•Improved reaction conditions for benzoyl chloride labeling for HPLC–MS/MS analysis.•Novel assay of 70 neurologically relevant compounds using benzoyl chloride labeling.•Analysis of rat dialysate, ...fly tissue homogenate and hemolymph, human CSF and serum.•Stable-isotope labeled internal standard for all analytes for quantification.
Widely targeted metabolomic assays are useful because they provide quantitative data on large groups of related compounds. We report a high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method that utilizes benzoyl chloride labeling for 70 neurologically relevant compounds, including catecholamines, indoleamines, amino acids, polyamines, trace amines, antioxidants, energy compounds, and their metabolites. The method includes neurotransmitters and metabolites found in both vertebrates and insects. This method was applied to analyze microdialysate from rats, human cerebrospinal fluid, human serum, fly tissue homogenate, and fly hemolymph, demonstrating its broad versatility for multiple physiological contexts and model systems. Limits of detection for most assayed compounds were below 10nM, relative standard deviations were below 10%, and carryover was less than 5% for 70 compounds separated in 20min, with a total analysis time of 33min. This broadly applicable method provides robust monitoring of multiple analytes, utilizes small sample sizes, and can be applied to diverse matrices. The assay will be of value for evaluating normal physiological changes in metabolism in neurochemical systems. The results demonstrate the utility of benzoyl chloride labeling with HPLC–MS/MS for widely targeted metabolomics assays.
The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5'-phosphate as a cofactor. GAD ...exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5'-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.
Experimental efforts to understand how the brain represents, stores and processes information require high-fidelity recordings of multiple different forms of neural activity within functional ...circuits. Thus, creating improved technologies for large-scale recordings of neural activity in the live brain is a crucial goal in neuroscience. Over the past two decades, the combination of optical microscopy and genetically encoded fluorescent indicators has become a widespread means of recording neural activity in nonmammalian and mammalian nervous systems, transforming brain research in the process. In this review, we describe and assess different classes of fluorescent protein indicators of neural activity. We first discuss general considerations in optical imaging and then present salient characteristics of representative indicators. Our focus is on how indicator characteristics relate to their use in living animals and on likely areas of future progress.
Cellular and brain metabolism of dopamine can be correlated with a number of neurodegenerative disorders, and as such, in vivo analysis of dopamine in the presence of structurally related ...neurotransmitters (NT) represents a holy grail of neuroscience. Interference from those NTs generally does not allow selective electroanalysis of dopamine, which redox transformation overlaps with those of other catecholamines. In our previous work, we reported an electrochemical RNA-aptamer-based biosensor for specific analysis of dopamine (Analytical Chemistry, 2013; Vol. 85, p 121). However, the overall design of the biosensor restricted its stability and impeded its operation in serum. Here, we show that specific biorecognition and electroanalysis of dopamine in serum can be performed by the RNA aptamer tethered to cysteamine-modified gold electrodes via the alkanethiol linker. The stabilized dopamine aptasensor allowed continuous 20 h amperometric analysis of dopamine in 10% serum within the physiologically important 0.11 μM range and in the presence of catechol and such dopamine precursors and metabolites as norepinephrine and l-DOPA. In a flow-injection mode, the aptasensor response to dopamine was ∼1 s, the sensitivity of analysis, optimized by adjusting the aptamer surface coverage, was 67 ± 1 nA μM–1 cm–2, and the dopamine LOD was 62 nM. The proposed design of the aptasensor, exploiting both the aptamer alkanethiol tethering to the electrode and screening of the catecholamine-aptamer electrostatic interactions, allows direct monitoring of dopamine levels in biological fluids in the presence of competitive NT and thus may be further applicable in biomedical research.
•There is a PTSD-related block in ALLO synthesis at 3α-HSD.•The present study confirmed this finding in plasma.•Ability to measure ALLO in plasma is important for use in clinical care.
There is a ...need to identify new and more effective treatments for posttraumatic stress disorder (PTSD). Allopregnanolone and its stereoisomer pregnanolone (together termed ALLO) are metabolites of progesterone that positively and allosterically modulate GABA effects at GABAA receptors, thereby reducing anxiety and depression. Previous research revealed that women with PTSD had low cerebrospinal fluid (CSF) ALLO levels and a low ratio of ALLO to the allopregnanolone precursor 5α-DHP, consistent with deficient activity of the ALLO synthetic enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD). The current study examined ALLO and the ratio of ALLO to 5α-DHP in plasma at rest and in response to psychophysiological stressors in trauma-exposed, medication-free women with and without PTSD. Participants were examined twice in random order during the early follicular phase (eFP) and mid-luteal phase (mLP) of the menstrual cycle. Plasma neurosteroids were measured using gas chromatography-mass spectrometry. Results indicate that the ALLO to 5α-DHP ratio in plasma increases between the eFP and mLP. In addition, women with PTSD have a lower ratio of ALLO to 5α-DHP than trauma-exposed healthy women, as well as blunted increases in this ratio in response to a moderately stressful laboratory procedure, i.e., differential fear conditioning, across the menstrual cycle. Clinically feasible testing for 3α-HSD dysfunction is critical to translating this line of research into clinical care. Measurement of this ratio in plasma could facilitate patient stratification in clinical treatment trials, as well as precision medicine targeting of treatments that address ALLO synthesis deficits in women with PTSD.
Nicotinic acetylcholine receptors are ligand-gated ion channels that mediate fast chemical neurotransmission at the neuromuscular junction and have diverse signalling roles in the central nervous ...system. The nicotinic receptor has been a model system for cell-surface receptors, and specifically for ligand-gated ion channels, for well over a century. In addition to the receptors' prominent roles in the development of the fields of pharmacology and neurobiology, nicotinic receptors are important therapeutic targets for neuromuscular disease, addiction, epilepsy and for neuromuscular blocking agents used during surgery. The overall architecture of the receptor was described in landmark studies of the nicotinic receptor isolated from the electric organ of Torpedo marmorata. Structures of a soluble ligand-binding domain have provided atomic-scale insights into receptor-ligand interactions, while high-resolution structures of other members of the pentameric receptor superfamily provide touchstones for an emerging allosteric gating mechanism. All available high-resolution structures are of homopentameric receptors. However, the vast majority of pentameric receptors (called Cys-loop receptors in eukaryotes) present physiologically are heteromeric. Here we present the X-ray crystallographic structure of the human α4β2 nicotinic receptor, the most abundant nicotinic subtype in the brain. This structure provides insights into the architectural principles governing ligand recognition, heteromer assembly, ion permeation and desensitization in this prototypical receptor class.
Summary Background Refractory chronic cough causes substantial symptoms and quality-of-life impairment. Similarities between central reflex sensitisation in refractory chronic cough and neuropathic ...pain suggest that neuromodulators such as gabapentin might be effective for refractory chronic cough. We established the efficacy of gabapentin in patients with refractory chronic cough. Methods This randomised, double-blind, placebo-controlled trial was undertaken at an outpatient clinic in Australia. Adults with refractory chronic cough (>8 weeks' duration) without active respiratory disease or infection were randomly assigned to receive gabapentin (maximum tolerable daily dose of 1800 mg) or matching placebo for 10 weeks. Block randomisation was done with randomisation generator software, stratified by sex. Patients and investigators were masked to assigned treatment. The primary endpoint was change in cough-specific quality of life (Leicester cough questionnaire LCQ score) from baseline to 8 weeks of treatment, analysed by intention to treat. This study is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12608000248369. Findings 62 patients were randomly assigned to gabepentin (n=32) or placebo (n=30) and ten patients withdrew before the study end. Gabapentin significantly improved cough-specific quality of life compared with placebo (between-group difference in LCQ score during treatment period 1·80, 95% CI 0·56–3·04; p=0·004; number needed to treat of 3·58). Side-effects occurred in ten patients (31%) given gabapentin (the most common being nausea and fatigue) and three (10%) given placebo. Interpretation The treatment of refractory chronic cough with gabapentin is both effective and well tolerated. These positive effects suggest that central reflex sensitisation is a relevant mechanism in refractory chronic cough. Funding National Health and Medical Research Council of Australia and Hunter Medical Research Institute, Newcastle, Australia.
Type A γ-aminobutyric acid receptors (GABA
Rs) are the principal mediators of inhibitory neurotransmission in the human brain. Endogenous neurosteroids interact with GABA
Rs to regulate acute and ...chronic anxiety and are potent sedative, analgesic, anticonvulsant and anesthetic agents. Their mode of binding and mechanism of receptor potentiation, however, remain unknown. Here we report crystal structures of a chimeric GABA
R construct in apo and pregnanolone-bound states. The neurosteroid-binding site is mechanically coupled to the helices lining the ion channel pore and modulates the desensitization-gate conformation. We demonstrate that the equivalent site is responsible for physiological, heteromeric GABA
R potentiation and explain the contrasting modulatory properties of 3a versus 3b neurosteroid epimers. These results illustrate how peripheral lipid ligands can regulate the desensitization gate of GABA
Rs, a process of broad relevance to pentameric ligand-gated ion channels.
-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors important for synaptic plasticity, memory, and neuropsychiatric health. NMDAR hypofunction contributes to multiple disorders, ...including anti-NMDAR encephalitis (NMDARE), an autoimmune disease of the CNS associated with GluN1 antibody-mediated NMDAR internalization. Here we characterize the functional/pharmacological consequences of exposure to CSF from female human NMDARE patients on NMDAR function, and we characterize the effects of intervention with recently described positive allosteric modulators (PAMs) of NMDARs. Incubation (48 h) of rat hippocampal neurons of both sexes in confirmed NMDARE patient CSF, but not control CSF, attenuated NMDA-induced current. Residual NMDAR function was characterized by lack of change in channel open probability, indiscriminate loss of synaptic and extrasynaptic NMDARs, and indiscriminate loss of GluN2B-containing and GluN2B-lacking NMDARs. NMDARs tagged with N-terminal pHluorin fluorescence demonstrated loss of surface receptors. Thus, function of residual NMDARs following CSF exposure was indistinguishable from baseline, and deficits appear wholly accounted for by receptor loss. Coapplication of CSF and PAMs of NMDARs (SGE-301 or SGE-550, oxysterol-mimetic) for 24 h restored NMDAR function following 24 h incubation in patient CSF. Curiously, restoration of NMDAR function was observed despite washout of PAMs before electrophysiological recordings. Subsequent experiments suggested that residual allosteric potentiation of NMDAR function explained the persistent rescue. Further studies of the pathogenesis of NMDARE and intervention with PAMs may inform new treatments for NMDARE and other disorders associated with NMDAR hypofunction.
Anti-
-methyl-d-aspartate receptor encephalitis (NMDARE) is increasingly recognized as an important cause of sudden-onset psychosis and other neuropsychiatric symptoms. Current treatment leaves unmet medical need. Here we demonstrate cellular evidence that newly identified positive allosteric modulators of NMDAR function may be a viable therapeutic strategy.
PDF
