Microdialysis coupled with droplet microfluidics and mass spectrometry for determination of neurotransmitters in vivo with high temporal resolution.

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.

A microfluidic chip for on-line derivatization and application to in vivo neurochemical monitoring.

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 (R2 > 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 Signaling and NMDAR-Mediated Synaptic Plasticity Are Regulated by System A-Type of Glutamine/D-Serine Dual Transporters.

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 D-enantiomer. 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.SIGNIFICANCE STATEMENT Despite the plethora of roles attributed to D-serine, the regulation of its synaptic turnover is poorly understood. We identified the system A transporters Slc38a1 and Slc38a2 as the main pathway for neuronal reuptake of D-serine. These transporters are not saturated with glutamine in vivo and provide an unexpected link between the serine shuttle pathway, responsible for regulating D-serine synaptic turnover, and the glutamine-glutamate cycle. Our observations suggest that Slc38a1 and Slc38a2 have a dual role in regulating neurotransmission. In addition to their classical role as the glutamine providers, the system A transporters regulate extracellular D-serine and therefore affect NMDAR-dependent synaptic plasticity. Higher glutamine export from astrocytes would increase extracellular D-serine, providing a feedforward mechanism to increase synaptic NMDAR activation.

Electrokinetic Convection-Enhanced Delivery of Solutes to the Brain.

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.

Incentive and dopamine sensitization produced by intermittent but not long access cocaine self-administration.

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.

Electrokinetic infusions into hydrogels and brain tissue: Control of direction and magnitude of solute delivery.

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.

ASCT1 (Slc1a4) transporter is a physiologic regulator of brain d-serine and neurodevelopment.

d-serine is a physiologic coagonist of NMDA receptors, but little is known about the regulation of its synthesis and synaptic turnover. The amino acid exchangers ASCT1 (Slc1a4) and ASCT2 (Slc1a5) are candidates for regulating d-serine levels. Using ASCT1 and ASCT2 KO mice, we report that ASCT1, rather than ASCT2, is a physiologic regulator of d-serine metabolism. ASCT1 is a major d-serine uptake system in astrocytes and can also export l-serine via heteroexchange, supplying neurons with the substrate for d-serine synthesis. ASCT1-KO mice display lower levels of brain d-serine along with higher levels of l-alanine, l-threonine, and glycine. Deletion of ASCT1 was associated with neurodevelopmental alterations including lower hippocampal and striatal volumes and changes in the expression of neurodevelopmental-relevant genes. Furthermore, ASCT1-KO mice exhibited deficits in motor function, spatial learning, and affective behavior, along with changes in the relative contributions of d-serine vs. glycine in mediating NMDA receptor activity. In vivo microdialysis demonstrated lower levels of extracellular d-serine in ASCT1-KO mice, confirming altered d-serine metabolism. These alterations are reminiscent of some of the neurodevelopmental phenotypes exhibited by patients with ASCT1 mutations. ASCT1-KO mice provide a useful model for potential therapeutic interventions aimed at correcting the metabolic impairments in patients with ASCT1 mutations.

In Vivo Chemical Monitoring at High Spatiotemporal Resolution Using Microfabricated Sampling Probes and Droplet-Based Microfluidics Coupled to Mass Spectrometry.

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.