In vivo monitoring of amino acids by direct sampling of brain extracellular fluid at ultralow flow rates and capillary electrophoresis
Introduction
The extracellular compartment of the brain is a complex, dynamic microenvironment containing chemical signals, metabolites, and nutrients. Chemical analysis of this environment in vivo has led to important insights into neuronal and glia communication and function. The interest in in vivo monitoring of the extracellular fluid (ECF) has been heightened by the discovery of volume transmission in which neurotransmitters escape the confines of the synaptic cleft and activate other receptors in the vicinity of the release site (Zoli et al., 1998).
Both sensor and sampling approaches have been employed to monitor neurotransmitters and related compounds in the ECF of the brain. Sensors are advantageous in that they can provide high spatial and temporal resolution (Kawagoe et al., 1992, Kulagina et al., 1999, Burmeister and Gerhardt, 2001, Georganopoulou et al., 2000). In the best case, sensors have response times ≤0.1 s with spatial resolution of a few micrometers. Sensors with these capabilities are available for only a few analytes and are usually restricted to monitoring one analyte at a time. An alternate strategy involves collecting samples from the ECF and analyzing fractions either off-line (Ungerstedt and Hallstrom, 1987) or on-line (Chen and Lunte, 1995, Zhou et al., 1995, Lada et al., 1997).
One approach to in vivo sampling is push–pull perfusion in which two concentric tubes with a total diameter of 500 μm are implanted in or near the brain region of interest. Sampling is accomplished by flowing in one tube and out the other tube at a flow rate of ∼10 μl/min (Gaddum, 1961, Izquierdo and Izquierdo, 1971, Myers, 1972). This method has fallen out of favor, except for neuropeptide monitoring, because of the large perturbation of the tissue caused by the perfusion of the tissue. Push–pull perfusion has been largely supplanted by microdialysis sampling (Ungerstedt and Hallstrom, 1987, Ungerstedt, 1991). In microdialysis, molecules in the ECF diffuse across a semi-permeable membrane into a flowing stream of solution which is collected for analysis. Microdialysis has emerged as the most important method for in vivo sampling because tissue perturbation is considerably reduced since the tissue is not directly bathed with a perfusate. Despite the popularity of the technique, microdialysis is not without shortcomings. Microdialysis has poor spatial resolution because the probes are usually 1–4 mm long and 200 μm in diameter. Therefore, it is of limited use for small brain nuclei or heterogeneous brain regions. In addition, temporal resolution can be poor because sufficient sample must be collected for analysis. Typical sampling times are 5–15 min (Kissinger, 1991, Herrera-Marschitz et al., 1996); however, the recent implementation of sensitive analytical methods coupled to microdialysis has pushed the temporal resolution to a few seconds (Tucci et al., 1997, Robert et al., 1998, Lada et al., 1998, Boyd et al., 2001). Finally, obtaining quantitative estimates of extracellular concentrations by microdialysis is problematic because the relative recovery obtained by in vitro experiments does not match that obtained in vivo (Marsden et al., 1986, Lerma et al., 1986, Benveniste, 1989). In vivo calibration methods (Justice, 1993) such as the no-net flux (Lonnroth et al., 1987, Parsons et al., 1991) and low-flow rate method (Wages et al., 1986) are available, but problems persist in their use (Peters and Michael, 1998, Bungay et al., 2001).
In an attempt to improve spatial resolution and quantification for in vivo monitoring we have explored direct sampling of brain ECF using miniaturized probes that are 60–90 μm in diameter and remove fluids at 1–50 nl/min without the use of a ‘push’ fluid. In this work, we have examined the feasibility of using this sampling approach to determine the extracellular levels of neuroactive amino acids. We have investigated: (1) the effect of sampling flow rate on the measurement, (2) the possibility of monitoring changes in transmitter levels in vivo, and (3) the spatial and temporal resolution of direct sampling.
Section snippets
Reagents and materials
Amino acids, derivatization reagents, KCl, l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and chloral hydrate were from Sigma (St. Louis, MO) and were used as received. MgSO4, CaCl2, NaCl, NaOH and boric acid were from Fisher Scientific (Pittsburgh, PA). Hydroxypropyl β-cyclodextrin was from Aldrich Chemical Company (Milwaukee, WI). Artificial cerebral spinal fluid (aCSF) consisted of 145 mM NaCl, 2.68 mM KCl, 1.01 mM MgSO4, and 1.22 mM CaCl2. High K+ CSF consisted of 27 mM NaCl, 120 mM KCl,
In vitro characterization of direct sampling
Initial experiments were conducted to investigate the feasibility of quantitative sampling and analyzing nanoliter volume samples. In these experiments, sample was pulled from a vial into the sampling capillary by vacuum and pumped into the analysis system for read-out as described in Section 2. The observed signal, measured as peak area for each separated compound, was plotted as a function of time in order to assess the temporal response as well as compare the signal intensity to standards.
Quantification of neurotransmitters in ECF and comparison to microdialysis
One goal of this work was to determine if extracellular levels of neuroactive compounds could be quantified using a direct sampling approach. When fluid is removed at 10–50 nl/min, the measured levels of the neurotransmitters are lower than those found at the lower sampling rates. This result suggests that at higher sampling rates depletion of these compounds occurs in the vicinity of the sampling probe. To achieve an accurate measurement of the levels of transmitters in vivo it is necessary to
Conclusions
Direct sampling at 1–50 nl/min appears to be a viable method for accessing the ECF of the brain in the living animals. Measurements of neuroactive substances yield results that are in general similar to calibrated microdialysis measurements when sampling rates of 1–2 nl/min are used. The ability to observe changes in response to depolarizing agents and uptake inhibitors demonstrates the ability to record dynamic changes and to sample from active neurons. The technique produces only nanoliter
Acknowledgements
This work was supported by NIH (NS38476). We thank Dr Michael T. Bowser for helpful discussions.
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