EXPERIMENTAL

NEUROLOGY

Distribution Motor

48,

542-556 (1975)

and Density of Cholinergic Endplates of a Denervated

Receptors at the Mouse Muscle

C. W. PORTERAND E. A. BARNARD l Departments

of Biochemistry

and Pathology, State Buffalo, Received

New

York

University of New

York,

14214

March 3, 1975

The localization and quantitation of the acetylcholine receptor at the normal and S-day denervated mouse diaphragm endplate has been evaluated by means of labeling with tritiated or-bungarotoxin followed by electron microscope autoradiography. The toxin binds specifically and irreversibly to the acetylcholine receptor molecules of skeletal muscle. The distribution of silver grains over denervated endplates labeled to saturation with this toxin is consistent with a receptor location on the postjunctional membrane at a mean density similar to that of the innervated endplate, 8,SOO/pm’. As in the innervated endplate, the receptors are not located uniformly along the membrane, but are concentrated at the fold crests, i.e., that area nearest to the axonal membrane when it is present, at a density near to 22,030/rm9 Since morphometric measurements show that the total postjunctional membrane at the denervated endplate is unchanged at S days, we deduce from the mean receptor site density that the total receptor population at the endplate remains the same. On the basis of these findings, we conclude that only a very small population of nicotinic receptors could be associated with the terminal axon, and perhaps that none at all are present there. Further, the receptors on the postjunctional membrane are unaffected in either density or distribution following the removal of neural influences by denervation even though these same influences have a profound effect on extrajunctional receptors and endplate cholinesteraee. Additional studies on junctional receptors at later periods of denervation were not possible due to degenerative tissue changes associated with denervation. 1 Send reprint requests to Dr. E. A. Barnard, Department of Biochemistry. This work was supported by Grant GM-11754 of the National Institutes of Health. Dr. Porter was supported by N.I.H. Pathology Training Grant 01500; his present address is Department of Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo, N.Y. 14263. The authors are especially grateful to Dr. Tieh Chiu who prepared the [SH]bungarotoxin used in this study. 542 Copyright All rights

9

1975 by reproduction

Academic Press, in any

form

Inc. reserved.

CHOLINERGIC

RECEPTORS

543

INTRODUCTION The development of extrajunctional high sensitivity to acetylcholine (ACh) in skeletal muscle after nerve section is well known (4, 22, 27) and has been shown to be associated, as expected, with the appearance of new sites along the sarcolemma that have the binding properties of the ACh receptor (9, 18, 28). In a preceding paper (9), these binding properties of the extrajunctional receptors with respect to d-tubocurarine and to a specific neurotoxin, a-bungarotoxin (BuTX) , were shown to be generally similar to those of normal endplate ACh receptors. It is of interest to enquire whether, during the earlier stages of the denervation process (but after complete disappearance of the nerve terminal), there is any migration of receptors away from the enclplate or any other change in their density or distribut;on there. Such questions would be difficult to address by methods with lower resolution in space. i.e. based upon light microscope autoradiography or upon scintillation counting of endplatecontaining regions of muscle, due to the large increase in surrounding extrajunctional receptors. We approach this problem here by applying to denervated endplates a method we have developed previously on normal muscle (3-l-36), namely high resolution quantitative autoradiography of muscle labeled to saturation with a tritiated form of BuTX. The latter has been shown in a variety of pharmacological and biochemical studies (reviewed by Lee : 23), to be a sufficiently specific and irreversible blocker of the nicotinic acetylcholine (ACh) receptor. Previously, it was reported that only SC-65% of the BuTSreactive sites at the endplate are such receptor sites, the remainder being d-tubocurarine-insensitive ( 1, 9). but further studies have shown that, in fact, at least 905% of the endplate sites (and their equivalent in denervated muscle ) have the d-tubocurarine sensitivity and other properties of the ACh receptor (5, 11). In applying this autoradiographic methodology, we have found it convenient to introduce tritium into the BuTX molecule by [3H] acetylation. The monoacetyl and diacetyl forms ( [3H]BuTX) of the toxin, used in the present work, hare been shown by several criteria to retain fully the native binding activity of the toxin to the receptor, as described in full in the preceding study (9). In earlier studies with the electron microscope using this autoradiographic approach (33-36 ) , [“H] BuTX was introduced into the diaphragm muscles of the mouse and of the bat, E~trsir~s ~USCIIS. Autoradiographs were obtained thus that provided a dirrrt determination of the cholinergic receptor distribution at the motor endplate. Thus, the grain distribution found was consistent with a location of the labeled sites on the postjunctional membrane, at an average density of 8,500 receptor active centers/pm’. Other approaches to determining ACh receptor den-

544

PORTER

AND

BARNARD

sity have been made by several investigators, with differing results, and are reviewed by Porter et al. (35). The density noted is an average density over the entire postjunctional membrane, and had the same value in each of the three different muscles studied so far (34). However, detailed studies have shown (2, 34) that these receptor active centers are not uniformly dispersed over the postjunctional membrane, but are at a density of 20,000 to 25,000/~m2 in the crest regions of the membrane (i.e. at and close to the juxtaneuronal area of the folds) and at a very much lower density in the depths and on the lower walls of the folds. A qualitatively similar location of such sites at the juxtaneuronal zone of the postjunctional membrane has also been noted by Fertuck and Salpeter ( 15). We now apply this approach to evaluate changes in ACh receptor density and distribution at the mouse diaphragm endplate following motor nerve section. The removal of the presynaptic membrane as a result of denervation offers an opportunity, also, for us to examine the possibility of an original presynaptic location of some fraction of the receptors. MATERIALS

AND

METHODS

Toxin and Labeling. Pure a-BuTX, and its [3H]monoacetyl and [SH]diacetyl derivatives, were prepared and characterized as previously (6, 36). The diacetyl derivative gave the same behavior in its reaction at the mouse endplate, as described (9 )for the monoacetyl derivative, having the same specificity as native BuTX. The diacetyl form was preferred for the autoradiographic work, since it introduces twice the amount of radioactivity. Rockefeller albino (RR) strain male mice (24 g) were used. Denervation was achieved by removal of a thoracic segment of the left phrenic nerve under ether anesthesia. The labeled BuTX was injected into the caudal vein at a supralethal dose of 1.5 pg/g, body wt; death occurred in 5-7 min. The entire diaphragm with rib cage attached was removed, and denervation confirmed by the presence of the nerve stump. Endplate-rich regions from each hemidiaphragm (the innervated side serving as the control) were dissected out, and thoroughly washed in Tyrode solution at 4 C. Fixation was then applied for 2 hr at 4 C in 3% glutaraldehyde/ 0.1 M phosphate (pH 7.4) containing 0.2 M sucrose. Subsequent buffer washes, postfixation in buffered 1% OSO~, and Epon/Araldite were as before (35). Autoradiography. Autoradiographic methods, based upon those of Salpeter and Bachman (37) were followed as described (34, 35). Briefly, sections about 1,000 A in thickness were mounted on glass slides, stained with 2% aqueous uranyl acetate, layered with carbon, and coated (using

CIIOLISI

Distribution and density of cholinergic receptors at the motor endplates of a denervated mouse muscle.

EXPERIMENTAL NEUROLOGY Distribution Motor 48, 542-556 (1975) and Density of Cholinergic Endplates of a Denervated Receptors at the Mouse Muscle...
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