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.

Techniques for Measurement of Serotonin: Implications in Neuropsychiatric Disorders and Advances in Absolute Value Recording Methods.

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).

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.

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 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) with measuring tonic levels of striatal DA. Nor-BNI (3 mg/kg; s.c.) did not lead to functional restoration in the DA-depleted state, but a change in 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-hydroxydopamine (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 dosing with 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 saw an increase of tonic DA levels in the moderately lesioned dorsolateral striatum, there was no tonic DA change following administration of nor-BNI.

Neurochemical Concentration Prediction Using Deep Learning vs Principal Component Regression in Fast Scan Cyclic Voltammetry: A Comparison Study.

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.

Design and Synthesis of Brain Penetrant Glycopeptide Analogues of PACAP With Neuroprotective Potential for Traumatic Brain Injury and Parkinsonism.

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.

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.

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.

Preclinical evidence in support of repurposing sub-anesthetic ketamine as a treatment for L-DOPA-induced dyskinesia.

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.

Moving Fast-Scan Cyclic Voltammetry toward FDA Compliance with Capacitive Decoupling Patient Protection.

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.

A pain-induced tonic hypodopaminergic state augments phasic dopamine release in the nucleus accumbens.

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.

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.

Highly-selective µ-opioid receptor antagonism does not block L-DOPA-induced dyskinesia in a rodent model.

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.

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.

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.

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.

Tracking tonic dopamine levels in vivo using multiple cyclic square wave voltammetry.

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 ±â€¯18 nM (n = 7 rats, ±â€¯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.

The combination of the opioid glycopeptide MMP-2200 and a NMDA receptor antagonist reduced l-DOPA-induced dyskinesia and MMP-2200 by itself reduced dopamine receptor 2-like agonist-induced dyskinesia.

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 (D1R)-induced LAO AIMs. In contrast, it markedly reduced dopamine receptor 2 (D2R)-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 D2R-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.