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Cognitive decline in Parkinson's Disease (PD) is a prevalent and undertreated aspect of disease. Currently, no therapeutics adequately improve this aspect of disease. It has been previously shown that MAS receptor agonism via the glycosylated Angiotensin (1-7) peptide, PNA5, effectively reduces cognitive decline in models of vascular contributions to cognitive impairment and dementia (VCID). PNA5 has a brain/plasma ratio of 0.255 indicating good brain penetration. The goal of the present study was to determine if (1) systemic administration of PNA5 rescued cognitive decline in a mouse model of PD, and (2) if improvements in cognitive status could be correlated with changes to histopathological or blood plasma-based changes. Mice over-expressing human, wild-type α-synuclein (αSyn) under the Thy1 promoter (Thy1-αSyn mice, "line 61") were used as a model of PD with cognitive decline. Thy1-αSyn mice were treated with a systemic dose of PNA5, or saline (1 mg/kg/day) beginning at 4 months of age and underwent behavioral testing at 6 months, compared to WT. Subsequently, mice brains were analyzed for changes to brain pathology, and blood plasma was examined with a Multiplex Immunoassay for peripheral cytokine changes. Treatment with PNA5 reversed cognitive dysfunction measured by Novel Object Recognition and spontaneous alteration in a Y-maze in Thy1-αSyn mice. PNA5 treatment was specific to cognitive deficits, as fine-motor disturbances were unchanged. Enhanced cognition was associated with decreases in hippocampal inflammation and reductions in circulating levels of Macrophage Induced Protein (MIP-1β). Additionally, neuronal loss was blunted within the CA3 hippocampal region of PNA5-treated αsyn mice. These data reveal that PNA5 treatment reduces cognitive dysfunction in a mouse model of PD. These changes are associated with decreased MIP-1β levels in plasma identifying a candidate biomarker for target engagement. Thus, PNA5 treatment could potentially fill the therapeutic gap for cognitive decline in PD., Competing Interests: Declaration of competing interest Dr. Meredith Hay is the founder and CEO of ProNeurogen, Inc. a company that holds exclusive rights to the clinical development of the Ang-(1-7)/MasR agonist described herein. This interest played no role in the design of the studies; in the collection, analyses, or interpretation of data; in the writing of the Paper, or in the decision to present the results. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Sub-anesthetic ketamine treatment has been shown to be an effective therapy for treatment-resistant depression and chronic pain. Our group has previously shown that sub-anesthetic ketamine produces acute anti-parkinsonian, and acute anti-dyskinetic effects in preclinical models of Parkinson's disease (PD). Ketamine is a multifunctional drug and exerts effects through blockade of N-methyl-d-aspartate receptors but also through interaction with the opioid system. In this report, we provide detailed pharmacokinetic rodent data on ketamine and its main metabolites following an intraperitoneal injection, and second, we explore the pharmacodynamic properties of ketamine in a rodent PD model with respect to the opioid system, using naloxone, a pan-opioid receptor antagonist, in unilateral 6-hydroxydopamine-lesioned male rats, treated with 6 mg/kg levodopa (l-DOPA) to establish a model of l-DOPA-induced dyskinesia (LID). As previously reported, we showed that ketamine (20 mg/kg) is highly efficacious in reducing LID and now report that the magnitude of this effect is resistant to naloxone (3 and 5 mg/kg). The higher naloxone dose of 5 mg/kg, however, led to an extension of the time-course of the LID, indicating that opioid receptor activation, while not a prerequisite for the anti-dyskinetic effects of ketamine, still exerts an acute modulatory effect. In contrast to the mild modulatory effect on LID, we found that naloxone added to the anti-parkinsonian activity of ketamine, further reducing the akinetic phenotype. In conclusion, our data show opioid receptor blockade differentially modulates the acute anti-parkinsonian and anti-dyskinetic actions of ketamine, providing novel mechanistic information to support repurposing ketamine for individuals with LID., Competing Interests: Declaration of competing interest SJS and TF have a patent for the use of ketamine as a novel treatment for levodopa-induced dyskinesia associated with Parkinson's disease that was licensed to PharmaTher Inc. in 2020. They have consulted for PharmaTher Inc. in 2021 and 2023. TF also received travel support in 2022 and 2023., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Opioids acting at the mu opioid receptor (MOR) remain the most effective treatment for moderate to severe pain, but their use is limited by serious side effects. We have shown that a cyclized analog of endomorphin-1 provided pain relief comparable to that of morphine with reduction or absence of several side effects, including abuse liability. Glycosylation can promote penetration of cellular barriers. Here we developed cyclic glycopeptide endomorphin (glycoEM) analogs as drug candidates for potent and long-lasting analgesia. The analogs were assessed in receptor binding and functional assays and for blood-brain barrier penetration by microdialysis and MS. Two of the analogs showed MOR selectivity and more potent and longer lasting antinociception than morphine in male and female mice. Comparable antinociception occurred at A2 doses 5-fold lower (20-fold on a molar basis) than morphine doses. The results support further study of the glycoEMs for clinical application., Competing Interests: The authors declare the following competing financial interest(s): JZ, LS and RP are inventors on patents for compounds described here., (© 2024 The Authors. Published by American Chemical Society.)

Electrical brain stimulation has been used in vivo and in vitro to investigate neural circuitry. Historically, stimulation parameters such as amplitude, frequency, and pulse width were varied to investigate their effects on neurotransmitter release and behavior. These experiments have traditionally employed fixed-frequency stimulation patterns, but it has previously been found that neurons are more precisely tuned to variable input. Introducing variability into the interpulse interval of stimulation pulses will inform on how dopaminergic release can be modulated by variability in pulse timing. Here, dopaminergic release in rats is monitored in the nucleus accumbens (NAc), a key dopaminergic center which plays a role in learning and motivation, by fast-scan cyclic voltammetry. Dopaminergic release in the NAc could also be modulated by stimulation region due to differences in connectivity. We targeted two regions for stimulation─the medial forebrain bundle (MFB) and the medial prefrontal cortex (mPFC)─due to their involvement in reward processing and projections to the NAc. Our goal is to investigate how variable interpulse interval stimulation patterns delivered to these regions affect the time course of dopamine release in the NAc. We found that stimulating the MFB with these variable stimulation patterns saw a highly responsive, frequency-driven dopaminergic response. In contrast, variable stimulation patterns applied to the mPFC were not as sensitive to the variable frequency changes. This work will help inform on how stimulation patterns can be tuned specifically to the stimulation region to improve the efficiency of electrical stimulation and control dopamine release.

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 .

Serotonin (5-HT) is a monoamine neurotransmitter in the peripheral, enteric, and central nervous systems (CNS). Within the CNS, serotonin is principally involved in mood regulation and reward-seeking behaviors. It is a critical regulator in CNS pathologies such as major depressive disorder, addiction, and schizophrenia. Consequently, in vivo serotonin measurements within the CNS have emerged as one of many promising approaches to investigating the pathogenesis, progression, and treatment of these and other neuropsychiatric conditions. These techniques vary in methods, ranging from analyte sampling with microdialysis to voltammetry. Provided this diversity in approach, inherent differences between techniques are inevitable. These include biosensor size, temporal/spatial resolution, and absolute value measurement capabilities, all of which must be considered to fit the prospective researcher's needs. In this review, we summarize currently available methods for the measurement of serotonin, including novel voltammetric absolute value measurement techniques. We also detail serotonin's role in various neuropsychiatric conditions, highlighting the role of phasic and tonic serotonergic neuronal firing within each where relevant. Lastly, we briefly review the present clinical application of these techniques and discuss the potential of a closed-loop monitoring and neuromodulation system utilizing deep brain stimulation (DBS).

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Acute and chronic pain is often treated with opioids despite the negative side effects of constipation, physical dependence, respiratory depression, and overdose. The misuse of opioid analgesics has given rise to the opioid crisis/epidemic, and alternate nonaddictive analgesics are urgently needed. Oxytocin, a pituitary hormone, is an alternative to the small molecule treatments available and has been used as an analgesic as well as for the treatment and prevention of opioid use disorder (OUD). Clinical implementation is limited by its poor pharmacokinetic profile, a result of the labile disulfide bond between two cysteine residues in the native sequence. Stable brain penetrant oxytocin analogues have been synthesized by replacement of the disulfide bond with a stable lactam and glycosidation of the C-terminus. These analogues show exquisite selectivity for the oxytocin receptor and potent in vivo antinociception in mice following peripheral (i.v.) administration, supporting further study of their clinical potential., Competing Interests: The authors declare the following competing financial interest(s): J.M.S., M.L.H., T.F., and R.P. are co-founders of Teleport Pharmaceuticals, LLC, a company for the development and commercialization of glycosylated peptides for neurodegenerative disease and other neurological conditions. J.M.S. is an equity holder in Botanical Results, LLC, a local cannabidiol company; no company products or interests were tested here., (© 2023 American Chemical Society.)

Neurotransmitters, such as dopamine and serotonin, are responsible for mediating a wide array of neurologic functions, from memory to motivation. From measurements using fast scan cyclic voltammetry (FSCV), one of the main tools used to detect synaptic efflux of neurochemicals in vivo , principal component regression (PCR), has been commonly used to predict the identity and concentrations of neurotransmitters. However, the sensitivity and discrimination performance of PCR have room for improvement, especially for analyzing mixtures of similar oxidizable neurochemicals. Deep learning may be able to address these challenges. To date, there have been a few studies to apply machine learning to FSCV, but no attempt to apply deep learning to neurotransmitter mixture discrimination and no comparative study have been performed between PCR and deep learning methods to demonstrate which is more accurate for FSCV analysis so far. In this study, we compared the neurochemical identification and concentration estimation performance of PCR and deep learning in an analysis of FSCV recordings of catecholamine and indolamine neurotransmitters. Both analysis methods were tested on in vitro FSCV data with a single or mixture of neurotransmitters at the desired concentration. In addition, the estimation performance of PCR and deep learning was compared in incorporation with in vivo experiments to evaluate the practical usage. Pharmacological tests were also conducted to see whether deep learning would track the increased amount of catecholamine levels in the brain. Using conventional FSCV, we used five electrodes and recorded in vitro background-subtracted cyclic voltammograms from four neurotransmitters, dopamine, epinephrine, norepinephrine, and serotonin, with five concentrations of each substance, as well as various mixtures of the four analytes. The results showed that the identification accuracy errors were reduced 5-20% by using deep learning compared to using PCR for mixture analysis, and the two methods were comparable for single analyte analysis. The applied deep-learning-based method demonstrated not only higher identification accuracy but also better discrimination performance than PCR for mixtures of neurochemicals and even for in vivo testing. Therefore, we suggest that deep learning should be chosen as a more reliable tool to analyze FSCV data compared to conventional PCR methods although further work is still needed on developing complete validation procedures prior to widespread use.

There is an unmet clinical need for curative therapies to treat neurodegenerative disorders. Most mainstay treatments currently on the market only alleviate specific symptoms and do not reverse disease progression. The Pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide hormone, has been extensively studied as a potential regenerative therapeutic. PACAP is widely distributed in the central nervous system (CNS) and exerts its neuroprotective and neurotrophic effects via the related Class B GPCRs PAC1, VPAC1, and VPAC2, at which the hormone shows roughly equal activity. Vasoactive intestinal peptide (VIP) also activates these receptors, and this close analogue of PACAP has also shown to promote neuronal survival in various animal models of acute and progressive neurodegenerative diseases. However, PACAP's poor pharmacokinetic profile (non-linear PK/PD), and more importantly its limited blood-brain barrier (BBB) permeability has hampered development of this peptide as a therapeutic. We have demonstrated that glycosylation of PACAP and related peptides promotes penetration of the BBB and improves PK properties while retaining efficacy and potency in the low nanomolar range at its target receptors. Furthermore, judicious structure-activity relationship (SAR) studies revealed key motifs that can be modulated to afford compounds with diverse selectivity profiles. Most importantly, we have demonstrated that select PACAP glycopeptide analogues ( 2LS80Mel and 2LS98Lac ) exert potent neuroprotective effects and anti-inflammatory activity in animal models of traumatic brain injury and in a mild-toxin lesion model of Parkinson's disease, highlighting glycosylation as a viable strategy for converting endogenous peptides into robust and efficacious drug candidates., Competing Interests: Conflict of Interest: CA, LS, JS, MH, TF, and RP hold patents related to the content. JS, MH, TF, and RP have equity in Teleport Pharmaceuticals, LLC, a UArizona biotech startup. This interest played no role in the design of the studies; in the collection, analyses, or interpretation of data; in the writing of the Poster, or in the decision to present the results. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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.

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., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

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., Competing Interests: Declaration of competing interest The authors declare no competing financial interest.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Pharmacotherapy with L-DOPA remains the gold-standard therapy for PD, but is often limited by the development of the common side effect of L-DOPA-induced dyskinesia (LID), which can become debilitating. The only effective treatment for disabling dyskinesia is surgical therapy (neuromodulation or lesioning), therefore effective pharmacological treatment of LID is a critical unmet need. Here, we show that sub-anesthetic doses of ketamine attenuate the development of LID in a rodent model, while also having acute anti-parkinsonian activity. The long-term anti-dyskinetic effect is mediated by brain-derived neurotrophic factor-release in the striatum, followed by activation of ERK1/2 and mTOR pathway signaling. This ultimately leads to morphological changes in dendritic spines on striatal medium spiny neurons that correlate with the behavioral effects, specifically a reduction in the density of mushroom spines, a dendritic spine phenotype that shows a high correlation with LID. These molecular and cellular changes match those occurring in hippocampus and cortex after effective sub-anesthetic ketamine treatment in preclinical models of depression, and point to common mechanisms underlying the therapeutic efficacy of ketamine for these two disorders. These preclinical mechanistic studies complement current ongoing clinical testing of sub-anesthetic ketamine for the treatment of LID by our group, and provide further evidence in support of repurposing ketamine to treat individuals with PD. Given its clinically proven therapeutic benefit for both treatment-resistant depression and several pain states, very common co-morbidities in PD, sub-anesthetic ketamine could provide multiple therapeutic benefits for PD in the future., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Diseases and disorders such as Parkinson disease, schizophrenia, and chronic pain are characterized by altered mesolimbic dopaminergic neurotransmission. Dopamine release in the nucleus accumbens influences behavior through both tonic and phasic signaling. Tonic dopamine levels are hypothesized to inversely regulate phasic signals through dopamine D2 receptor feedback inhibition. We tested this hypothesis directly in the context of ongoing pain. Tonic and phasic dopamine signals were measured using fast-scan controlled-adsorption voltammetry and fast-scan cyclic voltammetry, respectively, in the nucleus accumbens shell of male rats with standardized levels of anesthesia. Application of capsaicin to the cornea produced a transient decrease in tonic dopamine levels. During the pain-induced hypodopaminergic state, electrically evoked phasic dopamine release was significantly increased when compared to baseline, evoked phasic release. A second application of capsaicin to the same eye had a lessened effect on tonic dopamine suggesting desensitization of TRPV1 channels in that eye. Capsaicin treatment in the alternate cornea, however, again produced coincident decreased dopaminergic tone and increased phasic dopamine release. These findings occurred independently of stimulus lateralization relative to the hemisphere of dopamine measurement. Our data show that (1) the mesolimbic dopamine circuit reliably encodes acute noxious stimuli; (2) ongoing pain produces decreases in dopaminergic tone; and (3) pain-induced decreases in tonic dopamine correspond to augmented evoked phasic dopamine release. Enhanced phasic dopamine neurotransmission resulting from salient stimuli may contribute to increased impulsivity and cognitive deficits often observed in conditions associated with decreased dopaminergic tone, including Parkinson disease and chronic pain.

Carbon-fiber microelectrodes allow for high spatial and temporal measurements of electroactive neurotransmitter measurements in vivo using fast-scan cyclic voltammetry (FSCV). However, common instrumentation for such measurements systems lack patient safety precautions. To add safety precautions as well as to overcome chemical and electrical noise, a two-electrode FSCV headstage was modified to introduce an active bandpass filter on the electrode side of the measurement amplifier. This modification reduced the measured noise and ac-coupled the voltammetric measurement and moved it from a classical direct current response measurement. ac-coupling not only reduces the measured noise, but also moves FSCV toward compliance with IEC-60601-1, enabling future human trials. Here, we develop a novel ac-coupled voltammetric measurement method of electroactive neurotransmitters. Our method allows for the modeling of a system to then calculate a waveform to compensate for added impedance and capacitance for the system. We describe how first by measuring the frequency response of the system and modeling the analogue response as a digital filter we can then calculate a predicted waveform. The predicted waveform, when applied to the bandpass filter, is modulated to create a desired voltage sweep at the electrode interface. Further, we describe how this modified FSCV waveform is stable, allowing for the measurement of electroactive neurotransmitters. We later describe a 32.7% sensitivity enhancement for dopamine with this new measurement as well as maintaining a calibration curve for dopamine, 3,4-dihydroxyphenylacetic acid, ascorbic acid, and serotonin in vitro . We then validate dopamine in vivo with stimulated release. Our developed measurement method overcame the added capacitance that would traditionally make a voltammetric measurement impossible, and it has wider applications in electrode sensor development, allowing for measurement with capacitive systems, which previously would not have been possible.

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.

Objectives: Dopamine-replacement utilizing L-DOPA is still the mainstay treatment for Parkinson's disease (PD), but often leads to development of L-DOPA-induced dyskinesia (LID), which can be as debilitating as the motor deficits. There is currently no satisfactory pharmacological adjunct therapy. The endogenous opioid peptides enkephalin and dynorphin are important co-transmitters in the direct and indirect striatofugal pathways and have been implicated in genesis and expression of LID. Opioid receptor antagonists and agonists with different selectivity profiles have been investigated for anti-dyskinetic potential in preclinical models. In this study we investigated effects of the highly-selective μ-opioid receptor antagonist CTAP (> 1200-fold selectivity for μ- over δ-opioid receptors) and a novel glycopeptide congener (gCTAP5) that was glycosylated to increase stability, in the standard rat LID model., Results: Intraperitoneal administration (i.p.) of either 0.5 mg/kg or 1 mg/kg CTAP and gCTAP5 completely blocked morphine's antinociceptive effect (10 mg/kg; i.p.) in the warm water tail-flick test, showing in vivo activity in rats after systemic injection. Neither treatment with CTAP (10 mg/kg; i.p.), nor gCTAP5 (5 mg/kg; i.p.) had any effect on L-DOPA-induced limb, axial, orolingual, or locomotor abnormal involuntary movements. The data indicate that highly-selective μ-opioid receptor antagonism alone might not be sufficient to be anti-dyskinetic.

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.

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., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

For over two decades, fast-scan cyclic voltammetry (FSCV) has served as a reliable analytical method for monitoring dopamine release in near real-time in vivo. However, contemporary FSCV techniques have been limited to measure only rapid (on the order of seconds, i.e. phasic) changes in dopamine release evoked by either electrical stimulation or elicited by presentation of behaviorally salient stimuli, and not slower changes in the tonic extracellular levels of dopamine (i.e. basal concentrations). This is because FSCV is inherently a differential method that requires subtraction of prestimulation tonic levels of dopamine to measure phasic changes relative to a zeroed baseline. Here, we describe the development and application of a novel voltammetric technique, multiple cyclic square wave voltammetry (M-CSWV), for analytical quantification of tonic dopamine concentrations in vivo with relatively high temporal resolution (10 s). M-CSWV enriches the electrochemical information by generating two dimensional voltammograms which enable high sensitivity (limit of detection, 0.17 nM) and selectivity against ascorbic acid, and 3,4-dihydroxyphenylacetic acid (DOPAC), including changes in pH. Using M-CSWV, a tonic dopamine concentration of 120 ± 18 nM (n = 7 rats, ± SEM) was determined in the striatum of urethane anethetized rats. Pharmacological treatments to elevate dopamine by selectively inhibiting dopamine reuptake and to reduce DOPAC by inhibition of monoamine oxidase supported the selective detection of dopamine in vivo. Overall, M-CSWV offers a novel voltammetric technique to quantify levels and monitor changes in tonic dopamine concentrations in the brain to further our understanding of the role of dopamine in normal behavior and neuropsychiatric disorders., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

Dopamine (DA)-replacement therapy utilizing l-DOPA is the gold standard symptomatic treatment for Parkinson's disease (PD). A critical complication of this therapy is the development of l-DOPA-induced dyskinesia (LID). The endogenous opioid peptides, including enkephalins and dynorphin, are co-transmitters of dopaminergic, GABAergic, and glutamatergic transmission in the direct and indirect striatal output pathways disrupted in PD, and alterations in expression levels of these peptides and their precursors have been implicated in LID genesis and expression. We have previously shown that the opioid glycopeptide drug MMP-2200 (a.k.a. Lactomorphin), a glycosylated derivative of Leu-enkephalin mediates potent behavioral effects in two rodent models of striatal DA depletion. In this study, the mixed mu-delta agonist MMP-2200 was investigated in standard preclinical rodent models of PD and of LID to evaluate its effects on abnormal involuntary movements (AIMs). MMP-2200 showed antiparkinsonian activity, while increasing l-DOPA-induced limb, axial, and oral (LAO) AIMs by ∼10%, and had no effect on dopamine receptor 1 (D 1 R)-induced LAO AIMs. In contrast, it markedly reduced dopamine receptor 2 (D 2 R)-like-induced LAO AIMs. The locomotor AIMs were reduced by MMP-2200 in all three conditions. The N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801 has previously been shown to be anti-dyskinetic, but only at doses that induce parkinsonism. When MMP-2200 was co-administered with MK-801, MK-801-induced pro-parkinsonian activity was suppressed, while a robust anti-dyskinetic effect remained. In summary, the opioid glycopeptide MMP-2200 reduced AIMs induced by a D 2 R-like agonist, and MMP-2200 modified the effect of MK-801 to result in a potent reduction of l-DOPA-induced AIMs without induction of parkinsonism., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The medial prefrontal cortex (mPFC) coordinates goal-directed behaviors, which may be mediated through mPFC regulation of dopamine release in the nucleus accumbens (NAc). Furthermore, frequency-specific oscillatory activity between the frontal cortex and downstream structures may facilitate inter-region communication. Although high-frequency (e.g., 60 Hz) mPFC stimulation is known to increase basal dopamine levels in the NAc, little is known about how phasic dopamine release is affected by mPFC stimulation. Understanding the frequency-specific control of phasic dopamine release by mPFC stimulation could elucidate mechanisms by which the mPFC modulates other regions. It could also inform optimization of deep brain stimulation for treatment of neurological disorders., Objective: The goal of this work was to characterize the frequency response of NAc dopamine release resultant from mPFC stimulation. We hypothesized that the magnitude of dopamine release in the NAc would increase with increasing stimulation frequency., Methods: Electrical stimulation of the mPFC of anesthetized rats was delivered at 4-60 Hz and at varying durations while measuring NAc dopamine release with fast-scan cyclic voltammetry., Results: mPFC stimulation resulted in phasic dopamine release in the NAc. Furthermore, 20 Hz stimulation evoked the largest peak response for stimulation intervals >5 s when compared to higher or lower frequencies., Conclusions: Activation of the mPFC drives dopamine release in the NAc in a complex frequency- and duration-dependent manner. This has implications for the use of deep brain stimulation treatment of disorders marked by dopaminergic dysregulation, and suggest that mPFC may exert more specialized control over neuromodulator release than previously understood., (Copyright © 2017. Published by Elsevier Inc.)

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.

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.

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.

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., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Tonic dopamine (DA) levels influence the activity of dopaminergic neurons and the dynamics of fast dopaminergic transmission. Although carbon fiber microelectrodes and fast-scan cyclic voltammetry (FSCV) have been extensively used to quantify stimulus-induced release and uptake of DA in vivo and in vitro, this technique relies on background subtraction and thus cannot provide information about absolute extracellular concentrations. It is also generally not suitable for prolonged (>90 s) recordings due to drift of the background current. A recently reported, modified FSCV approach called fast-scan controlled-adsorption voltammetry (FSCAV) has been used to assess tonic DA levels in solution and in the anesthetized mouse brain. Here we describe a novel extension of FSCAV to investigate pharmacologically induced, slowly occurring changes in tonic (background) extracellular DA concentration, and phasic (stimulated) DA release in brain slices. FSCAV was used to measure adsorption dynamics and changes in DA concentration (for up to 1.5 h, sampling interval 30 s, detection threshold < 10 nM) evoked by drugs affecting DA release and uptake (amphetamine, l-DOPA, pargyline, cocaine, Ro4-1284) in submerged striatal slices obtained from rats. We also show that combined FSCAV-FSCV recordings can be used for concurrent study of stimulated release and changes in tonic DA concentration. Our results demonstrate that FSCAV can be effectively used in brain slices to measure prolonged changes in extracellular level of endogenous DA expressed as absolute values, complementing studies conducted in vivo with microdialysis.

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.

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.

Tonic neurochemical dopamine activity underlies many brain functions; however a consensus on this important concentration has not yet been reached. In this work, we introduce in vivo fast-scan controlled-adsorption voltammetry to report tonic dopamine concentrations (90 ± 9 nM) and the dopamine diffusion coefficient (1.05 ± 0.09 × 10(-6) cm(2) s(-1)) in the mouse brain.

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.

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., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

A number of efforts have been made to understand how pituitary adenylate cyclase activating polypeptide (PACAP) functions as a neurotrophic and neuroprotective factor in Parkinson's disease (PD). Recently its effects on neurotransmission and underlying mechanisms have generated interest. In the present study, we investigate the effects of PACAP on catecholamine storage and secretion in PC12 cells with amperometry and transmission electron microscopy (TEM). PACAP increases quantal release induced by high K+ without significantly regulating the frequency of vesicle fusion events. TEM data indicate that the increased volume of the vesicle is mainly the result of enlargement of the fluidic space around the dense core. Moreover, the number of docked vesicles isn't modulated by PACAP. When cells are acutely treated with L-DOPA, the vesicular volume and quantal release both increase dramatically. It is likely that the characteristics of amperometric spikes from L-DOPA treated cells are associated with increased volume of individual vesicles rather than a direct effect on the mechanics of exocytosis. Treatment with PACAP versus L-DOPA results in different profiles of the dynamics of exocytosis. Release via the fusion pore prior to full exocytosis was observed with the same frequency following treatment with PACAP and L-DOPA. However, release events have a shorter duration and higher average current after PACAP treatment compared to L-DOPA. Furthermore, PACAP reduced the proportion of spikes having rapid decay time and shortened the decay time of both fast and slow spikes. In contrast, the distributions of the amperometric spike decay for both fast and slow spikes were shifted to longer time following L-DOPA treatment. Compared to L-DOPA, PACAP may produce multiple favorable effects on dopaminergic neurons, including protecting dopaminergic neurons against neurodegeneration and potentially regulating dopamine storage and release, making it a promising therapeutic agent for the treatment of PD.

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 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.

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.

Although our understanding of the actions of cocaine in the brain has improved, an effective drug treatment for cocaine addiction has yet to be found. Methylphenidate binds the dopamine transporter and increases extracellular dopamine levels in mammalian central nervous systems similar to cocaine, but it is thought to elicit fewer addictive and reinforcing effects owing to slower pharmacokinetics for different routes of administration between the drugs. This study utilizes the fruit fly model system to quantify the effects of oral methylphenidate on dopamine uptake during direct cocaine exposure to the fly CNS. The effect of methylphenidate on the dopamine transporter has been explored by measuring the uptake of exogenously applied dopamine. The data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter and the inhibition is concentration dependent. The peak height increased to 150% of control when cocaine was used to block the dopamine transporter for untreated flies but only to 110% for methylphenidate-treated flies. Thus, the dopamine transporter is mostly inhibited for the methylphenidate-fed flies before the addition of cocaine. The same is true for the rate of the clearance of dopamine measured by amperometry. For untreated flies the rate of clearance changes 40% when the dopamine transporter is inhibited with cocaine, and for treated flies the rate changes only 10%. The results were correlated to the in vivo concentration of methylphenidate determined by CE-MS. Our data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter for cocaine uptake, and the inhibition is concentration dependent.

Resolution of synaptic vesicle neurotransmitter content has mostly been limited to the study of stimulated release in cultured cell systems, and it has been controversial as to whether synaptic vesicle transmitter levels are saturated in vivo. We use electrochemical cytometry to count dopamine molecules in individual synaptic vesicles in populations directly sampled from brain tissue. Vesicles from the striatum yield an average of 33,000 dopamine molecules per vesicle, an amount considerably greater than typically measured during quantal release at cultured neurons. Vesicular content was markedly increased by L-DOPA or decreased by reserpine in a time-dependent manner in response to in vivo administration of drugs known to alter dopamine release. We investigated the effects of the psychostimulant amphetamine on vesicle content, finding that vesicular transmitter is rapidly depleted by 50% following in vivo administration, supporting the "weak base hypothesis" that amphetamine reduces synaptic vesicle transmitter and quantal size.

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.

Background current noise is often a significant limitation when using constant-potential amperometry for biosensor application such as amperometric recordings of transmitter release from single cells through exocytosis. In this paper, we fabricated thin-film electrodes of gold and conductive polymers and measured the current noise in physiological buffer solution for a wide range of different electrode areas. The noise measurements could be modeled by an analytical expression, representing the electrochemical cell as a resistor and capacitor in series. The studies revealed three domains; for electrodes with low capacitance, the amplifier noise dominated, for electrodes with large capacitances, the noise from the resistance of the electrochemical cell was dominant, while in the intermediate region, the current noise scaled with electrode capacitance. The experimental results and the model presented here can be used for choosing an electrode material and dimensions and when designing chip-based devices for low-noise current measurements.

Dopamine projections that extend from the ventral tegmental area to the striatum have been implicated in the biological basis for behaviors associated with reward and addiction. Until recently, it has been difficult to evaluate the complex balance of energy utilization and neural activity in the striatum. Many techniques such as electrophysiology, functional magnetic resonance imaging (fMRI), and fast-scan cyclic voltammetry have been employed to monitor these neurochemical and neurophysiological changes. In this brain region, physiological responses to cues and rewards cause local, transient pH changes. Oxygen and pH are coupled in the brain through a complex system of blood flow and metabolism as a result of transient neural activity. Indeed, this balance is at the heart of imaging studies such as fMRI. To this end, we measured pH and O(2) changes with fast-scan cyclic voltammetry in the striatum as indices of changes in metabolism and blood flow in vivo in three Macaca mulatta monkeys during reward-based behaviors. Specifically, the animals were presented with Pavlovian conditioned cues that predicted different probabilities of liquid reward. They also received free reward without predictive cues. The primary detected change consisted of pH shifts in the striatal extracellular environment following the reward predicting cues or the free reward. We observed three types of cue responses that consisted of purely basic pH shifts, basic pH shifts followed by acidic pH shifts, and purely acidic pH shifts. These responses increased with reward probability, but were not significantly different from each other. The pH changes were accompanied by increases in extracellular O(2). The changes in pH and extracellular O(2) are consistent with current theories of metabolism and blood flow. However, they were of sufficient magnitude that they masked dopamine changes in the majority of cases. The findings suggest a role of these chemical responses in neuronal reward processing.

In this paper we investigate the physical and electrochemical properties of micropatterned poly(3,4-ethylenedioxythiophene):tosylate (PEDOT:tosylate) microelectrodes for neurochemical detection. PEDOT:tosylate is a promising conductive polymer electrode material for chip-based bioanalytical applications such as capillary electrophoresis, high-performance liquid chromatography, and constant potential amperometry at living cells. Band electrodes with widths down to 3 μm were fabricated on polymer substrates using UV lithographic methods. The electrodes are electrochemically stable in a range between -200 mV and 700 mV vs. Ag/AgCl and show a relatively low resistance. A wide range of transmitters is shown to oxidize readily on the electrodes. Kinetic rate constants and half wave potentials are reported. The capacitance per area was found to be high (1670 ± 130 μF cm(-2)) compared to other thin film microelectrode materials. Finally, we use constant potential amperometry to measure the release of transmitters from a group of PC 12 cells. The results show how the current response decreases for a series of stimulations with high K(+) buffer.

Carbon-fiber-microelectrode arrays (MEAs) have been utilized to electrochemically image neurochemical secretion from individual pheochromocytoma (PC12) cells. Dopamine release events were electrochemically monitored from seven different locations on single PC12 cells using alternately constant-potential amperometry and fast-scan cyclic voltammetry (FSCV). Cyclic voltammetry, when compared to amperometry, can provide excellent chemical resolution; however, spatial and temporal resolution are both compromised. The spatial and temporal resolution of these two methods have been quantitatively compared and the differences explained using models of molecular diffusion at the nanogap between the electrode and the cell. A numerical simulation of the molecular flux reveals that the diffusion of dopamine molecules and electrochemical reactions both play important roles in the temporal resolution of electrochemical imaging. The simulation also reveals that the diffusion and electrode potential cause the differences in signal crosstalk between electrodes when comparing amperometry and FSCV.

Computational modeling and theoretical simulations have recently become important tools for the development, characterization, and validation of microfluidic devices. The recent proliferation of commercial user-friendly software has allowed researchers in the microfluidics community, who might not be familiar with computer programming or fluid mechanics, to acquire important information on microsystems used for sensors, velocimetry, detection for microchannel separations, and microfluidic fuel cells. We discuss the most popular computational technique for modeling these systems--the finite element method--and how it can be applied to model electrochemical processes coupled with hydrodynamic flow in microchannels. Furthermore, some of the limitations and challenges of these computational models are also discussed.