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Microdialysis and Advances for Sampling Synaptic and Extrasynaptic Pools

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Microdialysis Techniques in Neuroscience

Part of the book series: Neuromethods ((NM,volume 75))

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Abstract

Intercellular communication plays a key role in information processing in the nervous system, to immune response, to cellular growth and differentiation, and to most processes fundamental to life for multicellular organisms. As a sampling technique microdialysis enables in vivo studies of brain and other tissues and has advanced our understanding of intercellular signal processing. The first part of this chapter is an overview of microdialysis in the context of a how-to-guide with reference to sampling extrasynaptic glutamate and GABA using conventional methodology. The limitations and challenges associated with sampling the synaptic pool of fast neurotransmitters are then addressed. The last part of this chapter presents ideas of advancing the microdialysis technique that may bring the microdialysis membrane closer to the synapse.

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Author information

Authors and Affiliations

  1. Department of Mechanical Engineering, University of Alaska Fairbanks, Fairbanks, AK, USA

    Cheng-fu Chen

  2. Department of Chemistry and Biochemistry, University of Alaska Fairbanks, Fairbanks, AK, USA

    Brian T. Rasley, Tom K. Green & Kelly L. Drew

  3. School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Benjamin P. E. Warlick

  4. Institute for Systems Biology, Seattle, WA, USA

    Kristian E. Swearingen

Authors
  1. Cheng-fu Chen
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  2. Brian T. Rasley
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  3. Benjamin P. E. Warlick
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  4. Tom K. Green
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  5. Kristian E. Swearingen
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  6. Kelly L. Drew
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Corresponding author

Correspondence to Kelly L. Drew .

Editor information

Editors and Affiliations

  1. , Dept. of Physiology & Biochemistry, University of Malta, Biomedical Sciences Building, Msida MSD, 2080, Malta

    Giuseppe Di Giovanni

  2. Consorzio "Mario Negri" Sud, Via Nazionale, Santa Maria Imbaro, 66030, Chieti, Italy

    Vincenzo Di Matteo

Construction of Low Dead Volume Microdialysis Probes

Construction of Low Dead Volume Microdialysis Probes

  1. 1.

    Prepare capillary: Using a ceramic capillary tubing cutter, cut up to a 50 cm piece of capillary tubing1 (this will be the inlet to the probe, so adjust length as needed). Measure and cut another piece for the outlet. For acute, anesthetized preps this may be 10 cm. For awake animal preparations leave the outlet long enough to reach your fraction collector or collection vial. Note that longer outlets will decrease the flow rate that a probe can handle before breaking or seeping fluid (ultrafiltration). Ensure that cuts are smooth; check smoothness using a microscope. Smoothness means that the tube is not shattered and the cut does not leave a point on the tube.

  2. 2.

    Adjust offset between inlet and outlet that will define the length of dialyzing tip: On a glass slide with double-sided tape on the topside of the slide, lay the long capillary piece on the slide and next to it lay the short capillary piece. Bring the ends of the two pieces flush with one another, and then make the long capillary tube stick out further than the short capillary tube by 1–4 mm or longer depending on the desired length of your dialyzing tip. Put this in an area where it will not be disturbed.

  3. 3.

    Prepare membrane for sliding onto capillaries: Cut 0.5 cm of cellulose membrane2 from the roll at a 45° angle. Put the cut membrane on a glass slide with double-sided tape on the top. Using polyimide resin3, seal the cut membrane end that is not angled. The sealed part should not exceed a thickness of 0.5 mm (Fig. 7a). Leave this to dry for several hours.

    Fig. 7.
    figure 7

    Offset short and long piece of capillary to define the length of dialyzing tip (a). Feed into membrane, sealed on one end with polyimide resin (plug not to exceed 0.5 mm) and trimmed at a 45° angle on the other end. After inserting the capillary tubes into the membrane about 1 mm, apply polyimide resin to the capillaries at the opening of the membrane (b). Coat the membrane and capillary tubes with polyimide resin as shown leaving the dialyzing tip free of resin (c). These membrane-covered capillaries are now referred to as “the probe”.

    Full size image
  4. 4.

    Insert capillary tubes into membrane: Insert offset capillary tubes into a dry, sealed membrane tube. For easier insertion put the short tube on the long side of the angled membrane opening (Fig. 7b).

  5. 5.

    Coat non-dialyzing surface of membrane and define the final length of dialyzing tip: After inserting capillary tubes about 1 mm apply polyimide resin onto the capillaries and to the open end of the membrane (the end cut to 45°). Continue feeding the capillaries into the membrane. Adjust the capillaries so that they remain offset as before, but the long capillary is about 0.1 mm from the polyimide plug at the end of the membrane. Spread polyimide resin on the membrane entrance and the membrane itself. Stop spreading resin just before the opening of the furthest back capillary tube. Remove excess polyimide resin and let dry for several hours (Fig. 7c).

  6. 6.

    Insert the completed probe into a push–pull cannula assembly: The assembly consists of two pieces, the push–pull connector-no internal tube referred to here as the “cap” (C313ICP/NIT4 where NIT indicates “no internal tube”), and the push–pull guide cannula (C316GPIO/uncut4). Attach a 1 cm piece of Tygon tubing (0.020–0.025” id) to the stainless steel tube on the top of the cap that leads to a hole directly in the middle of the cap (Fig. 8a). Thread the probe through this piece of Tygon and stainless steel tube. Next insert the probe through the push–pull guide cannula and then screw the two pieces together for a tight, reproducible fit.

    Fig. 8.
    figure 8

    The probe is threaded through a piece of Tygon tubing affixed to the stainless steel tube that leads to the center of the “cap” that is the push–pull connector-no internal tube (Cat No. C313ICP/NIT) (a). The probe that is now connected to the cap is then threaded through the push–pull guide cannula (Cat No. C316GP/O) and the two pieces are screwed together and tightened for a reproducible fit. Adjust the position of the probe within the cannula assembly so that the short capillary just clears the cannula tip and the active portion of the membrane extends beyond the tip (b). Apply a dab of super glue where the probe enters the Tygon tubing (c) and let dry for at least 20 min.

    Full size image
  7. 7.

    Adjust and secure probe within cannula assembly: With the capillary mounted on sticky tack, adjust the push–pull cannula assembly so that active portion of the probe membrane extends just beyond the end of the cannula tip (Fig. 8b). When this is done, apply a little bit of super glue to the entrance of the Tygon to hold the probe in position. Let this dry for 20 min. The fully assembled probe is shown in Fig. 8c and Fig. 9.

    Fig. 9.
    figure 9

    Photograph of dialyzing tip of an assembled probe.

    Full size image
  8. 8.

    Prepare capillary inlet to connect to a syringe filled with perfusion fluid: To fit the capillary tubing onto a luer lock syringe you need to first put the capillary through the luer adapter fitting and then insert the capillary through a red NanoTight®sleeve5. After inserting the capillary through the red NanoTight® sleeve, pull the tubing a little further out and cut off about 3–4 cm of tubing to get rid of capillary tubing that might be obstructed, leaving 1 mm projecting from the red NanoTight® sleeve. Place a ferrule on the sleeve and connect using normal HPLC fittings.

  9. 9.

    Prepare cannula to fit probe holder needed for stereotaxic surgery: If probes will be used in an acute, anesthetized preparation, unscrew the cap and use a dremel tool to file the edges of the outer threads of the internal cannula to make two parallel, flat surfaces. Use a CMA clip designed to hold the flat body of a CMA 11 or 12 guide cannula6 for surgery. If probes will be used for an awake preparation that requires chronic guide cannula implantation, use a nut that threads onto the top of the guide cannula. Flatten the sides of the nut so that it fits securely within the CMA clip. To insure accurate depth of placement take care to adjust the length of capillary and dialyzing membrane to an exact length that is consistent for all probes.

  10. 10.

    Notes on performance: Due to the low internal diameter of the outlet capillary, these probes will sweat at relatively slow flow rates. Probes with short outlets (15 cm) sweat between 1.0 and 8.0 μL/min (mean of 4.7 μL/min, n  =  8). Probes with long outlets (100 cm) sweat between 0.3 and 0.8 μL/min (mean of 0.6 μL/min, n  =  8). In vitro tests show that when these probes are perfused at 0.1 μL/min the temporal resolution, or time to equilibrate to a new concentration of glu ranging from 0.5 to 2.0 μM, is about 1 min.

1.1 Supply List for Microdialysis Probe Construction

Manufacturer-Specific Items

  1. 1.

    Silica capillary Tubing; 105 μm od, 40 μm id (Polymicro Technologies; http://www.polymicro.com).

    Part No. TSP040105.

  2. 2.

    Regenerated cellulose membrane; 13 kDa cut-off (Spectrum Labs; http://www.spectrumlabs.com).

    Part No.132294.

  3. 3.

    Polyimide sealing resin; part No.5825 (W.R. Grace and Co.; http://www.discoverysciences.com).

  4. 4.

    Push–pull cannula and cannula guides (Plastics 1; http://www.plastics1.com).

    • Part No.C313ICP/NIT Push–pull connector-internal cannula.

    • Part No. C316GPIO/uncut (maximum length) push–pull guide cannula.

  5. 5.

    HPLC fittings and sleeves (Upchurch Scientific; http://www.upchurch.com).

    • Part No. F-237x NanoTight FEP sleeve (0.005”; red).

    • Part No. F-142Nx HPLC ferrule.

    • Part No. F-331Nx HPLC fitting.

  6. 6.

    CMA 11 and 12 clip (CMA Microdialysis AB; http://www.microdialysis.se).

General Items (Found in Hardware Store or Scientific Supply Catalog)

  • Small Tygon Tubing (0.020–0.025” id).

  • Super Glue.

  • Epoxy Glue.

  • 2-sided tape.

  • Microscope slides.

  • Ceramic capillary tubing cutter.

  • Poster putty.

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Chen, Cf., Rasley, B.T., Warlick, B.P.E., Green, T.K., Swearingen, K.E., Drew, K.L. (2013). Microdialysis and Advances for Sampling Synaptic and Extrasynaptic Pools. In: Di Giovanni, G., Di Matteo, V. (eds) Microdialysis Techniques in Neuroscience. Neuromethods, vol 75. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-173-8_4

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  • DOI: https://doi.org/10.1007/978-1-62703-173-8_4

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  • Publisher Name: Humana Press, Totowa, NJ

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  • Online ISBN: 978-1-62703-173-8

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