Understanding the different effects of fouling mechanisms on working and reference electrodes in fast-scan cyclic voltammetry for neurotransmitter detection.

Fast-scan cyclic voltammetry (FSCV) is a widely used technique for detecting neurotransmitters. However, electrode fouling can negatively impact its accuracy and sensitivity. Fouling refers to the accumulation of unwanted materials on the electrode surface, which can alter its electrochemical properties and reduce its sensitivity and selectivity. Fouling mechanisms can be broad and may include biofouling, the accumulation of biomolecules on the electrode surface, and chemical fouling, the deposition of unwanted chemical species. Despite individual studies discussing fouling effects on either the working electrode or the reference electrode, no comprehensive study has been conducted to compare the overall fouling effects on both electrodes in the context of FSCV. Here, we examined the effects of biofouling and chemical fouling on the carbon fiber micro-electrode (CFME) as the working electrode and the Ag/AgCl reference electrode with FSCV. Both fouling mechanisms significantly decreased the sensitivity and caused peak voltage shifts in the FSCV signal with the CFME, but not with the Ag/AgCl reference electrode. Interestingly, previous studies have reported peak voltage shifts in FSCV signals due to the fouling of Ag/AgCl electrodes after implantation in the brain. We noticed in a previous study that energy-dispersive spectroscopy (EDS) spectra showed increased sulfide ion concentration after implantation. We hypothesized that sulfide ions may be responsible for the peak voltage shift. To test this hypothesis, we added sulfide ions to the buffer solution, which decreased the open circuit potential of the Ag/AgCl electrode and caused a peak voltage shift in the FSCV voltammograms. Also, EDS analysis showed that sulfide ion concentration increased on the surface of the Ag/AgCl electrodes after 3 weeks of chronic implantation, necessitating consideration of sulfide ions as the fouling agent for the reference electrodes. Overall, our study provides important insights into the mechanisms of electrode fouling and its impact on FSCV measurements. These findings could inform the design of FSCV experiments, with the development of new strategies for improving the accuracy and reliability of FSCV measurements in vivo.

Tonic Serotonin Measurements In Vivo Using N-Shaped Multiple Cyclic Square Wave Voltammetry.

Here, we present the development of a novel voltammetric technique, N-shaped multiple cyclic square wave voltammetry (N-MCSWV) and its application in vivo. It allows quantitative measurements of tonic extracellular levels of serotonin in vivo with mitigated fouling effects. N-MCSWV enriches the electrochemical information by generating high dimensional voltammograms, which enables high sensitivity and selectivity against 5-hydroindoleacetic acid (5-HIAA), dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), histamine, ascorbic acid, norepinephrine, adenosine, and pH. Using N-MCSWV, in combination with PEDOT:Nafion-coated carbon fiber microelectrodes, a tonic serotonin concentration of 52 ± 5.8 nM (n = 20 rats, ±SEM) was determined in the substantia nigra pars reticulata of urethane-anesthetized rats. Pharmacological challenges with dopaminergic, noradrenergic, and serotonergic synaptic reuptake inhibitors supported the ability of N-MCSWV to selectively detect tonic serotonin levels in vivo. Overall, N-MCSWV is a novel voltammetric technique for analytical quantification of serotonin. It offers continuous monitoring of changes in tonic serotonin concentrations in the brain to further our understanding of the role of serotonin in normal behaviors and psychiatric disorders.

Biocompatible reference electrodes to enhance chronic electrochemical signal fidelity in vivo.

In vivo electrochemistry is a vital tool of neuroscience that allows for the detection, identification, and quantification of neurotransmitters, their metabolites, and other important analytes. One important goal of in vivo electrochemistry is a better understanding of progressive neurological disorders (e.g., Parkinson's disease). A complete understanding of such disorders can only be achieved through a combination of acute (i.e., minutes to hours) and chronic (i.e., days or longer) experimentation. Chronic studies are more challenging because they require prolonged implantation of electrodes, which elicits an immune response, leading to glial encapsulation of the electrodes and altered electrode performance (i.e., biofouling). Biofouling leads to increased electrode impedance and reference electrode polarization, both of which diminish the selectivity and sensitivity of in vivo electrochemical measurements. The increased impedance factor has been successfully mitigated previously with the use of a counter electrode, but the challenge of reference electrode polarization remains. The commonly used Ag/AgCl reference electrode lacks the long-term potential stability in vivo required for chronic measurements. In addition, the cytotoxicity of Ag/AgCl adversely affects animal experimentation and prohibits implantation in humans, hindering translational research progress. Thus, a move toward biocompatible reference electrodes with superior chronic potential stability is necessary. Two qualifying materials, iridium oxide and boron-doped diamond, are introduced and discussed in terms of their electrochemical properties, biocompatibilities, fabrication methods, and applications. In vivo electrochemistry continues to advance toward more chronic experimentation in both animal models and humans, necessitating the utilization of biocompatible reference electrodes that should provide superior potential stability and allow for unprecedented chronic signal fidelity when used with a counter electrode for impedance mitigation.

Mitigating the Effects of Electrode Biofouling-Induced Impedance for Improved Long-Term Electrochemical Measurements In Vivo.

Biofouling is a prevalent issue in studies that involve prolonged implantation of electrochemical probes in the brain. In long-term fast-scan cyclic voltammetry (FSCV) studies, biofouling manifests as a shift in the peak oxidative potential of the background signal that worsens over days to weeks, diminishing sensitivity and selectivity to neurotransmitters such as dopamine. Using open circuit potential (OCP) measurements, scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDX), and electrochemical impedance spectroscopy (EIS), we examined the biofouling-induced events that occur due to electrode implantation. We determined that the FSCV background signal shift results from cathodic polarization of the Ag/AgCl-wire reference electrode and increased electrochemical impedance of both the Ag/AgCl-wire reference electrode and carbon-fiber working electrode. These events are likely caused collectively by immune response-induced electrode encapsulation. A headstage utilizing a three-electrode configuration, designed to compensate for the impedance component of biofouling, reduced the FSCV background signal shift in vivo and preserved dopamine sensitivity at artificially increased impedance levels in vitro. In conjunction with a stable reference electrode, this three-electrode configuration will be critical in achieving reliable neurotransmitter detection for the duration of long-term FSCV studies.

Sensitive and Selective Measurement of Serotonin in Vivo Using Fast Cyclic Square-Wave Voltammetry.

Although N-shaped fast scan cyclic voltammetry (N-FSCV) is well-established as an electroanalytical method to measure extracellular serotonin concentrations in vivo, it is in need of improvement in both sensitivity and selectivity. Based on our previous studies using fast cyclic square-wave voltammetry (FCSWV) for in vivo dopamine measurements, we have modified this technique to optimize the detection of serotonin in vivo. A series of large amplitude square-shaped potentials was superimposed onto an N-shaped waveform to provide cycling through multiple redox reactions within the N-shaped waveform to enhance the sensitivity and selectivity to serotonin measurement when combined with a two-dimensional voltammogram. N-Shaped fast cyclic square-wave voltammetry (N-FCSWV) showed significantly higher sensitivity to serotonin compared to conventional N-FSCV. In addition, N-FCSWV showed better performance than conventional N-shaped FSCV in differentiating serotonin from its major interferents, dopamine and 5-hydroxyindoleascetic acid (5-HIAA). It was also confirmed that the large amplitude of the square waveform did not influence local neuronal activity, and it could monitor electrical stimulation evoked phasic release of serotonin in the rat substantia nigra pars reticulata (SNr) before and after systemic injection of escitalopram (ESCIT, 10 mg/kg i.p.), a serotonin selective reuptake inhibitor.

Evaluation of electrochemical methods for tonic dopamine detection in vivo.

Dysfunction in dopaminergic neuronal systems underlie a number of neurologic and psychiatric disorders such as Parkinson's disease, drug addiction, and schizophrenia. Dopamine systems communicate via two mechanisms, a fast "phasic" release (sub-second to second) that is related to salient stimuli and a slower "tonic" release (minutes to hours) that regulates receptor tone. Alterations in tonic levels are thought to be more critically important in enabling normal motor, cognitive, and motivational functions, and dysregulation in tonic dopamine levels are associated with neuropsychiatric disorders. Therefore, development of neurochemical recording techniques that enable rapid, selective, and quantitative measurements of changes in tonic extracellular levels are essential in determining the role of dopamine in both normal and disease states. Here, we review state-of-the-art advanced analytical techniques for in vivo detection of tonic levels, with special focus on electrochemical techniques for detection in humans.

The Delta-Specific Opioid Glycopeptide BBI-11008: CNS Penetration and Behavioral Analysis in a Preclinical Model of Levodopa-Induced Dyskinesia.

In previous work we evaluated an opioid glycopeptide with mixed µ/δ-opioid receptor agonism that was a congener of leu-enkephalin, MMP-2200. The glycopeptide analogue showed penetration of the blood-brain barrier (BBB) after systemic administration to rats, as well as profound central effects in models of Parkinson's disease (PD) and levodopa (L-DOPA)-induced dyskinesia (LID). In the present study, we tested the glycopeptide BBI-11008 with selective δ-opioid receptor agonism, an analogue of deltorphin, a peptide secreted from the skin of frogs (genus Phyllomedusa). We tested BBI-11008 for BBB-penetration after intraperitoneal (i.p.) injection and evaluated effects in LID rats. BBI-11008 (10 mg/kg) demonstrated good CNS-penetrance as shown by microdialysis and mass spectrometric analysis, with peak concentration levels of 150 pM in the striatum. While BBI-11008 at both 10 and 20 mg/kg produced no effect on levodopa-induced limb, axial and oral (LAO) abnormal involuntary movements (AIMs), it reduced the levodopa-induced locomotor AIMs by 50% after systemic injection. The N-methyl-D-aspartate receptor antagonist MK-801 reduced levodopa-induced LAO AIMs, but worsened PD symptoms in this model. Co-administration of MMP-2200 had been shown prior to block the MK-801-induced pro-Parkinsonian activity. Interestingly, BBI-11008 was not able to block the pro-Parkinsonian effect of MK-801 in the LID model, further indicating that a balance of mu- and delta-opioid agonism is required for this modulation. In summary, this study illustrates another example of meaningful BBB-penetration of a glycopeptide analogue of a peptide to achieve a central behavioral effect, providing additional evidence for the glycosylation technique as a method to harness therapeutic potential of peptides.

A Novel Angiotensin-(1-7) Glycosylated Mas Receptor Agonist for Treating Vascular Cognitive Impairment and Inflammation-Related Memory Dysfunction.

Increasing evidence indicates that decreased brain blood flow, increased reactive oxygen species (ROS) production, and proinflammatory mechanisms accelerate neurodegenerative disease progression such as that seen in vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer's disease and related dementias. There is a critical clinical need for safe and effective therapies for the treatment and prevention of cognitive impairment known to occur in patients with VCID and chronic inflammatory diseases such as heart failure (HF), hypertension, and diabetes. This study used our mouse model of VCID/HF to test our novel glycosylated angiotensin-(1-7) peptide Ang-1-6-O-Ser-Glc-NH2 (PNA5) as a therapy to treat VCID and to investigate circulating inflammatory biomarkers that may be involved. We demonstrate that PNA5 has greater brain penetration compared with the native angiotensin-(1-7) peptide. Moreover, after treatment with 1.0/mg/kg, s.c., for 21 days, PNA5 exhibits up to 10 days of sustained cognitive protective effects in our VCID/HF mice that last beyond the peptide half-life. PNA5 reversed object recognition impairment in VCID/HF mice and rescued spatial memory impairment. PNA5 activation of the Mas receptor results in a dose-dependent inhibition of ROS in human endothelial cells. Last, PNA5 treatment decreased VCID/HF-induced activation of brain microglia/macrophages and inhibited circulating tumor necrosis factor α, interleukin (IL)-7, and granulocyte cell-stimulating factor serum levels while increasing that of the anti-inflammatory cytokine IL-10. These results suggest that PNA5 is an excellent candidate and "first-in-class" therapy for treating VCID and other inflammation-related brain diseases.

Fast Cyclic Square-Wave Voltammetry To Enhance Neurotransmitter Selectivity and Sensitivity.

Although fast-scan cyclic voltammetry (FSCV) has been widely used for in vivo neurochemical detection, the sensitivity and selectivity of the technique can be further improved. In this study, we develop fast cyclic square-wave voltammetry (FCSWV) as a novel voltammetric technique that combines large-amplitude cyclic square-wave voltammetry (CSWV) with background subtraction. A large-amplitude, square-shaped potential was applied to induce cycling through multiple redox reactions within a square pulse to increase sensitivity and selectivity when combined with a two-dimensional voltammogram. As a result, FCSWV was significantly more sensitive than FSCV ( n = 5 electrodes, two-way ANOVA, p = 0.0002). In addition, FCSWV could differentiate dopamine from other catecholamines (e.g., epinephrine and norepinephrine) and serotonin better than conventional FSCV. With the confirmation that FCSWV did not influence local neuronal activity, despite the large amplitude of the square waveform, it could monitor electrically induced phasic changes in dopamine release in rat striatum before and after injecting nomifensine, a dopamine reuptake inhibitor.

Platform to Enable Combined Measurement of Dopamine and Neural Activity.

Complex behaviors depend on the coordination of the activities of ensembles of neurons and the release of neuromodulators such as dopamine. The mechanisms underlying such coordination are not well-understood due to a lack of instrumentation for combined and real-time monitoring of neuromodulator release and the activities of large ensembles of neurons. Here we describe a measurement platform that allows for the combined monitoring of electrophysiology from a high-density electrode array and dopamine dynamics from a carbon-fiber microelectrode. Integration of these two measurement systems was achieved through modification of the existing instrumentation. A shared grounded reference electrode was used in both systems to minimize electrical interference. Further, an optional solid-state-relay array positioned between the electrophysiological electrode array and amplifiers was added to provide additional electrical isolation. The capacity of the integrated measurement platform, termed DANA (Dopamine And Neural Activity), to measure action potentials (high frequency) and local-field oscillations (low frequency) was characterized in vitro using an artificial cerebral spinal fluid gelatin. In vivo recordings from the DANA platform in anesthetized rats demonstrated the ability of the system for near-simultaneous measurement of dopamine release and activity from multiple neurons both in distant brain regions (striatum and hippocampus) and within the same brain region (striatum). Furthermore, this system was shown to be sufficiently compact to measure activity in freely moving animals through recording of single-neuron activity, high-frequency local-field oscillations, and dopamine release.

In Vivo Ambient Serotonin Measurements at Carbon-Fiber Microelectrodes.

The mechanisms that control extracellular serotonin levels in vivo are not well-defined. This shortcoming makes it very challenging to diagnose and treat the many psychiatric disorders in which serotonin is implicated. Fast-scan cyclic voltammetry (FSCV) can measure rapid serotonin release and reuptake events but cannot report critically important ambient serotonin levels. In this Article, we use fast-scan controlled adsorption voltammetry (FSCAV), to measure serotonin's steady-state, extracellular chemistry. We characterize the "Jackson" voltammetric waveform for FSCAV and show highly stable, selective, and sensitive ambient serotonin measurements in vitro. In vivo, we report basal serotonin levels in the CA2 region of the hippocampus as 64.9 ± 2.3 nM (n = 15 mice, weighted average ± standard error). We electrochemically and pharmacologically verify the selectivity of the serotonin signal. Finally, we develop a statistical model that incorporates the uncertainty in in vivo measurements, in addition to electrode variability, to more critically analyze the time course of pharmacological data. Our novel method is a uniquely powerful analysis tool that can provide deeper insights into the mechanisms that control serotonin's extracellular levels.

Rapid Voltammetric Measurements at Conducting Polymer Microelectrodes Using Ultralow-Capacitance Poly(3,4-ethylenedioxythiophene):Tosylate.

UNLABELLED: We use a vapor-phase synthesis to generate conducting polymer films with low apparent capacitance and high conductance enabling rapid electrochemical measurements. Specifically, oxidative chemical vapor deposition was used to create thin films of poly(3,4-ethylenedioxythiophene):tosylate ( PEDOT: tosylate). These films had a conductance of 17.1 ± 1.7 S/cm. Furthermore, they had an apparent capacitance of 197 ± 14 µF/cm(2), which is an order of magnitude lower than current commercially available and previously reported PEDOT. Using a multistage photolithography process, these films were patterned into PEDOT: tosylate microelectrodes and were used to perform fast-scan cyclic voltammetry (FSCV) measurements. Using a scan rate of 100 V/s, we measured ferrocene carboxylic acid and dopamine by FSCV. In contrast to carbon-fiber microelectrodes, the reduction peak showed higher sensitivity when compared to the oxidation peak. The adsorption characteristics of dopamine at the polymer electrode were fit to a Langmuir isotherm. The low apparent capacitance and the microlithographic processes for electrode design make PEDOT: tosylate an attractive material for future applications as an implantable biosensor for FSCV measurements. Additionally, the integration of PEDOT: tosylate electrodes on plastic substrates enables new electrochemical measurements at this polymer using FSCV.

Biocompatible PEDOT:Nafion composite electrode coatings for selective detection of neurotransmitters in vivo.

A Nafion and poly(3,4-ethylenedioxythiophene) (PEDOT) containing composite polymer has been electropolymerized on carbon-fiber microelectrodes with the goal of creating a mechanically stable, robust, and controllable electrode coating that increases the selectivity and sensitivity of in vivo electrochemical measurements. The coating is deposited on carbon-fiber microelectrodes by applying a triangle waveform from +1.5 V to -0.8 V and back in a dilute solution of ethylenedioxythiophene (EDOT) and Nafion in acetonitrile. Scanning electron microscopy demonstrated that the coating is uniform and ∼100 nm thick. Energy-dispersive X-ray spectroscopy demonstrated that both sulfur and fluorine are present in the coating, indicating the incorporation of PEDOT (poly(3,4-ethylenedioxythiophene) and Nafion. Two types of PEDOT:Nafion coated electrodes were then analyzed electrochemically. PEDOT:Nafion-coated electrodes made using 200 µM EDOT exhibit a 10-90 response time of 0.46 ± 0.09 s versus 0.45 ± 0.11 s for an uncoated fiber in response to a 1.0 µM bolus of dopamine. The electrodes coated using a higher EDOT concentration (400 µM) are slower with a 10-90 response time of 0.84 ± 0.19 s, but display increased sensitivity to dopamine, at 46 ± 13 nA/µM, compared to 26 ± 6 nA/µM for the electrodes coated in 200 µM EDOT and 13 ± 2 nA/µM for an uncoated fiber. PEDOT:Nafion-coated electrodes were lowered into the nucleus accumbens of a rat, and both spontaneous and electrically evoked dopamine release were measured. In addition to improvements in sensitivity and selectivity, the coating dramatically reduces acute in vivo biofouling.

Microwave-plasma dry-etch for fabrication of conducting polymer microelectrodes.

An inexpensive dry etch technology based on a low-pressure microwave plasma generated in a countertop microwave oven is characterized for the patterning of a conductive polymer microelectrode. The etch process is described, and the microwave-generated plasma is characterized by emission spectroscopy. The plasma is generated with an atmospheric mixture of mostly nitrogen and oxygen. A 10 µm wide band microelectrode composed of PEDOT:Tosylate, an optically transparent conductive polymer, is fabricated on a plastic substrate. Conductive polymer etch rates are approximately 280-300 nm/minute. A patterned microelectrode is characterized by atomic force microscopy. The horizontal distance of a 10-90% height of a plasma-etched 150 nm thick electrode was measured to be 360 ± 200 nm (n = 5). Electrodes are further characterized using steady-state cyclic voltammetry, and they have an electroactive area congruent with their geometric area. Finally, a complete device is assembled and used as a separation platform for biogenic amines. A microwave-etched 250 µm PEDOT:PSS electrode is employed for end-channel electrochemical detection on this microchip, where an electrophoretic separation of dopamine and catechol and a micellar electrokinetic chromatography separation of dopamine and serotonin are performed. Both mass and concentration LODs are comparable to other electrochemical detectors in an end-channel configuration. With the added advantages of easy processing, robustness, optical transparency, and low cost, we expect microwave-etched polymer films to be a viable alternative to traditional electrodes.

Fast voltammetry of metals at carbon-fiber microelectrodes: copper adsorption onto activated carbon aids rapid electrochemical analysis.

Rapid, in situ trace metal analysis is essential for understanding many biological and environmental processes. For example, trace metals are thought to act as chemical messengers in the brain. In the environment, some of the most damaging pollution occurs when metals are rapidly mobilized and transported during hydrologic events (storms). Electrochemistry is attractive for in situ analysis, primarily because electrodes are compact, cheap and portable. Electrochemical techniques, however, do not traditionally report trace metals in real-time. In this work, we investigated the fundamental mechanisms of a novel method, based on fast-scan cyclic voltammetry (FSCV), that reports trace metals with sub-second temporal resolution at carbon-fiber microelectrodes (CFMs). Electrochemical methods and geochemical models were employed to find that activated CFMs rapidly adsorb copper, a phenomenon that greatly advances the temporal capabilities of electrochemistry. We established the thermodynamics of surface copper adsorption and the electrochemical nature of copper deposition onto CFMs and hence identified a unique adsorption-controlled electrochemical mechanism for ultra-fast trace metal analysis. This knowledge can be exploited in the future to increase the sensitivity and selectivity of CFMs for fast voltammetry of trace metals in a variety of biological and environmental models.

Electrochemical generation of hydroxyl radicals for examining protein structure.

The use of hydroxyl radicals to covalently label the solvent-exposed surface of proteins has been shown to be a powerful tool to examine the structure of proteins and intermolecular interfaces. Current methods to generate hydroxyl radicals for footprinting experiments rely on the laser photolysis of H2O2 or the synchrotron radiolysis of water, which adds significant costs and/or complexity to the experiments. In this work, we develop the electro-Fenton reaction as a means to generate hydroxyl radicals for structural footprinting mass spectrometry experiments to complement current laser and synchrotron-based methods, while reducing the costs and complexity of initiating such experiments. The use of an electrochemical flow cell also enables control of the timing and extent of the radical generation process, while reducing the complexity typically associated with radical footprinting experiments. Ubiquitin, a model protein, was labeled with electro-Fenton generated hydroxyl radicals and top-down proteomics was used to verify oxidation sites on the protein surface.

Rethinking data collection and signal processing. 2. Preserving the temporal fidelity of electrochemical measurements.

Direct electrochemical measurements of biological events are often challenging because of the low signal relative to the magnitude of the background and noise. When choosing a data processing approach, the frequency and phase content of the data must be considered. Here, we employ a zero-phase (infinite impulse response (IIR)) filter to remove the noise from the analytical signal, while preserving the phase content. In fast-scan cyclic voltammetry, the frequency content of the signal is a function of the scan rate of the applied waveform. Fourier analysis was used to develop a relationship between scan rate and the filter cutoff frequency to maximize the reduction in noise, while not altering the true nature of the analytical signal. The zero-phase filter has the same effect as traditional filters with regards to increasing the signal-to-noise ratio. Because the zero-phase filter does not introduce a change to ΔEpeak, the heterogeneous electron rate transfer constant (0.10 cm/s) for ferrocene is calculated accurately. The zero-phase filter also improves electrochemical analysis of signaling molecules that have their oxidation potential close to the switching potential. Lastly, a quantitative approach to filtering amperometric traces of exocytosis based on the rise time was developed.

Fast-scan controlled-adsorption voltammetry for the quantification of absolute concentrations and adsorption dynamics.

Fast-scan cyclic voltammetry has depended on background subtraction to quantify small changes in neurotransmitter concentration. Because of this requirement, measurements of absolute concentrations using fast-scan cyclic voltammetry have been limited. Here we develop and characterize fast-scan controlled-adsorption voltammetry (FSCAV), which enables direct measurements of absolute concentrations in vitro without the use of flow injection to change the concentration. This enables probing the diffusion-controlled adsorption dynamics of biogenic amines and other adsorbing species. An implicit finite-difference model of mass-transport-limited adsorption was developed and is in agreement with experimental results. Optimization of FSCAV yielded a sensitivity of 81 ± 11 nA/µM for dopamine, corresponding to a limit of detection of 3.7 ± 0.5 nM. Through the combination of novel instrumentation and validated computer simulations, we show that FSCAV is an important measurement tool that can be used to determine absolute concentrations and study mass-transport-limited adsorption.

Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation.

Mass spectrometry imaging has been used here to suggest that changes in membrane structure drive lipid domain formation in mating single-cell organisms. Chemical studies of lipid bilayers in both living and model systems have revealed that chemical composition is coupled to localized membrane structure. However, it is not clear if the lipids that compose the membrane actively modify membrane structure or if structural changes cause heterogeneity in the surface chemistry of the lipid bilayer. We report that time-of-flight secondary ion mass spectrometry images of mating Tetrahymena thermophila acquired at various stages during mating demonstrate that lipid domain formation, identified as a decrease in the lamellar lipid phosphatidylcholine, follows rather than precedes structural changes in the membrane. Domains are formed in response to structural changes that occur during cell-to-cell conjugation. This observation has wide implications in all membrane processes.

Antagonism of kappa opioid receptors accelerates the development of L-DOPA-induced dyskinesia in a preclinical model of moderate dopamine depletion.

Levels of the opioid peptide dynorphin, an endogenous ligand selective for kappa-opioid receptors (KORs), its mRNA and pro-peptide precursors are differentially dysregulated in Parkinson's disease (PD) and following the development of l-DOPA-induced dyskinesia (LID). It remains unclear whether these alterations contribute to the pathophysiological mechanisms underlying PD motor impairment and the subsequent development of LID, or whether they are part of compensatory mechanisms. We sought to investigate nor-BNI, a KOR antagonist, 1) in the dopamine (DA)-depleted PD state, 2) during the development phase of LID, and 3) via measuring of tonic levels of striatal DA. While nor-BNI (3 mg/kg; s.c.) did not lead to functional restoration in the DA-depleted state, it affected the dose-dependent development of abnormal voluntary movements (AIMs) in response to escalating doses of l-DOPA in a rat PD model with a moderate striatal 6-hydroxdopamine (6-OHDA) lesion. We tested five escalating doses of l-DOPA (6, 12, 24, 48, 72 mg/kg; i.p.), and nor-BNI significantly increased the development of AIMs at the 12 and 24 mg/kg l-DOPA doses. However, after reaching the 72 mg/kg l-DOPA, AIMs were not significantly different between control and nor-BNI groups. In summary, while blocking KORs significantly increased the rate of development of LID induced by chronic, escalating doses of l-DOPA in a moderate-lesioned rat PD model, it did not contribute further once the overall severity of LID was established. While we observed an increase of tonic DA levels in the moderately lesioned dorsolateral striatum, there was no tonic DA change following administration of nor-BNI.