Long-term effect of sub-anesthetic ketamine in reducing L-DOPA-induced dyskinesias in a preclinical model.

Low-dose sub-anesthetic ketamine infusion treatment has led to a long-term reduction of treatment-resistant depression and posttraumatic stress disorder (PTSD) symptom severity, as well as reduction of chronic pain states, including migraine headaches. Ketamine also is known to change oscillatory electric brain activity. One commonality between migraine headaches, depression, PTSD, Parkinson's disease (PD) and l-DOPA-induced dyskinesias (LID) is hypersynchrony of electric activity in the brain, including the basal ganglia. Therefore, we investigated the use of low-dose sub-anesthetic ketamine in the treatment of LID. In a preclinical rodent model of LID, ketamine (5-20mg/kg) led to long-term dose-dependent reduction of abnormal involuntary movements, only when low-dose ketamine was given for 10h continuously (5× i.p. injections two hours apart) and not after a single acute low-dose ketamine i.p. injection. Pharmacokinetic analysis of plasma levels showed ketamine and its major metabolites were not detectable any more at time points when a lasting anti-dyskinetic effect was seen, indicating a plastic change in the brain. This novel use of low-dose sub-anesthetic ketamine infusion could lead to fast clinical translation, and since depression and comorbid pain states are critical problems for many PD patients could open up the road to a new dual therapy for patients with LID.

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.

Improved Calibration of Voltammetric Sensors for Studying Pharmacological Effects on Dopamine Transporter Kinetics in Vivo.

The distribution and density of neurons within the brain poses many challenges when making quantitative measurements of neurotransmission in the extracellular space. A volume neurotransmitter is released into the synapse during chemical communication and must diffuse through the extracellular space to an implanted sensor for real-time in situ detection. Fast-scan cyclic voltammetry is an excellent technique for measuring biologically relevant concentration changes in vivo; however, the sensitivity is limited by mass-transport-limited adsorption. Due to the resistance to mass transfer in the brain, the response time of voltammetric sensors is increased, which decreases the sensitivity and the temporal fidelity of the measurement. Here, experimental results reveal how the tortuosity of the extracellular space affects the response of the electrode. Additionally, a model of mass-transport-limited adsorption is utilized to account for both the strength of adsorption and the magnitude of the diffusion coefficient to calculate the response time of the electrode. The response time is then used to determine the concentration of dopamine released in response to salient stimuli. We present the method of kinetic calibration of in vivo voltammetric data and apply the method to discern changes in the KM for the murine dopamine transporter. The KM increased from 0.32 ± 0.08 µM (n = 3 animals) prior to drug administration to 2.72 ± 0.37 µM (n = 3 animals) after treatment with GBR-12909.

Differential effects of the NMDA receptor antagonist MK-801 on dopamine receptor D1- and D2-induced abnormal involuntary movements in a preclinical model.

Dopamine-replacement therapy with l-DOPA is still the gold standard treatment for Parkinson's disease (PD). One drawback is the common development of l-DOPA-induced dyskinesia (LID) in patients, which can be as disabling as the disease itself. There is no satisfactory adjunct therapy available. Glutamatergic transmission in the basal ganglia circuitry has been shown to be an important player in the development of LID. The N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 has previously been shown to reduce l-DOPA-induced abnormal involuntary movements (AIMs) in a rat preclinical model but only at concentrations that worsen parkinsonism. We investigated the contribution of the direct and indirect striatofugal pathways to these effects. In the direct pathway, dopamine D1 receptors (D1R) are expressed, whereas in the indirect pathway, dopamine D2 receptors (D2R) are expressed. We used the 6-hydroxydopamine-lesioned hemi-parkinsonian rat model initially primed with l-DOPA to induce dyskinesia. When the rats were then primed and probed with the D1R agonist SKF81297, co-injection of MK-801 worsened the D1R-induced limb, axial, and orolingual (LAO) AIMs by 18% (predominantly dystonic axial AIMs) but did not aggravate parkinsonian hypokinesia as reflected by a surrogate measure of ipsiversive rotations in this model. In contrast, when the rats were then primed and probed with the D2R agonist quinpirole, co-injection of MK-801 reduced D2R-induced LAO AIMs by 89% while inducing ipsiversive rotations. The data show that only inhibition of the indirect striatopallidal pathway is sufficient for the full anti-dyskinetic/pro-parkinsonian effects of the NMDA receptor antagonist MK-801, and that MK-801 modestly worsens dyskinesias that are due to activation of the direct striatonigral pathway alone. This differential activation of the glutamatergic systems in D1R- and D2R-mediated responses is relevant to current therapy for PD which generally includes a mixture of dopamine agonists and l-DOPA.

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-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.

Rethinking data collection and signal processing. 1. Real-time oversampling filter for chemical measurements.

Minimizing noise in chemical measurements is critical to achieve low limits of detection and accurate measurements. We describe a real-time oversampling filter that offers a method to reduce stochastic noise in a time-dependent chemical measurement. The power of this technique is demonstrated in its application to the separation of dopamine and serotonin by micellar electrokinetic chromatography with amperometric detection. Signal-to-noise ratios were increased by almost an order of magnitude, allowing for limits of detection of 100 and 120 amol, respectively. Real-time oversampling filters can be implemented using simple software algorithms and require no change to existing experimental apparatus. The application is not limited to analytical separations, and this technique can be used to improve the signal-to-noise ratio in any experiment where the necessary sampling rate is less than the maximum sampling rate of the analog-to-digital converter. Theory, implementation, and the performance of this filter are described. We propose that this technique should be the default mode of operation for an analog-to-digital converter.