The Journal of Neuroscience, August 1989, 9(E): 2902-2906

Neurotransmission Regulates Stability of Receptors at the

Orlando L. Avila, Daniel 6. Drachman, and Alan Pestronk Department of Neurology, Johns Hopkins University, Baltimore, Maryland 21205

The majority of acetylcholine receptors (AChRs) at normally Loring and Salpeter, 1980; Bevan and Steinbach. 1983; Stanley innervated neuromuscular junctions are stable, with a half- and Drachman, 1983~). There is a growing body of evidence life averaging about 12 d in most rodent muscles. Following that the stability of thesejunctional AChRs dependson some denervation, the AChRs turn over much more rapidly after a function of the motor nerve. (1) Following surgicaldenervation, lag period. The mechanism by which motor nerves normally the rate of degradationof preexisting stableAChRs accclcrates maintain stabilization of junctional AChRs is not yet known. markedly after a lag period of severaldays, attaining a half-life In order to determine whether synaptic plays of 2-5.6 d (Loring and Salpcter, 1980:Levitt and Salpeter, 198I ; a role in this process, we have compared the effects of pre- Stanley and Drachman, 1981; Bcvan and Steinbach, 1983). (2) and postsynaptic chloinergic blockade with those of surgical After reinnervation of the denervatcd muscle, the AChRs are denervation. again stabilized (Salpeter et al., 1986). (3) Recently we have ‘251-a- was used to label junctional AChRs found that the initial stabilization of new AChRs after insertion and follow their loss over time. Presynaptic blockade of at normal mature neuromuscularjunctions also requites inncr- quanta1 ACh transmission was produced in the soleus (SOL) vation (Stanley and Drachman. 1983a.c;D. Ramsay and D. B. and flexor digitorum brevis muscles of mice by repeated Drachman, unpublishedobservations). Taken together, thisevi- injections of type A botulinum . Postsynaptic blockade dence indicates that the motor nerve provides some influence of quanta1 and nonquantal ACh transmission was produced that results in stabilization of junctional AChRs and maintc- by continuous infusion of a-bungarotoxin in the SOL. Our nanceof their stability. findings show that treatment with resulted The mechanismby which the nerve’s receptor-stabilizing in- in an accelerated loss of junctional AChRs that was similar fluence is mediated is not yet known. Sincecholincrgic synaptic to the effects of surgical denervation, though briefly delayed transmission has been shown to play a role in the neural rcg- in its onset. Treatment with a-bungarotoxin produced an ef- ulation of other properties of skeletal muscles, including the fect that was quantitatively equivalent to the accelerated synthesisand insertion of cxtrajunctional AChRs, and the rest- loss of junctional AChRs following surgical denervation, with ing membranepotential (Thesleff, 1960; Berg and Hall, I975a: an identical time course. These results support the concept Pcstronk et al., 1976; Mathers and Thesleff. 1978; Rubin et al., that synaptic transmission is a mediator of the 1980;Drachman et al., 1982) we wonderedifACh transmission neural control of stability of junctional AChRs. The possibility might also influence the stability of junctional AChRs. Other that receptor stabilization may represent a mechanism of hypothetical possibilitiesinclude physicalcontact bctwcen nerve long-term postsynaptic “memory” dependent on neural and musclemembranes (Steinbach, 1974;Anderson and Cohen, transmission is discussed. 1977; McMahan et al., 1984) or nontransmitter substancesre- leasedby the nerve (Albuquerque et al., 1972; Cangiano, 1973). Both the distribution and the turnover ofacetylcholine receptors In the present study, we have examined the role of ACh trans- (AChRs) of mammalian skeletalmuscles arc rcgulatcd to a large mission in the maintenanceof stability ofjunctional AChRs in extent by motor nerves (Fambrough, 1979: Drachman et al.. 2 musclesof the mouse,the soleus(SOL) and the flexor digi- 1984; Salpctcr and Loring, 1985; Schuetzeand Role, 1987). In torum brcvis (FDB). These 2 muscleswere usedbecause their normally innervated mature muscles,AChRs are presentalmost junctional AChRs have different ratesof turnover and different exclusively at the postsynaptic membranes of neuromuscular time coursesof denervation-induced destabilization. We stud- junctions (Milcdi, 1960; Albuquerque et al., 1974; Bevan and ied the change in turnover of preexisting junctional AChRs Steinbach, 1977; Fambrough, 1979; Salpeter, 1987). The ma- following pharmacologicblockade ofcholinergic neuromuscular jority of junctional AChRs are stable; their half-lives are re- transmission.To do this we used 2 specific , botu- ported to rangefrom 8 to 13 d in rodents (Berg and Hall, 1975b; linum toxin and a-bungarotoxin (cu-BuTx). Uotulinum toxin se- lectively blocks the quanta1release of ACh from motor nerve terminals, whereas n-BuTx blocks AChRs postsynaptically, Received Dec. 29. 1988; revised Feb. 22, 1989; accepted Feb. 28. 1989. thereby interrupting both quanta1and nonquantal ACh trans- This work was supported by the US Army. contract DAMD 17-85-C-5069, mission (Brooks, 1956; Thesleff. 1960; Changeux et al., 1970; grants from the Muscular Dystrophy Association and the NIH (I RO I AG07438. I ROI NS237 19). We are grateful to Dr. D. Ramsay for helpful discussion. to I’. Miledi and Potter, 1971; Lee, 1972: Kao et al., 1976). Shih for rechnicalassistance, and to C. F. Salemi for assistance with the manuscript. Our findings show that treatment with botulinum toxin re- Correspondence should be addressed to Daniel B. Drachman. M.D.. Depart- ment of Neurology. Johns Hopkins liniversity, School of Medicine. 600 N. Wolfe sulted in an acceleratedloss of stable AChRs that was similar Street. Baltimore. MD 2 1205. to the effect of surgicaldenervation, though delayed in its onset. Copyright C 1989 Society for Neuroscience 0270-6474;89/082902-05%02.00/O Treatment with tu-BuTx quantitatively reproduced the effect of The Journal of Neuroscience, August 1989, 9(8) 2903 surgicaldenervation, with an identical time course.These results points, i.e., 4 and 6 d, which were found to be critical in our denervation support the concept that cholinergic synaptic transmissionplays and botulinum experiments. After 4 and 6 d of treatment, the muscles were removed and the loss of stable junctional AChRs was evaluated an important role in mediating the neural control of turnover as described below (see Results). of stablejunctional AChRs. The implication that similar mech- Measurement of turnover of stable junctional AChRs. A baseline was anisms of transmitter-driven receptor stabilization may con- established by removing muscles with labeled stable AChRs from the tribute to synaptic “memory” is discussed. control group on day 0. Samples of 4-l 3 muscles were removed from the control and experimental groups at various times thereafter. Ra- dioactivity bound to junctional AChRs was measured by counting the Materials and Methods whole excised muscle in a gamma spectrometer (Micromedic Systems, Inc., Horsham, PA). The counts for each muscle were corrected for Experimental design. In order to compare the effects of pharmacologic decay and then cxprcssed as a fraction ofthe mean counts of the baseline blockade of neuromuscular transmission and denervation on the sta- (day 0) control group for that batch of experimental mice. bility ofjunctional AChRs, we studied the turnover ofjunctional AChRs In these experiments, lJ’I-a-BuTx bound to the whole SOL or FDB by monitoring the loss over time of ‘z’l-a-BuTx that was specifically was used as a measure of radioactivity bound to the junctional AChRs. bound to junctional AChRs in viva. The loss of bound ‘z’I-a-BuTx has We conducted experiments that showed that at 6 d or more after labeling, been widely used to follow the loss of AChRs in skeletal muscles (Berg nonspecific or extrajunctional binding was negligible. To assess the de- and Hall, 1975b; Devreotes and Fambrough, 1975; Merlie et al., 1976; gree of nonspecific binding, we washed the muscles extensively (I8 Bevan and Steinbach, 1977; Gardner and Fambrough, 1979; Loring and washes over I8 hr). This removed less than I% of the bound radioac- Salpeter, 1980; Stanley and Drachman, 1983c, 1987; Salpeter et al.. tivity (unpublished results). In order to assess the degree of cxtrajunc- 1986; Ramsay ct al., 1988). tional binding, we measured the radioactivity in the junctional region In each series of experiments, we first prepared a pure population of relative to extrajunctional regions. We found that 6 d after labeling more labeled stable junctional AChRs, as described below. We then measured than 98% of the total muscle radioactivity was bound at the junctional (I) the turnover of “control” stable junctional AChRs (i.e., without regions (unpublished observations). Thus. whole muscle radioactivity pharmacologic treatment or denervation); (2) the effect of surgical de- can be used as an accurate measure of junctional radioactivity. ncrvation on the turnover of junctional AChRs; (3) the effect of trcat- Analvsis of data. The data for each muscle (SOL or FDB) at each ment with botulinum toxin or a-BuTx on the turnover of junctional time point were pooled, means and SEM werc‘calculated, and in the AChRs. WC were thus able to compare the effects of denervation and experiments with sufficient numbers oftime points, plotted on a semilog pre- and postsynaptic pharmacologic blockade of ACh transmission on graph by the least-squares method. Data for groups of muscles were the loss of stable junctional AChRs. compared by the Student’s 2-tailed I test. Female Swiss mice were used throughout these experiments: the an- imals were anesthetized with chloral hydrate (0.5 gm/kg body weight) and ether for all procedures. Results Labeling of AChRs with “‘I-U-BuTx. AChRs of SOL muscles wcrc The resultsof this study show that treatment of the mouseSOL labeled by injecting “‘I-cu-BuTx (0.04 &gm body weight in IO ~1 PBS) and FDB muscles with botulinum toxin produced a significant under direct visualization. In the experiment on the FDB muscles, in- jections were made percutaneously into the sole of the right foot (0.5 denervation-like acceleration of degradation of labeled stable pg “‘I-u-BuTx in 20 ~1 PBS). All injections were made via a fine 30 junctional AChRs, although the onset of this effect was delayed gauge needle. comparedwith that of surgicaldenervation. Postsynaptic block- Preparation of muscles with pure populations of labeled stable AChRs. ade with cu-BuTx, which blocks both quanta1 and nonquantal We have previously found that neuromuscular junctions normally con- ACh transmission, produced an effect that was quantitatively tain 2 populations of AChRs: a subpopulation of rapidly turned over receptors (RTOs) with a half-life of approximately I d and a majority equivalent to that of denervation, with an identical time course. of stable receptors with a half-life averaging I2 d. In order to follow the turnover of a pure population of stable receptors, we waited 6 d after Presynaptic blockade of neuromusculartransmission with labeling the junctional AChRs, so as to allow virtually all the RTOs to botulinum toxin be degraded (Stanley and Drachman, 1983c, 1987; Ramsay et al., 1988). At this time point (designated day 0), the only remaining labeled AChRs Figure 1 showsthe degradation curves for stablejunctional re- are the stable ones. ceptors of the SOL muscle. In the control muscles,bound ra- Surgicaldenervation. The SOL and FDB muscles were denervated on dioactivity was lost with a half-life of approximately I I.5 d. day 0 by sectioning the sciatic nerve in the midthigh and avulsing its proximal end to avoid reinnervation. Within 4 d after denervation, there was a significantly greater Presynaptic blockade of synaptic transmission with botulinurn toxin. lossofjunctional AChRs compared with the controls (p < 0.0 l), Purified Tvoe A botulinum toxin (aenerouslv provided bv Dr. E. Schantz) and an increasein the rate of degradation with a half-life of 3.6 was freshly diluted in mammal&t Ringer solution before use. In the d. Following treatment with botulinum toxin, the lossofAChRs SOL muscle, injections were made under direct visualization (I .5 x first becamesignificantly different from controls at 6 d (i.e., 2 IO I0 gm on day 0, and I .O x 10. 1/Igm on days 6 and 12). In the FDB muscle, 1.5 x IO- I0 gm botulinum toxin was injected percutaneously d later than in the denervated group; p < 0.01). The rate of on day 0, and 1.0 x IO- lo gm was injected on days 5, 12, and 19. All degradation increased,after botulinum treatment, to a half-life control animals received injections of Ringer solution at the same times. of 3.6 d. However, at the 6 d time point, the loss of labeled A volume of IO @I was used for all injections. junctional AChRs in botulinum-treated muscleswas not asgreat Postsynaptic blockade of ACh transmission with a-BuTx. Continuous neuromuscular blockade was produced by perfusing soleus muscles with as that of the dencrvated musclesat 6 d (p < 0.01). purified u-BuTx via implantable osmotic pumps as previously described Figure 2 showsthe degradation curves for stablejunctional (Pestronk et al., 1980; Drachman et al., 1982; Pestronk and Drachman, receptors of the FDB muscle. In the control muscles,radioac- 1985). Neuromuscular blockade was initiated at day 0 by injecting I. I tivity waslost with a half-life of 16.2d. This rate of degradation rg a-BuTx in PBS directly into the muscle via a fine 30 gauge needle. was consistently slower than that of the junctional AChRs of Blockade was then maintained by continuous perfusion of a-BuTx in PBS (0.06 &PI at 0.46 pl/hr) by means of Alzet model 2002 mini- the SOL musclemeasured here and of the reported rate for the osmotic infusion pumps (Al72 Corp., Palo Alto, CA). The pumps were sternomastoid muscles(Loring and Salpeter, 1980; Salpeter et implanted subcutaneously into the back, and the solution was delivered al., 1986; Ramsay et al., 1988). Following denervation, there directly over the belly of the SOL via tapered PE60 polyethylene tubing was a trend toward more rapid degradation of AChRs by day (Clay Adams, Parsippany, NJ) sutured in place. Control animals were injected and perfused in the same manner with similar volumes of PBS. 16, but the difference betweendencrvatcd and control muscles The number of infusion pumps that could be implanted in a single first becamestatistically significant at day 20 0, < 0.001). The cxperimcnt was limited, and we therefore elected to study the 2 time half-life ofjunctional AChRs at this time was4.5 d. Botulinum- 2904 Avila et al. * Neural Control of Stability of Junctional AChRs

I .o l l CONTROL 0.9 o DENERVATION l CONTROL . BOTULINUM o DENERVATION 0.8 . BOTULINUM 0.7

E 0.6

F5 0.5 2 P 2 0.4

0 5 0.3

B

5 sF 0.2

E

0. I \ I 2 3 4 5 6 7 8 9 IO II 12 I3 14 \ ‘\ DAYS POST DENERVATION 4 0.060 2 4 6 s IO I2 I4 I6 I8 20 22 24 26 28 30 Figure 1. Effect of surgical denervation and botulinurn toxin treatment DAYS POST DENERVATION on turnover of stable junctional AChRs in the soleus muscle. SOL muscles of mice were either denervated, treated with botulinurn toxin, Figure 2. Effect of surgical denervation and botulinurn toxin treatment or treated with control injections as described. Note the accelerated loss on turnover of stable junctional AChRs in the flexor digitorum brevis ofjunctional AChRs in the denervated and botulinum-treated muscles. muscle. Flexor digitorum brevis muscles of mice were treated as in The difference between control and denervated muscles was significant Figure 1. Note the accelerated loss of junctional AChRs in the dener- at day 4 @ < 0.01); for botulinurn-treated muscles, the difference first vated muscles, reaching significance at day 20 and in the botulinum- became significant at day 6 @ < 0.01). treated muscles at day 29. treated musclesalso showeda greater lossof labeledjunctional portant, the effectsof ol-BuTx treatment did not differ from those AChRs, which first reached statistical significancelater, at day of denervation at either time point (p > 0.1). 29 (p < 0.01). As noted above, the acceleratedturnover of AChRs began Discussion earlier after denervation than after botulinum treatment; in the The present investigation was designedto examine the role of SOL muscle, the loss of stablejunctional AChRs was signifi- neuromuscularsynaptic transmissionin the maintenanceof sta- cantly greater at 4 and 6 d (p < 0.01). bility of junctional AChRs. Treatment with botulinum toxin, which blocks neuromusculartransmission presynaptically, and Postsynaptic blockade of ACh transmission with oc-BuTx oc-BuTx, which acts postsynaptically, resulted in “destabiliza- In order to comparethe effects of postsynaptic blockade of ACh tion” of preexisting AChRs at neuromuscularjunctions, similar transmissionwith thoseof denervation, we measuredjunctional to the effect of denervation. We first compared the effects of AChRs of SOL muscles at 4 and 6 d after the beginning of botulinum treatment and denervation in two muscles,the SOL oc-BuTx treatment and denervation. Table 1 showsthat both and the FDB. The normal rates of degradation of junctional proceduresproduced a highly significantloss ofjunctional AChRs AChRs and the time course of denervation-induced destabili- at 4 and 6 d, as compared with controls (p < 0.01). Most im- zation are consistently different in these 2 muscles.In both

Table 1. Rapid loss of junctional AChRs following cx-BuTx treatment or denervation

Experimental period Treatment 4d 6d Ringer’s infusion (controls) 0.84 k 0.03 (n = 6) 0.70 + 0.05 (n = 5) ol-Bungarotoxin infusion 0.59 k 0.04 (n = 7)o,* 0.48 k 0.03 (n = lip” Denervation 0.64 f 0.02 (n = 7p 0.46 k 0.02 (n = 6p The proportion of junctional AChRs remaining at each time point is expressedas the mean fraction of the day 0 control values + SEM. Note the loss ofjunctional AChRs in a-BuTx-treated and denervated muscles as compared with Ringer’s controls. n = number of animals. y Less than control p < 0.0 1. * Not different from denervation, p > 0.1. The Journal of Neuroscience, August 1989, 9(8) 2905

muscles, botulinum toxin treatment resulted in an unequivocal turnover is being followed in these experiments, since the Y- denervation-like acceleration of the turnover ofjunctional AChRs. c+BuTx used as a label itself blocks the ligand-binding sites of However, in both muscles, the onset of the effect of botulinum these AChRs. However, ACh transmission acting on non- began later than that of denervation. By contrast, postsynaptic blocked AChRs at the same junctions presumably is responsible blockade using c+BuTx quantitatively reproduced the effects of for mediating a stabilizing effect. Although the differences be- denervation on the destabilization of junctional AChRs, with tween stable and destabilized AChRs have as yet been defined an identical time course. only in terms of their turnover times, this undoubtedly reflects The interpretation of these results depends on an understand- some biochemical or structural differences between them. Pos- ing of the actions of the 2 neurotoxins used. Botulinum toxin sible differences include covalent modifications of the AChR is a specific presynaptic inhibitor of the quanta1 release of ACh molecules (for example, by phosphorylation, acylation, or meth- (both impulse-dependent and spontaneous) from cholinergic ylation) (reviewed by Salpeter and Loring, 1985) attachment nerves (Brooks, 1956; Thesleff, 1960; Kao et al., 1976; Simpson, ofAChRs to cytoskeletal elements (Bloch and Hall, 1983; Froeh- 1981; Stanley and Drachman, 1983b). It does not block the ner, 1986), or alterations in the surrounding microenvironment nonquantal release of ACh (Stanley and Drachman, 1983b) and of the synaptic membrane (McMahan et al., 1984). has no other known actions on nerves or muscles. In particular, Perhaps the most important implication of the present work it does not produce structural damage (Thesleff, 1960) nor does is its possible relation to synaptic memory processes. Previous it interfere with axonal transport (Pestronk et al., 1976). In studies of synaptic memory have focused for the most part on contrast, the action of a-BuTx is postsynaptic; numerous studies presynaptic mechanisms that modify (Kan- have demonstrated the specificity of action of a-BuTx in block- de1 et al., 1987). By contrast, stabilization of AChRs constitutes ing the ligand-binding sites of nicotinic junctional AChRs a long-term modification of the neuromuscular junction at a (Changeux et al., 1970; Miledi and Potter, 1971; Lee, 1972). postsynaptic level. AChR stabilization takes place over a much or-BuTx is therefore strategically located to interfere with both more extended time scale than the phenomenon of receptor quanta1 and nonquantal ACh transmission, in contrast to the “desensitization,” which has previously been proposed as a form quanta1 blockade produced by botulinum toxin. Although the of short-term postsynaptic memory (Changeux et al., 1984,1987). binding of cu-BuTx to AChRs is virtually irreversible, the rapid Thus, AChR stabilization represents a model of long-term post- synthesis and insertion of new AChRs at neuromuscular junc- synaptic “memory,” albeit at a peripheral between mo- tions (Ramsay et al., 1988) requires that or-BuTx be applied tor nerve and skeletal muscle cells. The present results dem- continuously to maintain persistent blockade of ACh transmis- onstrate that neurotransmission plays a key role in mediating sion. Prolonged application of cu-BuTx by the perfusion method this process. We suggest that similar mechanisms of transmitter- used here does not damage neuromuscular junctions morpho- driven receptor stabiization may be involved in memory pro- logically or interfere with fast axonal transport (Drachman et cesses in the CNS as well. al., 1982). 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