of diamond-like carbon (DLC) micro-gear by room temperature curing nanoimprint lithography (RTC-NIL) using glass-like carbon (GC) molds as applications to the DLC-based medical MEMS (Micro Electronic Mechanical Systems) was investigated. The DLC film which has excellent properties similar to chemical vapor deposited (CVD) diamond films was used as the patterning material. We propose GC as mold material because GC has higher etching selectivity than a diamond film. The etching selectivity of polysiloxane film against a GC substrate is about 5 times as high as that of a diamond film. Therefore we fabricated the GC molds that have micro-gear patterns with 30 µm-tip diameter and 500 nm-tooth thickness. We carried out the RTC-NIL process using the GC micro-gear molds under the following optimum conditions. 1 min-time from spin-coating to imprint: t1, 0.5 MPa-imprinting pressure: P and 5 min-holding time: t2, and then the imprinted polysiloxane pattern on DLC film was processed with an electron cyclotron resonance (ECR) oxygen ion shower. However, we were not able to fabricate micro-gear patterns in high accuracy because of a remaining residual layer on the DLC film. Therefore we propose the removing process for the residual layer with trifluoromethane (CHF3) ion shower under the optimum conditions of 300 eV-ion energy and 4 min-etching time. As a result, we succeeded to fabricate concave DLC-based micro-gear patterns in high accuracy which has 30 µm-tip diameter and 1 µm-depth.

TRPV1 in GABAergic Interneurons Mediates Neuropathic Mechanical Allodynia and Disinhibition of the Nociceptive Circuitry in the Spinal Cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Fabrication of micro-features and micro-tools using electrochemical micromachining

Electrochemical micromachining (ECMM) is an advanced machining process for the machining of electrically conducting materials. In the present work, an experimental setup has been designed and fabricated to machine micro-holes and micro-channels. Machining of these features is done using a sewing needle with 47 μm tip diameter as a tool. The effects of process parameters such as voltage, electrolyte concentration, pulse duty cycle, and feed rate on the machined hole diameter have been studied, and a mathematical model is developed. The fabrication of micro-tools is done on a different setup. A straight tool having a diameter of 80 μm is fabricated from a 1,000-μm steel wire using the ECMM process. Variations of wire diameter and the material removal rate with time are studied. Micro-feature measurements and photographic analysis are done using a digital microscope.

Fabrication of polymer micro-tip by optical lensing technique

Fabrication of polymer micro-tips using SU-8 negative photoresist for bio-applications is reported. The SU-8 processing technology and isotropic glass etching process have been developed and utilized to fabricate micro-tips on glass substrate by applying optical lensing effect during photolithography. Experimentally, micro-tips of 25 μm base diameter, ~1 μm tip diameter, and ~250 μm height, have been demonstrated.

A voltammetric flavin microelectrode for use in biofilms

Biofilms used in bioelectrochemical systems are expected to transfer electrons using electron transfer mediators. One mediator type, flavins, which includes flavin mononucleotide, riboflavin, and flavin adenine dinucleotide, has been found to be endogenously produced by Shewanella oneidensis MR-1. However, the presence and concentration of flavins inside a S. oneidensis MR-1 biofilm have never been reported. The goal of this study was to develop a flavin microelectrode capable of measuring flavins inside a living biofilm and apply it to a biofilm which produces flavins. Because flavins are electrochemically active molecules, the flavin microelectrode was based on detection via square-wave voltammetry. The microelectrode consisted of a carbon working electrode with a 10–30 μm tip diameter, a built-in platinum counter electrode, and a Ag/AgCl reference electrode, all enclosed in a glass outer case. The microelectrode was calibrated between 0.1 μM and 10 μM flavins and showed a linear correlation between flavin concentration and peak currents located at −424 mV Ag/AgCl on a square-wave voltammogram. We also developed a model to explain the electrochemical mechanism of flavin detection, and to determine the effective surface area of the microelectrode, the standard reduction potential, and the transfer coefficient. We found that the effective surface area of the microelectrode was close to 100 times the projected surface area. The model predicted a standard reduction potential for RF/RFH2 of −419 mV Ag/AgCl at 20 °C and a transfer coefficient of 0.45. Lastly, we measured flavin concentration inside a S. oneidensis MR-1 biofilm grown on a glass surface using oxygen as the electron acceptor. The flavin concentration reached 0.7 μM, increasing near the bottom of the biofilm, where no oxygen was present. This shows the possibility that flavins are produced in the anaerobic zone to act as intermediate electron acceptors in the deeper parts of the biofilm, where there is no oxygen.

Stretchable microelectrode array using room-temperature liquid alloy interconnects

In this paper, we present a stretchable microelectrode array for studying cell behavior under mechanical strain. The electrode array consists of gold-plated nail-head pins (250 µm tip diameter) or tungsten micro-wires (25.4 µm in diameter) inserted into a polydimethylsiloxane (PDMS) platform (25.4 × 25.4 mm2). Stretchable interconnects to the outside were provided by fusible indium-alloy-filled microchannels. The alloy is liquid at room temperature, thus providing the necessary stretchability and electrical conductivity. The electrode platform can withstand strains of up to 40% and repeated (100 times) strains of up to 35% did not cause any failure in the electrodes or the PDMS substrate. We confirmed biocompatibility of short-term culture, and using the gold pin device, we demonstrated electric field pacing of adult murine heart cells. Further, using the tungsten microelectrode device, we successfully measured depolarizations of differentiated murine heart cells from embryoid body clusters.

Lamotrigine inhibits postsynaptic AMPA receptor and glutamate release in the dentate gyrus

The dentate gyrus (DG) is a gateway that regulates seizure activity in the hippocampus. We investigated the site of action of lamotrigine (LTG), an effective anticonvulsant, in the regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptor-mediated excitatory synaptic transmission on DG. Evoked AMPA and NMDA receptor-mediated excitatory postsynaptic currents (eEPSCampa and eEPSCnmda) were recorded by whole-cell patch-clamp recording from the granule cells of DG in brain slice preparation of young Wistar rats (60-120 g). Exogenously applied AMPA and NMDA-induced currents and AMPA receptor-mediated miniature EPSC (mEPSCampa) were recorded in the presence of specific antagonists. LTG inhibited both eEPSCampa and eEPSCnmda, and had no effect on exogenously applied NMDA-induced current indicating LTG inhibited glutamate release. Previous studies demonstrated that alteration in glutamate concentration in synaptic cleft causes parallel changes of eEPSCampa and eEPSCnmda. Our results showed that LTG inhibited eEPSCampa significantly more than eEPSCnmda (p < 0.05), suggesting that LTG may also have blocked the postsynaptic AMPA receptor. The hypothesis is further supported by the facts that; (1) LTG (30-100 microM) inhibited direct exogenously applied AMPA-induced currents (to 90%), (2) LTG significantly reduced the amplitude, but not the frequency of mEPSCampa and asynchronous (EPSC), and (3) LTG-induced reduction of eEPSCampa was not associated with a modification of the paired-pulse ratio. To sum up, LTG exerts a postsynaptic inhibitory mechanism on the AMPA receptor. Our results demonstrate that LTG suppresses postsynaptic AMPA receptors and reduces glutamate release in granule cells of DG. The postsynaptic effect can be one of the underlying mechanisms of LTG's anticonvulsant action.

Analysis of the Heterogeneity of pO2 Dynamics During Photodynamic Therapy with Verteporfin¶

Photodynamic therapy (PDT) with verteporfin provides a reliable way to destroy malignant tissues. Changes in the blood flow and oxygen partial pressure (pO2) during verteporfin-PDT were studied here in the tumor tissue of the rat mammary R3230Ac carcinoma model. Oxygen microelectrodes (6–12 μm tip diameter) were used to measure the transients locally within tumors during intravenous injection of 1.0 mg/kg verteporfin followed by irradiation 15 min later with 690 nm light at 200 mW/cm2, for a cumulative dose of 144 J/cm2. The observed changes in pO2 were heterogeneous and there was a difference in the response of low-pO2 regions relative to higher-pO2 regions. The change in pO2 in hypoxic tissue regions (pO2 < 8 mmHg) had acute pO2 loss after treatment, whereas the response in regions of higher pO2 (>8 mm Hg) was more heterogeneous with some areas maintaining their pO2 value after treatment was completed. Blood flow measurements taken on a subset of the animals indicated a significant loss in flow during the initial light delivery that remained low after treatment, indicating some vascular stasis. The results suggest that hypoxic or poorly perfused vessels may be more susceptible to acute stasis than normoxic vessels in this treatment protocol.

A Taste Receptor Required for the Caffeine Response In Vivo

Caffeine is a methylxanthine present in the coffee tree, tea plant, and other naturally occurring sources and is among the most commonly consumed drugs worldwide. Whereas the pharmacological action of caffeine has been studied extensively, relatively little is known concerning the molecular mechanism through which this substance is detected as a bitter compound. Unlike most tastants, which are detected through cell-surface G protein-coupled receptors, it has been proposed that caffeine and related methylxanthines activate taste-receptor cells through inhibition of a cyclic nucleotide phosphodiesterase (PDE) . Here, we show that the gustatory receptor Gr66a is expressed in the dendrites of Drosophila gustatory receptor neurons and is essential for the caffeine response. In a behavioral assay, the aversion to caffeine was specifically disrupted in flies missing Gr66a. Caffeine-induced action potentials were also eliminated, as was the response to theophylline, the methylxanthine in tea. The Gr66a mutant exhibited normal tastant-induced action potentials upon presentation of theobromine, a methylxanthine in cocoa. Given that theobromine and caffeine inhibit PDEs with equal potencies , these data further support the role of Gr66a rather than a PDE in mediating the caffeine response. Gr66a is the first gustatory receptor shown to be essential for caffeine-induced behavior and activity of gustatory receptor cells in vivo.

Nitric Oxide Release during Evoked Neuronal Activity in Cerebellum Slices: Detection with Platinized Carbon-Fiber Microelectrodes

Nitric oxide is an important biological messenger that particularly induces the relaxation of smooth muscle cells surrounding vessels, and, hence, controls the flow of blood. This mechanism is essential for brain function, and its fine control, termed functional hyperemia, is supposed to be realized by certain neurons that may release bursts of NO*. The aim of the present study is to examine the advantages of platinized carbon-fiber microelectrodes (5-7 microm tip diameter) for the direct and in situ electrochemical detection of NO* released by neurons into ex vivo cerebellum slices. After establishing the different analytical properties of the platinized carbon-fiber microelectrodes in vitro on NO* solutions at 50 nM to 1 mM concentration, they were characterized using DEA-NONOate solutions that chemically decompose into NO*, and therefore mimic the measurement of transient variations of NO* concentration in biological samples. This validated the present approach, so that direct, in situ ex vivo measurements of nitric oxide released by neurons in a rat cerebellar slice are presented and discussed.