Metabolic Mapping

James S. McCasland1, Ginny M. Graczyk1

1 SUNY‐HSC at Syracuse, Syracuse, New York
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.6
DOI:  10.1002/0471142301.ns0106s11
Online Posting Date:  May, 2001
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Abstract

This unit describes a high‐resolution [3H]2‐deoxyglucose (2DG)/immunohistochemistry double labeling protocol which enables simultaneous visualization of metabolic and immunohistochemical markers (e.g., neurotransmitter‐specific epitopes) in the same tissue section. This approach generates single‐cell metabolic measures from large (many thousands) samples of neurons, and allows the assignment of putative neurotransmitter types to large samples of functionally assayed single neurons. Thus, it provides a unique opportunity to assay the metabolic activities of histochemically identified neural elements throughout an entire pathway. [3H]2DG injection and perfusion fixation of hamsters or mice is described first, followed by immunohistochemical staining of brain slices for a monoclonal antibody against glutamic acid decarboxylase‐6 (GAD‐6). Included are the steps for dipping and developing slides for autoradiography. An alternate protocol gives details of staining for cytochrome oxidase (CO).

     
 
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Table of Contents

  • Basic Protocol 1: [3H]2‐Deoxyglucose/Immunohistochemical Technique for Simultaneous Visualization of Metabolic and Antigenic Markers
  • Alternate Protocol 1: Histochemistry for Cytochrome Oxidase (CO)
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: [3H]2‐Deoxyglucose/Immunohistochemical Technique for Simultaneous Visualization of Metabolic and Antigenic Markers

  Materials
  • Mouse or golden Syrian hamster
  • 2.5 mCi/ml [3H]2‐deoxyglucose ([3H]2DG; American Radiolabeled Chemicals)
  • 1:9 (v/v) phenobarbital (see recipe)
  • 50 mg/ml sodium pentobarbital
  • PPPFL fixative, pH 6.5 and 7.4 (see recipe), 4°C
  • PPPFL fixative, pH 7.4 (see recipe), containing 30% (w/v) sucrose
  • 0.1 M sodium phosphate buffer, pH 7.4 containing 0.5% (w/v) glycogen (PB/0.5% glycogen; see recipe), 4°C
  • Blocking solution (see recipe)
  • GAD‐6 antiserum (see recipe)
  • Secondary antibody solution (see recipe)
  • 1:80 ABC reagent (see recipe)
  • 0.05 % DAB/0.01% hydrogen peroxide (see recipe)
  • 3% hydrogen peroxide (see recipe)
  • 50%, 70%, 95%, and 100% ethanol
  • Xylene
  • NTB‐2 autoradiography emulsion (Kodak)
  • Desiccant (e.g., mesh size 10‐12; Fisher)
  • D‐19 developer (Kodak)
  • Fixer (Kodak)
  • Nissl staining solution (see recipe)
  • 70% ethanol in glacial acetic acid
  • DPX mounting medium (Electron Microscopy Sciences)
  • Clean, novel cage
  • 1‐ml insulin syringe with 28‐G, 1/2‐in. needle (Fisher)
  • 3‐ml syringe and 25‐G, 5/8‐in. needle
  • Surgical tray
  • Dissection scissors
  • Peristaltic perfusion pump (Masterflex)
  • 21‐G needle
  • Scalpel with a size 15 blade
  • Microdissection scissors
  • 24‐well culture plates
  • Platform shaker
  • 52‐well honeycomb (see recipe)
  • 5.5‐in. suspension culture dishes (Fisher)
  • Gelatin subbed slides, subbed twice (unit 1.1)
  • Slide warmer, 37°C
  • 37°C oven
  • 37°C water bath in dark room
  • Dip miser (Electron Mircoscopy Sciences)
  • 15‐ml centrifuge tube
  • Drying rack
  • Light‐proof cabinet
  • Slide box
  • Desiccant bottles (Fisher)
  • Sticky labels
  • Foil
  • Slide rack
  • Additional reagents and equipment for sliding‐microtome sectioning of fixed tissue (unit 1.1) and mounting on gelatin‐subbed slides (unit 1.1)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Alternate Protocol 1: Histochemistry for Cytochrome Oxidase (CO)

  • Cytochrome oxidase (CO) solution (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
   Berod, A., Hartman, B.K., and Pujol, J.F. 1981. Importance of fixation in immunohistochemistry: Use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase. J. Histochem. Cytochem. 29:844‐850.
   Chang, Y.C. and Gottlieb, D.I. 1988. Characterization of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase. J. Neurosci. 8:2123‐2130.
   Dienel, G.A., Cruz, N.F., and Sokoloff, L. 1993. Metabolites of 2‐deoxy‐[14C]glucose in plasma and brain: Influence on rate of glucose utilization determined with deoxyglucose method in rat brain. J. Cereb. Blood Flow Metab. 13:315‐327.
   Durham, D., Woolsey, T.A., and Kruger, L. 1981. Cellular localization of 2‐[3H]deoxy‐D‐glucose from paraffin‐embedded brains. J. Neurosci. 1:519‐526.
   Kelly, P.A. and McCulloch, J. 1983. A potential error in modifications of the [14C]2‐deoxyglucose technique. Brain Res. 260:172‐177.
   McCasland, J.S. 1996. Metabolic activity in antigenically identified neurons: A double labeling method for high‐resolution 2‐deoxyglucose and immunohistochemistry. J. Neurosci. Methods. 68:113‐123.
   McCasland, J.S. and Hibbard, L.S. 1997. GABAergic neurons in barrel cortex show strong, whisker‐dependent metabolic activation during normal behavior. J. Neurosci. 17:5509‐5527.
   McCasland, J.S. and Woolsey, T.A. 1988a. New high‐resolution 2‐deoxyglucose method featuring double labeling and automated data collection. J. Comp. Neurol. 278:543‐554.
   McCasland, J.S. and Woolsey, T.A. 1988b. High‐resolution 2‐deoxyglucose mapping of functional cortical columns in mouse barrel cortex. J. Comp. Neurol. 278:555‐569.
   McCasland, J.S., Carvell, G.E., Simons, D.J., and Woolsey, T.A. 1991. Functional asymmetries in the rodent barrel cortex. Somatosens Mot. Res. 8:111‐116.
   Miao, F.J. and Lee, T.J. 1990. Cholinergic and VIPergic innervation in cerebral arteries: A sequential double‐labeling immunohistochemical study. J. Cereb. Blood Flow Metab. 10:32‐37.
   Mori, K., Schmidt, K., Jay, T., Palombo, E., Nelson, T., Lucignani, G., Pettigrew, K., Kennedy, C., and Sokoloff, L. 1990. Optimal duration of experimental period in measurement of local cerebral glucose utilization with the deoxyglucose method. J. Neurochem. 54:307‐319.
   Nelson, S.R., Schulz, D.W., Passonneau, J.V., and Lowry, O.H. 1968. Control of glycogen levels in brain. J. Neurochem. 15:1271‐1279.
   Nie, F. and Wong‐Riley, M.T. 1995. Double labeling of GABA and cytochrome oxidase in the macaque visual cortex: quantitative EM analysis. J. Comp. Neurochem. 15:115‐131.
   Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, O., and Shinohara, M. 1977. The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: Theory, procedure, and normal values in the conscious and anesthetized albino rat. J. Neurochem. 28:897‐916.
   Yokoyama, W.M. 1991. Monoclonal antibody supernatant and ascites fluid production. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 2.6.1‐2.6.7. John Wiley & Sons, New York.
Key References
  McCasland, 1996. See above.
  Provides more background information on the technique and additional examples of the labeling patterns obtained.
  McCasland and Hibbard, 1997. See above.
  Illustrates some computational strategies that can be applied to collecting and displaying data from the technique.
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