zeta-potentials of entities such as cells and synaptosomes have been determined, but zeta of brain tissue has never been measured. Electroosmotic flow, and the resulting transport of neuroactive substances, would result from naturally occurring and experimentally or clinically induced electric fields if zeta is significant. We have developed a simple method for determining zeta in tissue. An electric field applied across a rat organotypic hippocampal slice culture (OHSC) drives fluorescent molecules through the tissue by both electroosmotic flow and electrophoresis. Fluorescence microscopy is used to determine each molecule's velocity. Independently, capillary electrophoresis is used to measure the molecules' electrophoretic mobilities. The experiment yields zeta-potential and average tissue tortuosity. The zeta-potential of OHSCs is -22 +/- 2 mV, and the average tortuosity is 1.83 +/- 0.06. In a refined experiment, zeta-potential is measured in various subregions. The zeta-potentials of the CA1 stratum pyramidale, CA3 stratum pyramidal, and dentate gyrus are -25.1 +/- 1.6 mV, -20.3 +/- 1.7 mV, and -25.4 +/- 1.0 mV, respectively. Simple dimensional arguments show that electroosmotic flow is potentially as important as diffusion in molecular transport.
Electroosmotic sampling is a potentially powerful method for pulling extracellular fluid into a fused-silica capillary in contact with the surface of tissue. An electric field is created in tissue by passing current through an electrolyte-filled capillary and then through the tissue. The resulting field acts on the counter ions to the surface charges in the extracellular space to create electroosmotic fluid flow within the extracellular space of a tissue. Part of the development of this approach is to define conditions under which electroosmotic sampling minimizes damage to the tissue, in this case organotypic hippocampal slice cultures (OHSCs). We have assessed tissue damage by measuring fluorescence resulting from exposing sampled tissue to propidium iodide solution 16 -24 h after sampling. Sampling has been carried out with a variety of capillary diameters, capillary tip-tissue distance, and applied voltages. Tissue damage is negligible when the power (current × potential drop) created in the tissue is less than 120 µW. In practical terms, smaller capillary IDs, lower voltages, and greater tissue to capillary distances lead to lower power.Driven by the need to understand the chemistry of life processes better, the analysis of the extracellular space of brain tissue has emerged as an invaluable window into the mechanisms of signal transduction and processing of neuroactive substances in the brain. Methods such as push-pull perfusion 1 , microdialysis 2 , and direct sampling 3 have previously been developed to sample and control the environment in the extracellular space in vitro (cultures and slices) and in vivo. Each method has been significantly improved over its first introduction and major advances in the detection of neurotransmitters have occurred over the past decade 4 . Of particular note is the reduction in the damaging nature of early push-pull methods. The push-pull method was altered for compatibility with lower flow rates (10-50 nL/min) in order to minimize damage stemming from the higher flow rates and large fluid volume introduction in to the tissue 5 . This new improved method has since been applied to routine in vivo sampling [6][7][8] . Analysis of the low-volume samples has been improved by coupling it to online microfluidic devices 9 and creating selective membranes for the removal of proteins from biological samples in the interest of enhancing peptide collection and detection by mass spectrometry 10 . A further advantage of methods such as microdialysis and push-pull perfusion is the versatility and potential for introduction of pharmaceuticals into the sampling area via the inlet portion of the apparatus. immature rat pup, sectioning perpendicular to the septotemporal axis, and culturing the slices atop a porous membrane over medium. In vitro, these tissues can be maintained from a week up to 1-2 months, developing quite similarly to the intact tissue in the living animal and maintaining most hippocampal structural integrity and neuronal organization 12,13 . The well-organized la...
We hypothesize that peptide-containing solutions pulled through tissue should reveal the presence and activity of peptidases in the tissue. Using the natural ζ-potential in the organotypic hippocampal slice culture (OHSC), physiological fluids can be pulled through the tissue with an electric field. The hydrolysis of the peptides present in the fluid drawn through the tissue can be determined using capillary HPLC with electrochemical detection of the biuret complexes of the peptides following a postcolumn reaction. We have characterized this new sampling method by measuring the flow rate, examining the use of internal standards, and examining cell death caused by sampling. The sampling flow rate ranges from 60 to 150 nL/min with a 150 μm (ID) sampling capillary with an electric field (at the tip of the capillary) from 30 to 60 V/cm. Cell death can be negligible with controlled sampling conditions. Using this sampling approach, we have electroosmotically pulled Leu-enkephalin through OHSCs to identify ectopeptidase activity in the CA3 region. These studies show that a bestatin-sensitive aminopeptidase may be critical for the hydrolysis of exogenous Leu-enkephalin, a neuropeptide present in the CA3 region of OHSCs.Neuropeptides play a key role in brain and peripheral nervous system functions such as pain and learning 1 . They are mainly inactivated by ectopeptidases -outward-facing, membranebound peptidases that cleave the active peptides into inactive fragments 2-4 . Extracellular peptidases create active forms of BDNF 5 , substance P 6 , cholecystokinins 4 and alter the activity of dynorphins 7 . Recent work shows that peptidases are important in the degradation of amyloid 8,9 . Also, attenuated peptidase activity following stroke can contribute to neurotoxicity and an endogenous blocker of the ectopeptidase that cleaves enkephalin has powerful analgesic effects 10 . Thus, a deeper understanding of peptidase activity is necessary for understanding both normal and pathological brain function, as well as for the development of novel strategies for drug development.While the central focus of this paper is electroosmotic sampling, the determination of peptidase activity in tissue is an important direction. Peptidase activity has been a concern in microdialysis experiments. Microdialysis sampling of peptides is improved in the presence of peptidase inhibitors [11][12][13][14][15][16][17] . Recently, the Stenken group 18, 19 has developed microdialysis approaches to in vitro (enzyme solutions) determination of protease activity. In vitro (slice)
Iontophoresis uses electricity to deliver solutes into living tissue. Often, iontophoretic ejections from micropipettes into brain tissue are confined to millisecond pulses for highly localized delivery, but longer pulses are common. As hippocampal tissue has a ζ-potential of approximately –22 mV, we hypothesized that, in the presence of the electric field resulting from the iontophoretic current, electroosmotic flow in the tissue would carry solutes considerably farther than diffusion alone. A steady state solution to this mass transport problem predicts a spherically symmetrical solute concentration profile with the characteristic distance of the profile depending on the ζ-potential of the medium, the current density at the tip, the tip size and the solute electrophoretic mobility and diffusion coefficient. Of course, the ζ-potential of the tissue is defined by immobilized components of the extracellular matrix as well as cell-surface functional groups. As such, it cannot be changed at will. Therefore, the effect of the ζ-potential of the porous medium on ejections is examined using poly(acrylamide-co-acrylic acid) hydrogels with various magnitudes of ζ-potential, including that similar to hippocampal brain tissue. We demonstrated that nearly neutral fluorescent dextran (3 and 70 kD) solute penetration distance in the hydrogels and OHSCs depends on the magnitude of the applied current, solute properties, and, in the case of the hydrogels, the ζ-potential of the matrix. Steady state solute ejection profiles can be predicted semi-quantitatively.
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