Monitoring the concentration fluctuations of neurotransmitters in vivo is valuable for elucidating the chemical signals that underlie brain functions. Microdialysis sampling is a widely used tool for monitoring neurochemicals in vivo. The volume requirements of most techniques that have been coupled to microdialysis, such as HPLC, result in fraction collection times of minutes, thus limiting the temporal resolution possible. Further the time of analysis can become long for cases where many fractions are collected. Previously we have used direct analysis of dialysate by low-flow electrospray ionization-tandem mass spectrometry (ESI-MS/MS) on a triple quadrupole mass spectrometer to monitor acetylcholine, glutamate, and γ-amino-butyric acid to achieve multiplexed in vivo monitoring with temporal resolution of seconds. Here, we have expanded this approach to adenosine, dopamine, and serotonin. The method achieved limits of detection down to 2 nM, enabling basal concentrations of all these compounds, except serotonin, to be measured in vivo. Comparative analysis with LC-MS/MS showed accurate results for all compounds except for glutamate, possibly due to interference for this compound in vivo. Pairing this analysis with droplet microfluidics yields 11 s temporal resolution and can generate dialysate fractions down to 3 nL at rates up to 3 fractions per s from a microdialysis probe. The system is applied to multiplexed monitoring of neurotransmitter dynamics in response to stimulation by 100 mM K+ and amphetamine. These applications demonstrate the suitability of the droplet ESI-MS/MS method for monitoring short-term dynamics of up to six neurotransmitters simultaneously.

This study demonstrates how optimized liquid chromatography-mass spectrometry (LC-MS) conditions combined with a 50 µm internal diameter (I.D.) column operated at 100 nL/min enables high-sensitivity and high-throughput sample-limited proteomics analysis, including single-cell proteomics (SCP). Through systematic parameter evaluation, an optimized method was developed, capable of identifying ∼3,000 protein groups from 250 pg of HeLa protein digest using a 10-min gradient in the direct injection workflow using data-independent acquisition (DIA) from a library-free search method. Further improvements reduced the cycle time from 20 to 14.4 min by employing a trap-and-elute workflow, permitting 100 runs/day with 70% MS utilization. A proof of principle study indicated that ca . 1,700 protein groups were identified from single-cell samples without a library for label-free quantification (LFQ). In conclusion, we demonstrate a high-sensitivity LC-MS/MS configuration that serves the needs for limited sample analysis, permitting 100 runs/day throughout.

This work demonstrates the utility of high-throughput nanoLC-MS and label-free quantification (LFQ) for sample-limited bottom-up proteomics analysis, including single-cell proteomics (SCP). Conditions were optimized on a 50 μm internal diameter (I.D.) column operated at 100 nL/min in the direct injection workflow to balance method sensitivity and sample throughput from 24 to 72 samples/day. Multiple data acquisition strategies were also evaluated for proteome coverage, including data-dependent acquisition (DDA), wide-window acquisition (WWA), and wide-window data-independent acquisition (WW-DIA). Analyzing 250 pg HeLa digest with a 10-min LC gradient (72 samples/day) provided >900, >1,800, and >3,000 protein group identifications for DDA, WWA, and WW-DIA, respectively. Total method cycle time was further reduced from 20 to 14.4 min (100 samples/day) by employing a trap-and-elute workflow, enabling 70% mass spectrometer utilization. The method was applied to library-free DIA analysis of single-cell samples, yielding >1,700 protein groups identified. In conclusion, this study provides a high-sensitivity, high-throughput nanoLC-MS configuration for sample-limited proteomics.

Demonstrate the long-term robustness and consistent chromatographic performance of the next-generation Thermo Scientific ™ Vanquish ™ Neo UHPLC System under nanoLC conditions for bottom-up proteome profiling using a 75 µm I.D. × 50 cm Thermo Scientific ™ PepMap ™ Neo Column.

Demonstrate the superior performance of the Thermo Scientific ™ Vanquish ™ Neo next-generation low-flow UHPLC system for nanoLCMS bottom-up proteome profiling when coupled to a Thermo Scientific ™ Orbitrap Exploris ™ 480 mass spectrometer using the 75 µm I.D. × 75 cm Thermo Scientific ™ EASY-Spray ™ PepMap Neo column. Demonstrate system versatility and potential for new levels of proteomic depth coverage through the coupling of two 75 cm long nano-columns.

Demonstrate the performance of the Thermo Scientific ™ Vanquish ™ Neo UHPLC System, the next−generation nano-, capillary-and micro-flow LC, coupled to a Thermo Scientific ™ Orbitrap Exploris ™ 480 Mass Spectrometer for high-throughput bottom−up proteome profiling using a 75 µm I.D. × 15 cm Thermo Scientific ™ EASY-Spray ™ PepMap ™ Neo Column.

Introduction Nano-flow LC-MS analysis employing long columns and gradients at nano-flow rates has long been established as the method of choice for bottom-up, discovery proteomics. Nevertheless, the challenges associated with nano-flow LC have hitherto limited its application to the field of academic research. The main concerns associated with the technique include locating and eliminating the sources of dead volume, leaks, and capillary or column blockages, combined with the struggle to generate reproducible data. Together, such obstacles can result in the loss of precious time and irreplaceable samples. Another caveat of nanoLC-MS applications is their limited sample throughput and MS utilization. Lower flow rates result in proportionally long periods of MS idle time due to time-consuming sample loading and column equilibration particularly when long separation columns are employed. The latest low-flow UHPLC systems and columns have the potential to overcome many of these challenges and to usher in a new era of robustness and standardization in the field of lowflow LC-MS based analytics.

Introduction Nano-flow LC combined with mass spectrometry has been a mainstay for analyzing proteins and peptides for at least 20 years, particularly for proteomics research. The main advantage that nano-flow brings compared to analytical flow rates is increased sensitivity. Tryptic digestion of biological samples and tissues produces final sample mixtures of low volume, high complexity and with target peptide concentrations ranging from low attomole to high femtomole per microliter levels. In other areas of analytical science, such as clinical research applications, the adoption of nano-flow LC/MS has been limited. In such fields, limited robustness, repeatability, ease-of-use, and throughput are considered the main obstacles to nano-flow LC-MS adoption.1,2 The Vanquish Neo UHPLC system offers a flow range to support applications from nanoto micro-flow, which increases productivity for high sensitivity LC-MS workflows and produces the highest quality results for both LC-MS experts and novice users alike.

Goal Provide a customer application of the Thermo Scientific ™ Chromeleon ™ XPS Open Access software and discuss strengths and usability of a walk-up solution for synthetic chemists.

The splanchnic-chromaffin cell synapse is the site at which stimulus-secretion coupling in the adrenal medulla is regulated. However, since the discovery that acetylcholine underlies chemical signaling at this synapse, attention has been disproportionately placed on postsynaptic chromaffin cell function. As a result, the determinants of Ca-sensing and exocytosis from splanchnic nerves remain poorly understood. This study shows, for the first time, that a ubiquitous Ca-binding protein, synaptotagmin-7 (Syt7) is expressed within the neurons that innervate the adrenal medulla. In synapses that lack Syt7, evoked excitatory postsynaptic currents (EPSCs) are smaller in amplitude and decay with more rapid kinetics than wild-type synapses stimulated in an identical manner. EPSCs in Syt7-deficient adrenal slices also fail to facilitate, which is ordinarily a robust property of these synapses. These data are the first to implicate a role for Syt7 in regulating short-term synaptic plasticity in the peripheral nervous system.

Microfluidic chips can perform a broad range of automated fluid manipulation operations for chemical analysis including on-line reactions. Derivatization reactions carried out on-chip reduce manual sample preparation and improve experimental throughput. In this work we develop a chip for on-line benzoyl chloride derivatization coupled to microdialysis, an in vivo sampling technique. Benzoyl chloride derivatization is useful for the analysis of small molecule neurochemicals in complex biological matrices using HPLC-MS/MS. The addition of one or more benzoyl groups to small, polar compounds containing amines, phenols, thiols, and certain alcohols improves reversed phase chromatographic retention, electrospray ionization efficiency, and analyte stability. The current derivatization protocol requires a three-step manual sample preparation, which ultimately limits the utility of this method for rapid sample collection and large sample sets. A glass microfluidic chip was developed for derivatizing microdialysis fractions on-line as they exit the probe for collection and off-line analysis with HPLC-MS/MS. Calibration curves for 21 neurochemicals prepared using the on-chip method showed linearity (R-2 > 0.99), limits of detection (0.1-500 nM), and peak area RSDs (4-14%) comparable to manual derivatization. Method temporal resolution was investigated both in vitro and in vivo showing rapid rise times for all analytes, which was limited by fraction length (3 min) rather than the device. The platform was applied to basal measurements in the striatum of awake rats where 19 of 21 neurochemicals were above the limit of detection. For a typical 2 h study, a minimum of 120 pipetting steps are eliminated per animal. Such a device provides a useful tool for the analysis of small molecules in biological matrices which may extend beyond microdialysis to other sampling techniques.

D-serine is a physiologic coagonist of NMDA receptors (NMDARs) required for synaptic plasticity, but mechanisms that terminate D-serine signaling are unclear. In particular, the identity of unidirectional plasma membrane transporters that mediate D-serine reuptake has remained elusive. We report that D-serine and glutamine share the same neuronal transport system, consisting of the classic system A transporters Slc38a1 and Slc38a2. We show that these transporters are not saturated with glutamine in vivo and regulate the extracellular levels of D-serine and NMDAR activity. Glutamine increased the NMDAR-dependent long-term potentiation and the isolated NMDAR potentials at the Schaffer collateral-CA1 synapses, but without affecting basal neurotransmission in male mice. Glutamine did not increase the NMDAR potentials in slices from serine racemase knock-out mice, which are devoid of D-serine, indicating that the effect of glutamine is caused by outcompeting D-serine for a dual glutamine-D-serine transport system. Inhibition of the system A reduced the uptake of D-serine in synaptosomes and neuronal cultures of mice of either sex, while increasing the extracellular D-serine concentration in slices and in vivo by microdialysis. When compared with Slc38a2, the Slc38a1 transporter displayed more favorable kinetics toward the Denantiomer. Biochemical experiments with synaptosomes from Slc38a1 knock-down mice of either sex further support its role as a D-serine reuptake system. Our study identifies the first concentrative and electrogenic transporters mediating D-serine reuptake in vivo. In addition to their classical role in the glutamine-glutamate cycle, system A transporters regulate the synaptic turnover of D-serine and its effects on NMDAR synaptic plasticity.

Pressure-induced infusion of solutions into brain tissue is used both in research and in medicine. In medicine, convection enhanced delivery (CED) may be used to deliver agents to localized areas of the brain, such as with gene therapy to functional targets or with deep tumors not readily amenable to resection. However, clinical trials have demonstrated mixed results from CED. CED is limited by a lack of control of the infusion flow path and may cause damage or even neurological deficits due to neuronal distortion. In laboratory research, infusions may be achieved using pressure or using brief bursts of electrical current in iontophoresis. Electrokinetic convection enhanced delivery (ECED) has the potential to deliver drugs and other bioactive substances to local regions in the brain with improved control and lower applied pressures than pressure-based CED. ECED improves control over the infusion profile because the fluid follows the electrical current path and thus can be directed. Both small molecules and macromolecules can be delivered. Here we demonstrate proof-of-principal that electrokinetic (electroosmosis and electrophoresis) convection-enhanced delivery is a viable means for delivering solutes to the brain. We assessed the volume of tissue exposed to the infusates tris(2,2'-bipyridine)ruthenium(II) and fluorescent dextrans. Control of the direction of the transport was also achieved over distances ranging from several hundred micrometers to more than 4 mm. Electrokinetic delivery has the potential to improve control over infusions.

To identify neurons that specifically increase blood glucose from among the diversely-functioning cell types in the ventromedial hypothalamic nucleus (VMN), we studied the cholecystokinin (CCK) receptor-B (CCKBR)-expressing VMN targets of glucose-elevating parabrachial nucleus neurons. Activating these VMNCCKBR neurons increased blood glucose. Furthermore, while silencing the broader VMN decreased energy expenditure and promoted weight gain without altering blood glucose, silencing VMNCCKBR neurons decreased hepatic glucose production (HGP), insulin-independently decreasing blood glucose without altering energy balance. Silencing VMNCCKBR neurons also impaired the counter-regulatory response (CRR) to insulin-induced hypoglycemia and glucoprivation and replicated hypoglycemia-associated autonomic failure (HAAF). Hence, VMNCCKBR cells represent a specialized subset of VMN cells that function to elevate glucose. These cells not only mediate the allostatic response to hypoglycemia, but also insulin-independently modulate the homeostatic setpoint for blood glucose, consistent with a role for the brain in the insulin-independent control of glucose homeostasis.

The temporal pattern of drug use (pharmacokinetics) has a profound effect on the ability of self-administered cocaine to produce addiction-like behavior in rodents, and to change the brain. To further address this issue, we compared the effects of long access (LgA) cocaine self-administration, which is widely used to model the transition to addiction, with intermittent access (IntA), which is thought to better reflect the pattern of drug use in humans, on the ability of a single, self-administered injection of cocaine to increase dopamine (DA) overflow in the core of the nucleus accumbens (using in vivo microdialysis), and to produce addiction-like behavior. IntA experience was more effective than LgA in producing addiction-like behavior-a drug experience-dependent increase in motivation for cocaine assessed using behavioral economic procedures, and cue-induced reinstatement-despite much less total drug consumption. There were no group differences in basal levels of DA in dialysate [DA], but a single self-administered IV injection of cocaine increased [DA] in the core of the nucleus accumbens to a greater extent in rats with prior IntA experience than those with LgA or limited access experience, and the latter two groups did not differ. Furthermore, high motivation for cocaine was associated with a high [DA] response. Thus, IntA, but not LgA, produced both incentive and DA sensitization. This is consistent with the notion that a hyper-responsive dopaminergic system may contribute to the transition from casual patterns of drug use to the problematic patterns that define addiction.

BACKGROUND:Delivering solutes to a particular region of the brain is currently achieved by iontophoresis for very small volumes and by diffusion from a microdialysis probe for larger volumes. There is a need to deliver solutes to particular areas with more control than is possible with existing methods.NEW METHOD:Electrokinetic infusions of solutes were performed into hydrogels and organotypic hippocampal slice cultures. Application of an electrical current creates electroosmotic flow and electrophoresis of a dicationic fluorescent solute through organotypic hippocampal tissue cultures or larger hydrogels. Transport was recorded with fluorescence microscopy imaging in real-time.RESULTS:Electrokinetic transport in brain tissue slice cultures and hydrogels occurs along an electrical current path and allows for anisotropic delivery over distances from several hundred micrometers to millimeters. Directional transport may be controlled by altering the current path. The applied electrical current linearly affects the measured solute fluorescence in our model system following infusions.COMPARISON WITH EXISTING METHODS:Localized drug delivery involves iontophoresis, with diffusion primarily occurring beyond infusion capillaries under current protocols. Pressure-driven infusions for intraparenchymal targets have also been conducted. Superfusion across a tissue surface provides modest penetration, however is unable to impact deeper targets. In general, control over intraparenchymal drug delivery has been difficult to achieve. Electrokinetic transport provides an alternative to deliver solutes along an electrical current path in tissue.CONCLUSIONS:Electrokinetic transport may be applied to living systems for molecular transport. It may be used to improve upon the control of solute delivery over that of pressure-driven transport.

An essential approach for in vivo chemical monitoring is to use sampling probes coupled with analytical methods; however, this method traditionally has limited spatial and temporal resolution. To address this problem, we developed an analytical system that combines microfabricated push-pull sampling probes with droplet-based microfluidics. The microfabricated probe provides spatial resolution approximately 1000-fold better than that of common microdialysis probes. Microfabrication also facilitated integration of an extra channel into the probe for microinjection. We created microfluidic devices and interfaces that allowed manipulation of nanoliter droplet samples collected from the microfabricated probe at intervals of a few seconds. Use of droplet-based microfluidics prevented broadening of collected zones, yielding 6 s temporal resolution at 100 nL/min perfusion rates. Resulting droplets were analyzed by direct infusion nanoelectrospray ionization (nESI) mass spectrometry for simultaneous determination of glutamine, glutamate, γ-aminobutyric acid, and acetylcholine. Use of low infusion rates that enabled nESI (50 nL/min) was critical to allowing detection in the complex samples. Addition of 13C-labeled internal standards to the droplet samples was used for improved quantification. Utility of the overall system was demonstrated by monitoring dynamic chemical changes evoked by microinjection of high potassium concentrations into the brain of live rats. The results showed stimulated neurochemical release with rise times of 15 s. This work demonstrates the potential of coupling microfabricated sampling probes to droplet-based mass spectrometric assays for studying chemical dynamics in a complex microenvironment at high spatiotemporal resolution.

Faraji, Amir H MD, PhD; Jaquins-Gerstl, Andrea PhD; Valenta, Alec C; Ou, Yangguang PhD; Richardson, R. Mark MD; Weber, Stephen G PhD Author Information