Proc. Nati Acad. Sci. USAVol. 79, pp. 2714-2716, April 1982Neurobiology

Autoradiographic demonstration of the sodium-dependent high-affinity choline uptake system

(acetylcholine/cholinergic system/transport)

GERARD A. RUCH, GEORGE B. KOELLE, AND URSULA J. SANVILLEDepartment of Pharmacology, Medical School/G3, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Contributed by George B. Koelle, December 28, 1981

ABSTRACT Mouse phrenic nerve-hemidiaphragm prepara-tions were incubated for 10 min at 37C in an oxygenated mediumcontaining all the constituents reported as essential for the sodium-dependent high-affinity choline uptake system, including [3H]-choline chloride, 3.1 Ci/mmol, 2.5 ImM. The phrenic nerve wasstimulated by a suction electrode with square waves of 4-msecduration, 3 V, at 10 Hz; some preparations were not stimulated.Similar preparations in which 25 ,uM hemicholinium was includedin the medium served as controls. After exposure of emulsion-coated coverslips apposed to the preparations for 53 days, devel-opment, and staining of the motor endplates for acetylcholin-esterase, there was a marked accumulation of silver grains overthe motor endplates of stimulated preparations. Unstimulatedpreparations showed considerably lighter deposits; accumulationwas blocked by hemicholinium.

The sodium-dependent high-affinity choline uptake (SDHACU)system, which is uniquely characteristic of cholinergic nerveterminals, has been described as an active, carrier-mediatedtransport system (1). Its ionic and energy requirements, distri-bution, and other features have been studied extensively, asreviewed recently (2, 3). Its salient properties include a Km inthe micromolar range, requirements for sodium, potassium,and chloride ions, the coupling of its capacity with neuronalactivity, and its selective inhibition by low concentrations ofhemicholinium (HC-3). These characteristics have guided thedevelopment of a procedure for the autoradiographic demon-stration of the SDHACU system in the electrically stimulatedmouse phrenic nerve-hemidiaphragm preparation.

METHODSYoung mice (13-16 g) were sacrificed by cervical spinal dislo-cation; the hemidiaphragm, with 0.5-1 cm of phrenic nerveattached, was removed as rapidly as possible, trimmed at thecostal margin, and placed in cold (10°C) Krebs-Ringer solution.Within a few minutes it was transferred to a vessel containing6.5 ml of a solution of the following composition, based on pub-lished reports (4-9): NaCl, 140 mM; KCl, 5 mM; CaCl2, 1.3mM; MgCl2, 0.5 mM; D-glucose, 11.1 mM; L-ascorbic acid,0.02 mM; Na2HPO4, 9.5 mM; NaH2PO4, 0.5 mM; ATP, 0.8mM; [3H]choline chloride, 2.5 ,M; the pH was adjusted to 7.2.[methyl-3H]Choline chloride was obtained from Amersham; thesample had a specific activity of77 Ci/mmol (1 Ci = 3.7 x 1010becquerels). It was diluted with unlabeled choline chloride toproduce final activities in the solution of 15.4, 3.1, and 0.62 Ci/mmol. The vessel was placed in a waterbath at 37°C and the

solution was bubbled with 2. The phrenic nerve was impaledin a suction electrode with internal and external chlorided silverwires; it was stimulated with a Grass S5 stimulator with squarewave pulses of 4 msec duration, 3 V, and 10 Hz for 10 min. (Itwas found that similar preparations with the ribs and completecentral tendon still attached continued to respond consistentlyunder these conditions with muscle contractions for more than30 min; however, when the muscle fibers were cut their ensuingdepolarization frequently resulted in a Aessation ofcontractionswithin a few minutes.) At the end of 10 min, stimulation wasdiscontinued and the tissue was transferred rapidly to the rinsesolution; this was identical with the incubation solution exceptthat unlabeled choline chloride, 2.5 ,AM, was substituted for[3H]choline chloride. Periods of rinsing were varied; for theresults described here, tissues were rinsed by rapid swirling for15 sec each in two successive rinse solutions. Controls consistedof preparations treated identically but with the inclusion of 25AuM HC-3 bromide in the incubation solution (10). This con-centration is far in excess of what should be required to inhibitSDHACU under these conditions because we wished to ensureessentially complete inhibition when the tissue was exposedsimultaneously to HC-3 and choline. Tissues were also incu-bated similarly under static conditions, without phrenic nervestimulation, in the absence and presence of HC-3.The procedure used for autoradiography was similar to that

described by Young and Kuhar (11). Preparations were spreadon subbed (treated with a solution of 0.5% gelatin and 0.05%chrome alum) slides and allowed to dry at room temperaturefor 2-3 hr.

Acid-washed coverslips (24 X 60 mm, Corning no. 1-1/2)were coated by dipping into Kodak NTB 3 emulsion (diluted1:1 with water) at 430C, air dried for 3 hr, and stored at 5YC overdesiccant before use. The emulsion-coated coverslips were at-tached to slides with hemidiaphragms in the dark with cyanoac-rylate glue. Squares of Teflon [1/8 inch (3 mm) thick] wereplaced on top of the coverslips and the assembly was clampedtogether with no. 20 binder clips. The assemblies were storeddesiccated at 50C.

After various periods of exposure, the coverslips were gentlybent away from the tissue by toothpick spacers and the emulsionwas developed as described by Young and Kuhar except thatregular Kodak fixer for 5 min and a 5-min rinse in distilled waterwere employed. The entire assembly was allowed to air-dryovernight, after which the spacers were removed. Prior to theirassembly, the slides were scored on the subbed side verticallyat the midpoint so as to allow later separation of the tissue from

Abbreviations: AcCho, acetylcholine; HC-3, hemicholinium; MEP,motor endplate; SDHACU, sodium-dependent high-affinity cholineuptake.

2714

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Proc. Natl. Acad. Sci. USA 79 (1982) 2715

the emulsion-coated coverslip. Additional etchings on the re-verse side of the slide were made in this area so as to ensurevertical and lateral repositioning after their separation. The tis-sue-bearing slide section was then carefully snapped off at thispoint. This allowed for the localization of acetylcholine (AcCho)esterase activity in the tissues without damage to the developedemulsion layer.The slide segments, to which the hemidiaphragm prepara-

tions remained attached, were stained for AcCho esterase withthe standard copper thiocholine method (12, 13) by incubationat 370C for 60 min. In spite of the prolonged periods of storageat 50C and previous exposure to the developing and fixing so-lutions, this resulted in clear selective staining of the motorendplates (MEPs). After dehydration of the tissues with in-creasing ethanol concentrations followed by xylene, the tissue-bearing slide sections were reapposed with the emulsion-coatedcoverslips with Permount and precisely aligned to their originalposition by aid of the scribe marks made previously. The slideswere examined with a light microscope by focusing successivelyon the stained MEPs of the tissue and on the silver grains de-veloped on the overlying emulsion-coated coverslip. Exami-nation of emulsions apposed to hemidiaphragms treated withnonradioactive choline revealed minimal discernable pressure

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RESULTS

Best results to date have been obtained with stimulated prep-arations incubated with [3H]choline at 3.1 Ci/mmol and ex-posed for 53 days prior to development. Fig. 1A shows a portionof such a preparation, focused on a group of AcCho esterase-stained MEPs. The identical area is shown in Fig. 1B, whichis focused on the silver grains of the overlying coverslip; theseare distinctly concentrated in the areas of the MEPs, which in-clude the terminations of the cholinergic motor nerve fibers.Fig. 1 C and D illustrates MEPs and overlying silver grains,respectively, in a preparation incubated similarly but understatic conditions, without stimulation of the phrenic nerve.Here there is a much lower concentration of silver grains overthe MEPs than in the preceding set. In Fig. 1 E and F is shownan area ofMEPs from a stimulated preparation incubated underconditions identical with those for Fig. 1 A and B but with theinclusion of 25 AM HC-3 in the medium; there is no evidenceof accumulation of silver grains over that of the backgrounddeposit.

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FIG. 1. Accumulation of silver grains over MEPs of mouse hemidiaphragms after incubation with [3H]choline chloride, 2.5 AM, 3.1 Ci/mmol,at 37TC for 10 min, followed by exposure of emulsion-coated coverslips for 53 days, and staining of MEPs for AcCho esterase. A, C, andE are focusedon the AcCho esterase-stained MEPs; B, D, and F, on the overlying coverslips. (x 165.) (A and B) Phrenic nerve stimulated at 10 Hz, 3 V, 4 msecduring incubation. (C and D) Phrenic nerve not stimulated. (E and F) Phrenic nerve stimulated, 25 AM HC-3 included in medium.

Neurobiology: Ruch et al.

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Proc. Natl. Acad. Sci. USA 79 (1982)

DISCUSSIONThe mouse phrenic nerve-hemidiaphragm is an ideal prepa-ration for the type of study described here, just as it has servedpreviously for the localization oftubocurarine-binding sites (14),AcCho esterase (15), organophosphate anticholinesterase agents(16), and nicotinic cholinoreceptors (17). It is sufficiently thinthat ordinary stretch preparations are satisfactory for many pro-cedures and hence the biochemical damage entailed by freezingor embedding for sectioning can be avoided. For the demon-stration of SDHACU activity, for which it is advantageous tostimulate constantly throughout the incubation period, this isreadily accomplished; the failure ofthe muscle fibers, which aredepolarized to various degrees by cutting, to respond consis-tently in no way interferes with the reaction, because it has beenshown that SDHACU activity is confined to the cholinergicnerve terminals and is absent from the muscle fibers (18). Re-cently Waser et al. (19) have utilized the same preparation forthe autoradiographic demonstration of the uptake of [14C]cholineinto MEPs and muscle fibers in vivo after its intravenous admin-istration; under these conditions, in the absence of informationconcerning its local concentration, this procedure did not per-mit distinction between the uptake of choline by the high- orlow-affinity system (2, 3) or its accumulation at postjunctionalsites. For the application of the present method to thin slicesofbrain and other nervous tissue, the procedures developed byMcIlwain (20) for metabolic studies of free-floating electricallystimulated sections should prove satisfactory.

It has been postulated that there is a physical (21) or kinetic(22) coupling between SDHACU and choline acetyltransferase,which results in the immediate acetylation of choline after itsintracellular uptake. The present findings ofcourse do not per-mit distinction as to whether the radioisotope accumulated atthe MEPs represents mainly choline or AcCho. If choline isindeed immediately converted to AcCho this would explain inpart our failure to develop a colorimetric histochemical methodfor SDHACU based on the trapping of analogs of the nonacety-lated substrate after its intracellular accumulation during theincubation period. It should be possible to establish experi-

mental conditions that improve resolution of the localization ofSDHACU by the autoradiographic technique.

We thank Mrs. Kathleen Kitto Rickard for valuable assistance, andDr. C. P. Bianchi, Mr. Sirini Narayan, and Dr. S. D. Erulkar for adviceon stimulation procedures. This investigation was supported by Re-search Grant NS 00282-29,30 from the National Institute of Neurolog-ical and Communicative Disorders and Stroke, and by contributionsfrom the Foundation for Vascular-Hypertension Research, Philadelphia.

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564-575.7. Simon, J. R. & Kuhar, M. J. (1976)J. Neurochem. 27, 93-99.8. Suszkiw, J. B. & Pilar, G. (1976)J. Neurochem. 26, 1133-1138.9. Kuo, C.-H. & Yoshida, H. (1980)Jpn.J. Pharmacol. 30, 481-492.

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