An Update of the Classical and Novel Methods Used For Measuring Fast Neurotransmitters during Normal and Brain Altered Function (original) (raw)
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Electrochemical Detection of Neurotransmitters
Biosensors, 2020
Neurotransmitters are important chemical messengers in the nervous system that play a crucial role in physiological and physical health. Abnormal levels of neurotransmitters have been correlated with physical, psychotic, and neurodegenerative diseases such as Alzheimer’s, Parkinson’s, dementia, addiction, depression, and schizophrenia. Although multiple neurotechnological approaches have been reported in the literature, the detection and monitoring of neurotransmitters in the brain remains a challenge and continues to garner significant attention. Neurotechnology that provides high-throughput, as well as fast and specific quantification of target analytes in the brain, without negatively impacting the implanted region is highly desired for the monitoring of the complex intercommunication of neurotransmitters. Therefore, it is crucial to develop clinical assessment techniques that are sensitive and reliable to monitor and modulate these chemical messengers and screen diseases. This r...
Journal of Neuroscience Methods, 1992
We have attempted to demonstrate the technical requirements and performance of a microscopical assay using a densitometric application of image analysis to measure immunohistochemical stain intensity. Not surprisingly, the techniques required were more demanding than those used for the quantification of field and object parameters in the nervous system. The following areas of methodology have been shown to be important: (1) use of buffers free of metallic ions for tissue processing, (2) selection and titration of first and second layer antibodies, (3) reduction and control of fading of fluorescence, (4) selection of microscopical and imaging equipment to give accurate, sensitive and uniform representations of low-light biological images, and (5) use of appropriate image analysis algorithms in order to generate binary images that match the spatial and intensity distributions of immunostaining. Incorporation of these techniques into our assay system gave sensitive measurements of the time-scale of uptake of 5-hydroxytryptamine (5-HT) into sympathetic nerve terminals. The microscopical assay appears to have advantages over alternative approaches used for studies of neurotransmitter dynamics, particularly in small, heterogenous tissue samples.
Neuroimage, 2003
Over the last decade, it has become possible to study theories of cognition using positron emission tomography (PET) and functional magnetic resonance imaging (fMRI). These methods yield statistical parametric maps of changes in cerebral blood flow (CBF) elicited by cognitive tasks. A limitation of these studies is that they provide no information about the underlying neurochemistry. However, it is possible to extend the concept of activation studies to include measurements targeting neurotransmitters and specific receptor populations. Cognitive activation increases neuronal firing rate, increasing the endogenous neurotransmitter level. The increased neurotransmitter level can be used to alter the kinetics of specifically bound radioligands. We describe a new approach to the design and analysis of neuromodulation experiments. This approach uses PET, a single-scan session design, and a linear extension of the simplified reference region model (LSSRM) that accounts for changes in ligand binding induced by cognitive tasks or drug challenge. In the LSSRM, an "activation" parameter is included that represents the presence or absence of change in apparent dissociation rate. Activation of the neurotransmitter is detected statistically when the activation parameter is shown to violate the null hypothesis. Simulation was used to explore the properties of the LSSRM with regard to model identifiability, effect of statistical noise, and confounding effects of CBF-related changes. Simulation predicted that it is possible to detect and map neuromodulatory changes in single-subject designs. A human study was conducted to confirm the predictions of simulation using 11 C-raclopride and a motor planning task. Parametric images of transport, binding potential, areas of significant dopamine release, and statistical parameters were computed. Examination of the kinetics of activation demonstrated that maximum dopamine release occurred immediately following task initiation and then decreased with a half-time of about 3 min. This method can be extended to explore neurotransmitter involvement in other behavioral and cognitive domains.
Journal of Neuroscience Methods, 2007
High performance liquid chromatography (HPLC) is widely used to determine neurotransmitter concentrations in the central nervous system (CNS). Finding the optimal methods to sample from CNS tissue poses a challenge for neuroscientists. Here we describe a method that allows assay of neurotransmitters (or other chemicals) in small regions (down to 180 μm in diameter) in in vitro preparations concurrently with electrophysiological recordings. The efficiency for measuring small amounts of chemicals is enhanced by a sample collecting pipette with filter paper at the tip that makes close contact with the target region in CNS tissue. With a wire plunger in the calibrated pipette controlled by a microsyringe pump, there is virtually no dead volume. Samples in a volume of 10 μl (taken, e.g., at 2 μL/min over 5 minutes) can be injected into a HPLC machine with microbore columns. We demonstrate the effectiveness of this method by measuring acetylcholine (ACh) in the ventral horn and its surrounding areas of the spinal cord in en bloc brainstem-spinal cord preparations. In control conditions, endogenous ACh levels in these regions were detectable. Application of neostigmine (an inhibitor of acetylcholinesterases (AChEs)) increased ACh concentrations, and at the same time, induced tonic/seizure-like activity in efferent motor output recorded from cervical ventral nerve roots. Higher ACh concentrations in the ventral horn were differentiated from nearby regions: the lateral and midline aspects of the ventral spinal cord. In addition, ACh in the preBötzinger Complex (preBötC) and the hypoglossal nucleus in medullary slice preparations can also be measured. Our results indicate that the method proposed in this study can be used to measure neurotransmitters in small and localized CNS regions. Correlation between changes in neurotransmitters in target regions and the neuronal activities can be revealed in vitro. Our data also suggest that there is endogenous ACh release in spinal ventral motor columns at 4 th cervical (C4) level that regulates the respiratory-related motor activity.
Medycyna Pracy, 2017
Background: Glutamate (Glu) and γ-aminobutyric acid (GABA) are the main neurotransmitters in the central nervous system for excitatory and inhibitory processes, respectively. Monitoring these neurotransmitters is an essential tool in establishing pathological functions, among others in terms of occupational exposure to toxic substances. Material and Methods: We present modification of the HPLC (high-performance liquid chromatography) to the UPLC (ultra-performance liquid chromatography) method for the simultaneous determination of glutamate and γ-aminobutyric acid in a single injection. The isocratic separation of these neurotransmitter derivatives was performed on Waters Acquity BEH (ethylene bridged hybrid) C18 column with particle size of 1.7 μm at 35°C using a mobile phase consisting of 0.1 M acetate buffer (pH 6.0) and methanol (60:40, v/v) at a flow rate of 0.3 ml/min. The analytes were detected with the fluorescence detector (FLD) using derivatization with o-phthaldialdehyde (OPA), resulting in excitation at 340 nm and emission at 455 nm. Results: Several validation parameters including linearity (0.999), accuracy (101.1%), intra-day precision (1.52-1.84%), inter-day precision (2.47-3.12%), limit of detection (5-30 ng/ml) and quantification (100 ng/ml) were examined. The developed method was also used for the determination of these neurotransmitters in homogenates of selected rat brain structures. Conclusions: The presented UPLC-FLD is characterized by shorter separation time (3.5 min), which is an adaptation of the similar HPLC methods and is an alternative for more expensive references techniques such as liquid chromatography coupled with tandem mass-spectrometry (LC-MS/MS) methods.
Journal of Pharmaceutical and Biomedical Analysis, 2014
Quantification of amino acid based neurotransmitters in extracellular fluids, such as those in the neuron synapse, presents a challenge to the analytical chemistry because of the absence of UV-or fluorescencedetectable functional groups and the low sensitivity in mass spectrometric detection. This report describes a novel use of the succinimide reagent, N-␣-Boc-l-tryptophan hydroxysuccinimide ester (Boc-TRP), for the pre-column derivatization to simultaneously quantify multiple neurotransmitters in the rat brain microdialysis samples. The Boc-TRP derivatization was rapid and quantitative in phosphate the buffer (pH 7.4) at room temperature. The derivatized neurotransmitters were suitable for rapid LC/MS quantification with less than 3-min chromatographic separation. The Boc-group in the derivatized product generated unique fragmentation patterns in the triple quadrupole mass spectrometric analysis under Multiple Reaction Monitoring mode and significantly increased the specificity and sensitivity. The derivatization and rapid LC/MS quantification method developed in this study showed a linear dynamic range from single digit nM to 1000 nM with coefficient greater than 0.990. At the LOQ, the accuracy ranged from 95 to 108% and the precision (CV%) was less than 20%. Since there was no concentration and reconstitution in the sample workup process, this derivatization approach simplified the neurotransmitter quantification of the brain microdialysis samples.