Microcolumn separations and the analysis of single cells.

Capillary zone electrophoresis and open tubular liquid chromatography are two examples of an emerging area of analytical instrumentation known as microcolumn separations. The high resolution and small sample requirements of these methods make them suitable for the quantitative, multicomponent chemical analysis of single cells. Appropriate instrumentation for the analysis of nanoliter and subnanoliter samples is discussed. Data from the analysis of individual neurons are presented, including amino acid and neurotransmitter content.

Up-regulation of GLT1 expression increases glutamate uptake and attenuates the Huntington's disease phenotype in the R6/2 mouse.

The striatum, which processes cortical information for behavioral output, is a key target of Huntington's disease (HD), an autosomal dominant condition characterized by cognitive decline and progressive loss of motor control. Increasing evidence implicates deficient glutamate uptake caused by a down-regulation of GLT1, the primary astroglial glutamate transporter. To test this hypothesis, we administered ceftriaxone, a beta-lactam antibiotic known to elevate GLT1 expression (200 mg/kg, i.p., for 5 days), to symptomatic R6/2 mice, a widely studied transgenic model of HD. Relative to vehicle, ceftriaxone attenuated several HD behavioral signs: paw clasping and twitching were reduced, while motor flexibility, as measured in a plus maze, and open-field climbing were increased. Assessment of GLT1 expression in striatum confirmed a ceftriaxone-induced increase relative to vehicle. To determine if the change in behavior and GLT1 expression represented a change in striatal glutamate handling, separate groups of behaving mice were evaluated with no-net-flux microdialysis. Vehicle treatment revealed a glutamate uptake deficit in R6/2 mice relative to wild-type controls that was reversed by ceftriaxone. Vehicle-treated animals, however, did not differ in GLT1 expression, suggesting that the glutamate uptake deficit in R6/2 mice reflects dysfunctional rather than missing GLT1. Our results indicate that impaired glutamate uptake is a major factor underlying HD pathophysiology and symptomology. The glutamate uptake deficit, moreover, is present in symptomatic HD mice and reversal of this deficit by up-regulating the functional expression of GLT1 with ceftriaxone attenuates the HD phenotype.

The rate of intravenous cocaine administration alters c-fos mRNA expression and the temporal dynamics of dopamine, but not glutamate, overflow in the striatum.

The rapid entry of drugs into the brain is thought to increase the propensity for addiction. The mechanisms that underlie this effect are not known, but variation in the rate of intravenous cocaine delivery does influence its ability to induce immediate early gene expression (IEG) in the striatum, and to produce psychomotor sensitization. Both IEG induction and psychomotor sensitization are dependent upon dopamine and glutamate neurotransmission within the striatum. We hypothesized, therefore, that varying the rate of intravenous cocaine delivery might influence dopamine and/or glutamate overflow in the striatum. To test this we used microdialysis coupled to on-line capillary electrophoresis and laser-induced fluorescence, which allows for very rapid sampling, to compare the effects of a rapid (5 s) versus a slow (100 s) intravenous cocaine infusion on extracellular dopamine and glutamate levels in the striatum of freely moving rats. An acute injection of cocaine had no effect on extracellular glutamate, at either rate tested. In contrast, although peak levels of dopamine were unaffected by infusion rate, dopamine levels increased more rapidly when cocaine was administered over 5 versus 100 s. Moreover, c-fos mRNA expression in the region of the striatum sampled was greater when cocaine was administered rapidly than when given slowly. These data suggest that small differences in the temporal dynamics of dopamine neurotransmission may have a large effect on the subsequent induction of intracellular signalling cascades that lead to immediate early gene expression, and in this way influence the ability of cocaine to produce long-lasting changes in brain and behavior.

Biochemical indicators of implantation success of tissue-engineered oral mucosa.

Real-time (RT) determination of the health of in vitro tissue-engineered constructs prior to grafting is essential for prediction of success of the implanted tissue-engineered graft. In addition, the US Food and Drug Administration requires specific release criteria in RT prior to the release of tissue-engineered devices for human use. In principle, assessing the viability and functionality of the cellular component can be achieved by quantifying the secretion of growth factors and chemokines of tissue-engineered constructs. Ex vivo-produced oral mucosa equivalents (EVPOMEs) were fabricated under thermally stressed conditions at 43 °C for 24 h to create a functionally compromised EVPOME. We used microchannel enzyme-linked immunosorbent assay to evaluate the functionality of the cellular component, oral keratinocytes, of stressed and unstressed EVPOMEs by measuring the release of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), human ß-defensin 1 (hBD-1), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) into the spent medium, which was collected on the same day prior to graft implantation into severe combined immunodeficiency mice. Implanted EVPOMEs' histology on the seventh postimplantation day was used to correlate outcomes of grafting to secreted amounts of IL-8, hBD-1, VEGF, TIMP-1, and TIMP-2 from corresponding EVPOMEs. Our findings showed that significantly higher levels of IL-8, hBD-1, and TIMP-2 were secreted from controls than from thermally stressed EVPOMEs. We also found a direct correlation between secreted VEGF and IL-8 and blood vessel counts of implanted EVPOMEs. We concluded that measuring the constitutive release of these factors can be used as noninvasive predictors of healthy tissue-engineered EVPOMEs in RT, prior to their implantation.

Evoked neuronal activity accompanied by transmitter release increases oxygen concentration in rat striatum in vivo but not in vitro.

Dopamine and oxygen (O2) were measured in the caudate nucleus of anesthetized rats and in striatal slices during electrical stimulation. Simultaneous electrochemical detection of dopamine and O2 was accomplished with fast-scan cyclic voltammetry at a Nafion-coated carbon-fiber microelectrode. Stimulation of the medial forebrain bundle resulted in synaptic overflow of dopamine in the caudate nucleus. At the same time, O2 concentration increased in the extracellular fluid with two separate phases. The amplitude of the initial increase directly correlated with the frequency of the stimulus, with the time of maximum concentration reproducible across a range of frequencies. The second increase occurred at later times with a more random amplitude and with a broad, variable shape. Agents which blocked vasodilation affected both phases: atropine attenuated the initial increase, while the second feature was nearly absent after theophylline. Yohimbine and alpha-methyl-p-tyrosine did not affect the O2 responses. Local electrical stimulation of the slice preparation also resulted in dopamine overflow, but a prolonged decrease in O2 concentration accompanied this event. Striatal field stimulation in vivo produced changes in O2 concentration dependent on the relative position of the stimulating and working electrodes, but none of the responses resembled that seen in the caudate slice. Thus, while measurements in brain slices show O2 consumption as a result of stimulated neuronal activity, an apparent elevation of local cerebral blood flow during and after stimulation dominate the in vivo response.

Simultaneous measurement of oxygen and dopamine: coupling of oxygen consumption and neurotransmission.

Fast-scan cyclic voltammetry was used to simultaneously measure increases in dopamine concentration and decreases in O2 concentration evoked by brief electrical stimulation (two pulses at 10 Hz) in slices of rat caudate nucleus. Dopamine concentration began increasing immediately after the first pulse and reached a maximum within 200 ms of stimulation. The O2 concentration began to decrease 300-700 ms after onset of stimulus. Responses for both dopamine and O2 were dependent on external Ca2+ and were Cd2+ and tetrodotoxin sensitive. Only the O2 response was sensitive to CN- (0.15 mM). At short times after exposure to 50 microM ouabain, electrically stimulated dopamine overflow was increased by 150% and electrically stimulated changes in O2 concentration were unaffected. Maximum dopamine concentration was increased 28% by sulpiride (2 microM), 78% by L-DOPA (60 microM), 105% by nomifensine (10 microM) and unaffected by nialamide (10 microM). Maximum decrease in O2 concentration was increased by 25% by sulpiride and unaffected by nialamide, L-DOPA, or nomifensine. The decreases in O2 concentration are indicative of increased O2 consumption and are a measure of oxidative energy production evoked by electrical stimulation. The increase in dopamine is due to the release of dopamine balanced by uptake and serves as an indication of neurotransmitter activity. The results indicate that increases in oxidative energy production following electrical stimulation are dependent on external Ca2+ entry through Cd(2+)-sensitive channels. Possible mechanisms for this coupling are discussed.

Localized exocytosis detected by spatially resolved amperometry in single pancreatic beta-cells.

Spatially resolved measurements of exocytosis in pancreatic beta-cells were made using amperometry with 1-microm radius electrodes. These measurements revealed that certain portions of a cell actively undergo exocytosis following stimulation with depolarizing agents, but other regions are inactive. The amperometric measurements were compared to measurements made with the membrane indicator dye, FM1-43, which showed uneven increases in fluorescence around the surface of the cell, with amperometric secretion being detected only at the brightest regions. In some instances, a large number of exocytotic events were detected from one electrode position. The number of events was larger than what would be expected based on the number of vesicles that could fit under an electrode of the dimensions used. These results suggest a mechanism of vesicle traffic that allows multiple fusions at a small membrane area.

Secretion from islets and single islet cells following cryopreservation.

The ability to cryopreserve pancreatic islets has allowed the development of low-temperature banks that permit pooling of islets from multiple donors and allows time for sterility and viability testing. However, previous studies have shown that during cryopreservation and thawing there is a loss of islet mass and a reduction in islet function. The aim of this study was to measure and compare insulin secretion from cultured nonfrozen and frozen-thawed canine islets and beta-cells. Canine islets were isolated from mongrel dogs using intraductal collagenase distention, mechanical dissociation, and EuroFicoll purification. One group of purified islets was cultured overnight before dissociation into single cells and subsequent analysis. Remaining islets were cultured overnight (22 degrees C) and then cryopreserved in 2 M dimethyl sulfoxide (DMSO) solution using a slow stepwise addition protocol with slow cooling to -40 degrees C before storage in liquid nitrogen (-196 degrees C). Frozen islets were rapidly thawed (200 degrees C/min) and the DMSO removed using a sucrose dilution. From a series of seven consecutive canine islet isolations, islet recovery following postcryopreservation tissue culture was 81.5 +/- 4.8% compared to precryopreservation counts. In vitro islet function was equivalent between cultured nonfrozen and frozen-thawed islets with a calculated stimulation index of 10.4 +/- 1.5 (mean +/- SEM) for the frozen-thawed islets, compared with 12.4 +/- 1.2 for the cultured nonfrozen controls (p = ns, n = 7 paired experiments). Amperometric detection of secretion from single beta-cells in vitro has the sensitivity and temporal resolution to detect single exocytotic events and allows secretion to be monitored from single beta-cells in real time. Secretion from single beta-cells elicited by chemical stimulation was detected using a carbon fiber microelectrode. The frequency of exocytosis events was equivalent between the cultured nonfrozen and frozen-thawed beta-cells with an average of 7.0 +/- 1.32 events per stimulation for the cultured nonfrozen group compared with 6.0 +/- 1.45 events from the frozen then thawed preparations (minimum of 10 cells per run per paired experiment, p = ns) following stimulation with tolbutamide. The average amount of insulin released per individual exocytosis event was equivalent for the cultured nonfrozen and frozen-thawed islets. In addition, beta-cells responded to both tolbutamide and muscarinic stimulation following cryopreservation. It was determined that beta-cells recovered following cryopreservation are capable of secreting insulin at levels and frequencies comparable to those of cultured nonfrozen islet preparations.

Quantitative in vivo measurements using microdialysis on-line with capillary zone electrophoresis.

A system which couples microdialysis with capillary zone electrophoresis (CZE) on-line is used to monitor ascorbate and lactate in the caudate nucleus of rat brain. On-line interface of microdialysis probe and electrophoresis capillary, along with the high mass sensitivity of CZE, allows the probe to be operated at flow rates as low as 40 nl/min. Under these conditions, the relative recovery is nearly 100% and quantitative monitoring is possible. The microscale system also facilitates calibration by the low flow rate method. In spite of the low flow rate, temporal resolution in the 45-125 s range is possible for these compounds. The system is demonstrated by observing changes in ascorbate due to infusions of elevated K+ through the dialysis probe and systemic injections of amphetamine and an anesthetic (ketamine/xylazine/acepromazine mixture). Lactate is monitored in response to elevated K+ infusions.

In vivo monitoring of glutathione and cysteine in rat caudate nucleus using microdialysis on-line with capillary zone electrophoresis-laser induced fluorescence detection.

A fully-automated method for monitoring thiols in vivo using microdialysis coupled on-line with capillary zone electrophoresis with laser-induced fluorescence detection was developed. Dialysates were derivatized on-line with monobromobimane and automatically transferred to the separation capillary by a flow-gated interface. Analytes were detected on-column using the 2 mW, 354 nm line of a He-Cd laser for excitation. Dialysis probes were perfused at 79 nl/min resulting in relative recoveries of nearly 100%, which allowed quantitative monitoring. On-line detection limits for these analytes were in the 20-40 nM range and the response was linear up to 20 microM. The system was applied to the measurement of glutathione and cysteine in the extracellular space of the caudate nucleus of anesthetized rats. The measured basal concentrations of glutathione and cysteine were 2.0 +/- 0.1 microM and 2.3 +/- 0.3 microM, respectively which agree well with literature values. Increases in glutathione and cysteine were monitored with 180 s temporal resolution during stimulation by infusion of potassium. The average concentration of glutathione and cysteine during stimulation was 3.0 +/- 0.9 and 3.3 +/- 0.5 microM (n = 3), respectively. This system is the first to obtain high relative recoveries and high temporal resolution simultaneously for multiple thiols with microdialysis sampling in the brain.

Rapid simultaneous determination of glucagon and insulin by capillary electrophoresis immunoassays.

A rapid capillary electrophoresis (CE) with laser-induced fluorescence (LIF) competitive immunoassay has been developed for the determination of glucagon in biological mixtures. In the assay, fluorescein-conjugated glucagon is mixed with the sample followed by addition of anti-glucagon. Free and antibody-bound, tagged glucagon could be separated in 3 s using CE to obtain quantitative determination of glucagon with a concentration detection limit of 760 pM. The assay was combined with a previously developed competitive immunoassay for insulin to produce a simultaneous immunoassay for both peptides. The method was used to determine glucagon content of islets of Langerhans.

Selective preconcentration for capillary zone electrophoresis using protein G immunoaffinity capillary chromatography.

Capillaries with 150 microns inner diameter were packed with a perfused protein G chromatographic support and used as immunoaffinity preconcentrators for capillary zone electrophoresis. Antibody was loaded onto the protein G support to form an immunoaffinity stationary phase. Injection of samples onto the column caused selective retention and preconcentration of antigen. Injection of appropriate buffers onto the column caused desorption of the antibody and antigen which were then separated by capillary zone electrophoresis. The combination was used on-line and off-line. For on-line combination, a flow-gated interface coupled the two columns and allowed injection of desorbed zones onto the electrophoresis system. Off-line coupling required collection of desorbed fractions and then injection onto the electrophoresis system. Flow rates as high as 100 microL/min were used to load sample onto the affinity column. Desorbing flow rates had to be 1 microL/min or less to prevent excessive dilution during desorption. Using the system, 1 mL insulin samples could be loaded onto the affinity column and desorbed in volumes as small as 1 microL for 1000-fold preconcentration. The use of the preconcentrator with serum samples spiked with insulin was demonstrated.

Quantitative analysis of individual neurons by open tubular liquid chromatography with voltammetric detection.

The ability to analyze individual cells is often important in biology because of the heterogeneity of tissue; this is especially true in the area of neurobiology. A method is described for the determination of trace levels of organic compounds in individual cells by open tubular liquid chromatography with voltammetric detection. In the method, a cell is isolated, an internal standard is added, the cell is homogenized and centrifuged, and the supernatant is injected directly onto the chromatography column. Since data are collected in both the electrochemical and chromatographic domains, the resolution of the method is better than that obtained by using amperometric detection. The combination of voltammetry and chromatography also aids in the identification of compounds. By use of this method three different neurons, D2, E4, and F1, from the land snail Helix aspersa are analyzed. The data show that the cells give certain unique and repeatable chemical profiles. Dopamine, serotonin, tyrosine, and tryptophan were identified and quantified in two of the cells at the femtomole level. In the third cell, only the two amino acids were observed and measured. The quantitative data indicate that the method is at least as reliable as other methods that have been applied to single cells and considerably more sensitive. The combination of qualitative and quantitative information allows for the chemical mapping of cells.

Electrochemical detection of exocytosis at single rat melanotrophs.

Amperometry at a carbon fiber microelectrode was used to monitor secretion of peptide hormone from single melanotrophs of the intermediate lobe of the rat pituitary. The method is based on electrochemical oxidation of tryptophan and tyrosine residues of small proopiocortin-derived peptides secreted from these cells. For single-cell measurements, the electrode, which had a sensing diameter of approximately 9 microns and a total tip diameter of 30 microns, was positioned approximately 1 micron away from single melanotrophs. When cells were stimulated by application of 64 mM K+, a series of randomly occurring current spikes with an average area of 34 +/- 6 fC was observed. The current spikes were strongly dependent on the presence of Ca2+. Current spikes of nearly identical area and shape were also elicited by mechanical stimulation. Cyclic voltammograms obtained from cell releasates confirmed that the substance detected was a tryptophan- or tyrosine-containing peptide. On the basis of amperometric tests of the most abundant peptides in melanotrophs, it is concluded that the current spikes are due to detection of primarily alpha-melanocyte stimulating hormone. The spike area corresponds to 0.32 amol of alpha-melanocyte stimulating hormone. It is concluded that the current spikes represent detection of concentration pulses that are expected following exocytosis events.

Quantitative in vivo monitoring of primary amines in rat caudate nucleus using microdialysis coupled by a flow-gated interface to capillary electrophoresis with laser-induced fluorescence detection.

A method for monitoring primary amines in vivo using microdialysis coupled on-line with capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection was explored. Dialysates were derivatized on-line with o-phthaldialdehyde/beta-mercaptoethanol and automatically transferred to a separation capillary by a flow-gated interface. Analytes were detected on-column using the 2 mW, 354 nm line of a He-Cd laser for excitation. Dialysis probes were perfused at 79 nL/min, resulting in relative recoveries of nearly 100%, which allowed quantitative monitoring. On-line detection limits were in the 20-50 nM range, and the response was linear up to 50 microM. Temporal resolution was between 45 s and 3 min and was limited by separation time or broadening of sample zones during transfer to the separation capillary, depending on the operational parameters. The system was applied to measurement of primary amines in the caudate nucleus of anesthetized rats. Using CZE for separation, it was possible to resolve and monitor several compounds, including aspartate and glutamate. The measured basal concentrations of aspartate and glutamate were 1.2 +/- 0.1 and 5.0 +/- 0.4 microM, respectively, which agrees well with literature values. Increases in in vivo aspartate and glutamate were monitored with 90 s temporal resolution during K+ depolarization using dialysis flow rates of 79 nL/min; however, temporal resolution of 45 s was possible at the expense of lower relative recovery if the dialysis flow rate was increased to 155 nL/min. The use of MEKC as the separation mode significantly increased the number of compounds that could be resolved and detected. Using MEKC to separate the dialysate samples allowed aspartate, glutamate, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, tyrosine, and valine to be resolved and detected. The basal concentrations for these compounds using MEKC were 1.9 +/- 0.2, 4.1 +/- 0.2, 4.6 +/- 0.7, 2.6 +/- 0.3, 5.4 +/- 0.4, 1.8 +/- 0.2, 2.0 +/- 0.2, 11.3 +/- 1.3, 3.3 +/- 0.9, and 5.3 +/- 0.3 microM, respectively. The concentrations of these primary amines in the striatum were monitored after K+ depolarization with 3 min temporal resolution. This is the first microdialysis system to generate high relative recoveries and good temporal resolution simultaneously for multiple neurotransmitters.

On-line competitive immunoassay for insulin based on capillary electrophoresis with laser-induced fluorescence detection.

An on-line competitive immunoassay for insulin has been developed and applied to monitoring insulin concentration in a flowing stream. In the assay, solutions of fluorescein-labeled insulin (FITC-insulin), monoclonal anti-insulin, and sample containing insulin are pumped into a cross where they begin to mix. The mixture flows through a fused silica reactor capillary to a flow-gated interface. During transfer to the interface, insulin and FITC-insulin compete to form a complex with the antibody. At the interface, a plug of the mixture is injected into a separation capillary, where the bound and free FITC-insulin are separated and detected by capillary electrophoresis with laser-induced fluorescence detection. The amount of bound FITC-insulin, amount of free FITC-insulin, or bound/free ratio can be used to quantify insulin concentration. Typical relative standard deviations of bound over free ratio are 5%. The detection limit of the immunoassay in the on-line mode is < 0.3 nM. Each separation requires as little as 3 s, and over 1600 consecutive assays can be acquired with no need to rinse the separation capillary. Thus, the system can be used to monitor insulin in a flowing stream for flow injection analysis or for sensor-like monitoring. Dilution and zone broadening during transfer of sample to the interface limit the response time of the on-line system to about 25 s. As a demonstration of the on-line immunoassay, the insulin content of single islets of Langerhans was determined by flow injection analysis.

Determination of trace level gamma-aminobutyric acid using an improved OPA pre-column derivatization and on-column preconcentration capillary liquid chromatography with electrochemical detection.

An improved pre-column derivatization with o-phthalaldehyde/tert-butylthiol and on-column preconcentration are used with packed capillary liquid chromatography and electrochemical detection to obtain low concentration detection limits for gamma-aminobutyric acid (GABA). Using derivatization procedures from the literature, it was found that the detection limits for GABA were 380 amol in 50 microns i.d. packed capillaries, which is over 10-fold worse than the detection limit possible with the instrumentation. The higher detection limit was directly the result of electroactive interferences generated by the derivatization chemistry. Derivatization was improved by scavenging excess reagents with excess amine and iodoacetamide. With these improvements, the interfering peaks were eliminated and the detection limit was improved to 50 amol. With injection volumes of 0.5 microL, under conditions that permitted on-column preconcentration, the concentration detection limit, that is the concentration at which analytes could be derivatized and detected, was 100 pM. The technique was applied to determination of GABA release from islets of Langerhans.

Identification, quantitation, and characterization of biomolecules by capillary electrophoretic analysis of binding interactions.

The high resolving power of capillary electrophoresis combined with the specificity of binding interactions may be used with advantage to characterize the structure-function relationship of biomolecules, to quantitate specific analytes in complex sample matrices, and to determine the purity of pharmaceutical and other molecules. We here review recent and innovative methodologies and applications of high resolution affinity electrophoresis within the fields of binding constant determination, structure-activity studies, quantitative microassays, analysis of drug purity and protein conformation, and immobilized affinity ligands. Despite the virtues of these approaches with respect to applicability, resolving power, speed, and low sample consumption, problems remain with respect to analyte identification and low concentration limits of detection. The ongoing development of new detector technologies for capillary electrophoresis such as mass spectrometry, and possibly nuclear magnetic resonance and other spectroscopic methods, is therefore very promising for the continued increased use of affinity capillary electrophoresis.