Efficiency of reinnervation of neonatal rat muscle of original and foreign nerves

Formation of neuromuscular connections in mammals may involve a hierarchy of efficiency of synapse formation at a stage when motor nerves have already contacted muscle fibers and during the transitional period of multiple innervation. In an attempt to test for such a hierarchy, we examined, in neonatal rats, the relative efficiency of reinnervation by foreign or original nerves implanted simultaneously in a large muscle so that competition for muscle fibers was minimized. The tibial nerve, containing gastrocnemius nerve fibers, and the “foreign” peroneal nerve were implanted into the denervated lateral gastrocnemius muscle. One to five months later, indirect tetanic tensions obtained upon stimulating the implanted nerves were measured by isometric techniques and were compared to contralateral control muscles. When both nerves were implanted side by side at the end-plate region, approximately equal tetanic tensions were obtained at the time of testing. The same result was also obtained when the tibial and common peroneal nerves were implanted into non-end-plate and end-plate regions, respectively. However, in the reverse experiment, the tibial nerve implanted at the end-plate region produced significantly higher tetanic tension than the peroneal nerve at the non-end-plate site in the same muscle. Thus, the original nerve, compared to a foreign nerve, appeared to reinnervate neonatal muscle more effectively, but this was only revealed under conditions where access to former end-plate regions was unequal.     

Effects of innervation on the distribution of acetylcholine receptors in regenerating skeletal muscles of adult chickens

In order to determine the roles of nerves in the formation of clusters of acetylcholine receptors (AChRs) during synaptogenesis, we examined the distribution of AChRs in denervated, nerve-transplanted (neurotized) muscles and in regenerated skeletal muscles of adult chickens by fluorescence microscopy using curaremimetic toxins. In the denervated muscles, many extrajunctional clusters developed at the periphery of some of the muscle nuclei of a single muscle fiber and continued to be present for up to 3 months. The AChR accumulations originally present at the neuromuscular junctions disappeared within 3 weeks. In the neurotized muscles, line-shaped AChR clusters developed at 4 days after transection of the original nerve, but no change in the distribution of AChRs had occurred even at 2 months after implantation of the foreign nerve. The line-shaped AChR clusters were found to be newly formed junctional clusters as they were associated with nerve terminals of similar shape and size. Some of both the line-shaped and extrajunctional clusters were formed at least partly by the redistribution of preexisting AChRs. Finally, based on the above observations, the regenerating muscle fibers in normal muscles and in denervated muscles were examined: The extrajunctional clusters appeared in both kinds of muscles at 2 weeks after injury. Afterward, during the innervation process, the line-shaped AChR clusters developed while the extrajunctional clusters disappeared in the innervated muscles. In contrast with this, in the absence of innervation, only the extrajunctional clusters continued to be present for up to 3 months. These results demonstrate clearly that the nerve not only induces the formation of junctional clusters at the contact site, but also prevents the formation of clusters at the extrajunctional region during synaptogenesis.     

Acetylcholine sensitivity of innervated and noninnervated Xenopus muscle cells in culture

The chemosensitivity of Xenopus muscle cells grown in culture to iontophoretically applied acetylcholine (ACh) in the presence or absence of neurons was examined. Muscle cells grown without nerve cells are sensitive to ACh over their entire surface (2.4 mV/pC) with occasional spots of high chemosensitivity (“hot spots”). In cultures containing neural tube cells, the ACh sensitivity of muscle cells increased by approximately 50% regardless of the presence of nerve contacts or functional synapses. A similar increase in the ACh sensitivity was observed in muscle cells cultured in medium conditioned by neural tube cells. The ACh sensitivity of the extrajunctional region in functionally innervated muscle cells was not different from that of noninnervated cells growing in the same cultures. However, the chemosensitivity at the junctional region was about fivefold higher than that of the extrajunctional area. This increase in junctional chemosensitivity may well account for the increase in miniature endplate potential amplitude which has previously been reported to occur during nerve-induced ACh receptor accumulation.     

Spatial and temporal expression of acetylcholine receptor RNAs in innervated and denervated rat soleus muscle

In adult vertebrate skeletal muscle acetylcholine receptors are localized to the neuromuscular junction. Upon denervation, this distribution changes, with new receptors appearing in extrajunctional regions of the muscle fiber. The location of acetylcholine receptors in innervated or denervated muscle may result, in part, from the distribution of their RNAs. This was tested by assaying for receptor RNAs in junctional and extrajunctional regions of innervated and denervated rat soleus muscle using in situ hybridization and RNAase protection assays. These experiments showed alpha, beta, and delta subunit RNAs concentrated beneath the endplates of innervated muscle fibers. Following denervation, there was an unequal distribution of receptor RNAs along the muscle fiber, with highest levels occurring in extrajunctional regions near the endplate. These data are consistent with a nonuniform pattern of gene expression in adult skeletal muscle fibers.     

Effects of α-Bungarotoxin and Reversible Cholinergic Ligands on Normal and Denervated Mammalian Skeletal Muscle

α-Bungarotoxin (BuTX; 5 μg/ml) completely blocked the endplate potential and extrajunctional acetylcholine (ACh) sensitivity of surface fibers in normal and chronically denervated mammalian muscles, respectively, in about 35 min. A 0.72 ± 0.033 mV amplitude endplate potential returned in normal muscle fibers after 6.5 hr. of washout of α-BuTX, and an ACh sensitivity of 41.02 ± 3.95 mV/nC was recorded in denervated muscle after 6.5 hr of wash (control being 1215 ± 197 mV/nC). A two-step reaction of BuTX with binding sites which may allosterically interact is postulated.

Several pharmacologic differences were noted between the ACh receptors at the normal endplate and those appearing extrajunctionally following denervation. In normal innervated muscles exposed to BuTX in the presence of 20 μM carbamylcholine or decamethonium, washout of both drugs restored twitch to control levels within 2 hr. Endplate potentials large enough to initiate action potentials were also recorded in most surface fibers. In contrast, these agents, in much higher concentrations (50 μM), were almost ineffective in preventing BuTX blockade of ACh sensitivity in denervated muscle. Hexamethonium (10 and 50 mM) depressed neuromuscular transmission and blocked the action of BuTX in normal muscle in a dose-dependent fashion. On the extrajunctional receptors, hexamethonium (50 mM) was ineffective in protecting against BuTX. We may conclude that at the normal endplate region there are two distinct populations of ACh receptors, both of which react with cholinergic ligands and BuTX, but that a small population (representing ± 1% of the total) reacts with BuTX reversibly. Our findings further suggest a clear distinction between ACh receptors located at the normal endplate region and those of the extrajunctional region of the chronically denervated mammalian muscle.     

Expression of myosin heavy chain isoforms in regenerating myotubes of innervated and denervated chicken pectoral muscle

Monoclonal antibodies were prepared to stage-specific chicken pectoral muscle myosin heavy chain isoforms. From comparison of serial sections reacted with these antibodies, the myosin heavy chain isoform composition of individual myofibers was determined in denervated pectoral muscle and in regenerating myotubes that developed following cold injury of normal and denervated muscle. It was found that the neonatal myosin heavy chain reappeared in most myofibers following denervation of the pectoral muscle. Regenerating myotubes in both innervated and denervated muscle expressed all of the myosin heavy chain isoforms which have thus far been characterized in developing pectoral muscle. However, the neonatal and adult myosin heavy chains appeared more rapidly in regenerating myotubes compared to myofibers in developing muscle. While the initial expression of these isoforms in the regenerating areas was similar in innervated and denervated muscles, the neonatal myosin heavy chain did not disappear from noninnervated regenerating fibers. These results indicate that innervation is not required for the appearance of fast myosin heavy chain isoforms, but that the nerve plays some role in the repression of the neonatal myosin heavy chain.     

Acetylcholine receptors in normal and denervated rat diaphragm muscle. II. Comparison of junctional and extrajunctional receptors.

Acetylcholine (ACh) receptors have been purified separately from normal rat diaphragm muscle (junctional receptors) and from extrajunctional regions of denervated diaphragm (extrajunctional receptors) in order to compare their properties. The toxin-receptor complexes of the two receptors were indistinguishable by gel filtration and by zone sedimentation in sucrose gradients, and showed identical precipitation curves with rabbit antiserum to the eel ACh receptor. Both toxin-receptor complexes bind concanavalin A and are therefore probably glycoproteins. Low concentrations of d-tubocuratine (dTC) were more effective in decreasing the rate of toxin binding to junctional than to extrajunctional receptors. The apparent dissociation constant for dTC binding to the junctional receptor was 4.5 X 10 minus 8 M, whereas the value for the extrajunctional receptor was 5.5 X 10 minus 7 M. When the complexes were analyzed by isoelectric focusing, the junctional complex focused at approximately 0.15 pH unit lower than the extrajunctional complex. This result was also found with crude preparations of receptor. We conclude that junctional and extrajunctional receptors are similar but distinct molecules. The properties of receptors present in neonatal diaphragm muscle were also examined and found to be similar to those of receptors in denervated muscle, as shown by dTC inhibition and isoelectric focusing.     

Single channel properties of newly synthesized acetylcholine receptors following denervation of mammalian skeletal muscle

We have examined the single channel properties of newly synthesized acetylcholine (ACh) receptors in denervated adult mouse muscle. Patch-clamp recordings were made on freshly isolated fibers from flexor digitorum brevis (fdb) muscles that had been denervated in vivo for periods up to 3 wk. Muscles were treated with alpha-bungarotoxin (alpha-BTX), immediately before denervation, in order to block pre-existing receptors. Denervated fibers exhibited two types of ACh receptor channels, which differed in terms of single channel conductance (45 and 70 pS) and mean channel open time (approximately 7 and 2.5 ms, respectively). In contrast to innervated muscle, where only 3% of the total openings were contributed by the low-conductance channel type, greater than 80% of the openings in the nonsynaptic membrane of denervated muscle were of this type. Importantly, a similar increase in the proportion of low-conductance channels was observed for recordings from synaptic membrane after denervation. These data argue against the proposal that, in denervated muscle, the low-conductance channels undergo continued conversion to the high-conductance type focally at the site of former synaptic contact. Rather, our findings provide additional support for the idea that the functional properties of ACh receptors are governed uniformly by the state of innervation of the fiber and not by proximity to the site of synaptic contact.     

Distribution and quantification of ACh receptors and innervation in diaphragm muscle of normal and mdg mouse embryos

Muscular dysgenesis (mdg) in the mouse is an autosomal recessive mutation, expressed in the homozygous state (in vivo and in vitro) as an absence of skeletal muscle contraction. The distribution of acetylcholine receptors (ACh R) in the diaphragms of phenotypically normal and dysgenic (mdg/mdg) embryos was studied from the 14th to 19th day of gestation by binding of 125I-alpha-bungarotoxin to the muscle, followed by autoradiography of longitudinally sectioned hemidiaphragms and/or of isolated muscle fibers. Localization of ACh R at putative motor end-plate regions begins 14 to 15 days in utero in both normal and dysgenic diaphragms. The distribution of high ACh R density patches is aberrantly scattered beyond the normal innervation pattern in dysgenic diaphragms. Isolated mutant fibers possess (1) multiple ACh R clusters, up to five per single fiber, (2) larger clusters of more variable morphology and variable receptor density than normal clusters, and (3) higher levels of extrajunctional receptors than normal fibers. These autoradiographic results correlate well with higher total level of toxin binding sites per diaphragm and per milligram protein in dysgenic vs normal muscle, as quantified from gamma counting of sucrose density gradient isolation of 125I-toxin-ACh R complexes. The dispersed distribution of ACh R patches on dysgenic muscle may be correlated with extensive phrenic nerve branching as demonstrated by silver impregnation technique. We suggest that the aberrant ACh R cluster distribution is a result of multiple innervation of single fibers from the branched nerve terminals. Possible causes of the excessive nerve branching in the mutant are discussed in light of generalized nerve sprouting found in paralyzed muscle.     

Neural- and endocrine control of flight muscle degeneration in the adult cricket,Gryllus bimaculatus

Neural- and endocrine mechanisms controlling degeneration of a dorsal longitudinal flight muscle, M112a, have been studied in adult Gryllus bimaculatus (Orthoptera: Gryllidae). Decapitation completely prevented muscle degeneration. Implantation of a pair of corpora allata or injection of juvenile hormone III into decapitated crickets caused muscle degeneration. Denervation of M112a resulted in reduction of muscle mass compared with that in sham-operated crickets. Denervation of M112a in decapitated crickets, however, did not affect muscle mass. Birefringence and ultrastructure of M112a showed an obvious regional difference in the onset of degeneration. Fibrillar structures of M112a always disappeared from the ventral to dorsal part. Distribution of axon terminals of motor neurons and mechanical responses to the motor nerve stimuli showed that M112a is composed of five motor units with similar twitch properties. When M112a was fully denervated, regional differences in degeneration disappeared. Partial denervation resulted in denervated muscle fibers losing birefringence earlier than innervated fibers. These results suggest that juvenile hormone causes breakdown of flight muscles, and neural factors control degeneration of flight muscles to some extent under the presence of the juvenile hormone.     

Acetylcholine receptor degradation in adult rat diaphragms in organ culture and the effect of anti-acetylcholine receptor antibodies.

Acetylcholine receptor located at the neuromuscular synapse of normal innervated adult muscle fibers is extremely stable metabolically. We have studied the kinetics of receptor degradation in both normal innervated and denervated rat diaphragms in organ culture. These studies show that degradation of receptor-bound 125I-alpha-bungarotoxin is a valid measure of junctional receptor degradation. Degradation of junctional receptor is similar or identical to degradation of extrajunctional receptor in many ways: 1) both require energy, 2) both are inhibited by specific lysosomal protease inhibitors, 3) both are inhibited by treatment with colchicine, and 4) both are stimulated by treatment with anti-acetylcholine receptor antibodies. The one important distinction between degradation of junctional and extrajunctional receptor is a 10-fold difference in rate constant for the process.     

The developmental change in immunological properties of the acetylcholine receptor in rat muscle

We have used serum from a patient with myasthenia gravis containing antibodies that recognize unique determinants on the extrajunctional acetylcholine receptor (AChR) to characterize the AChR in extracts of developing rat muscle. Using mixtures of extrajunctional and junctional AChR from denervated and normal adult muscle, respectively, as standards, we estimated the proportion of each receptor type in muscle extracts of embryonic and neonatal rats. The presence of the immunologically adult form of the AChR was first detected during the first postnatal week. Analysis by two methods showed that this is also the time during which the proportion of the total muscle receptor that is at end plates increases.     

"Donor" muscle structure and function after end-to-side neurorrhaphy

End-to-end nerve coaptation is the preferred surgical technique for peripheral nerve reconstruction after injury or tumor extirpation. However, if the proximal nerve stump is not available for primary repair, then end-to-side neurorrhaphy may be a reasonable alternative. Numerous studies have demonstrated the effectiveness of this technique for muscle reinnervation. However, very little information is available regarding the potential adverse sequelae of end-to-side neurorrhaphy on the innervation and function of muscles innervated by the "donor" nerve. End-to-side neurorrhaphy is hypothesized to (1) acutely produce partial donor muscle denervation and (2) chronically produce no structural or functional deficits in muscles innervated by the donor nerve. Adult Lewis rats were allocated to one of two studies to determine the acute (2 weeks) and chronic (6 months) effects of end-to-side neurorrhaphy on donor muscle structure and function. In the acute study, animals underwent either sham exposure of the peroneal nerve (n = 13) or end-to-side neurorrhaphy between the end of the tibial nerve and the side of the peroneal nerve (n = 7). After a 2-week recovery period, isometric force (F(0) was measured, and specific force (sF(0) was calculated for the extensor digitorum longus muscle ("donor" muscle) for each animal. Immunohistochemical staining for neural cell adhesion molecule (NCAM) was performed to identify populations of denervated muscle fibers. In the chronic study, animals underwent either end-to-side neurorrhaphy between the end of the peroneal nerve and the side of the tibial nerve (n = 6) or sham exposure of the tibial nerve with performance of a peroneal nerve end-to-end nerve coaptation approximately 6), to match the period of anterior compartment muscle denervation in the end-to-side neurorrhaphy group. After a 6-month recovery period, contractile properties of the medial gastrocnemius muscle ("donor" muscle) were measured. Acutely, a fivefold increase in the percentage of denervated muscle fibers (1 +/0 0.7 percent to 5.4 +/-2.7 percent) was identified in the donor muscles of the animals with end-to-side neurorrhaphy (p < 0.001). However, no skeletal muscle force deficits were identified in these donor muscles. Chronically, the contractile properties of the medial gastrocnemius muscles were identical in the sham and end-to-side neurorrhaphy groups. These data support our two hypotheses that end-to-side neurorrhaphy causes acute donor muscle denervation, suggesting that there is physical disruption of axons at the time of nerve coaptation. However, end-to-side neurorrhaphy does not affect the long-term structure or function of muscles innervated by the donor nerve.     

Acetylcholinesterase in singly and multiply innervated muscles of normal and dystrophic chickens. II. Effects of denervation.

Neural regulation of mature normal fast twitch muscle of the chicken suppresses high activity, extrajunctional localization, and isozyme forms of acetylcholinesterase (AChE) characteristic of embryonic, denervated and dystrophic muscle. Normal adult slow tonic muscle ofthe chicken retains intermediate levels of activity and embryonic isozyme forms but not extrajunctional activity; it is not affected by muscular dystrophy. The hypothesis that neural regulation of the AChE system is lacking in slow tonic muscle and thus not affected by dystrophy was tested by denervating the fast twitch posterior latissimus dorsi and slow tonic anterior latissimus dorsi muscles of normal and dystrophic chickens. Extrajunctional AChE activity and embryonic isozyme forms increased, then declined, in both muscles. The results suggest that ocntrol of AChE is qualitatively similar in slow tonic and fast twitch muscle of the chicken.     

Ganglia implantation as a means of supplying neurotrophic stimulation to the newt regeneration blastema: cell-cycle effects in innervated and denervated limbs.

Regulation of blastema cell proliferation during amphibian limb regeneration is poorly understood. One unexplained phenomenon is the relatively low level of active cell cycling in the adult newt blastema compared to that of larval axolotls. In the present study, we used ganglia implantation as a means of "superinnervating" normally innervated adult newt blastemas to test whether blastema cell subpopulations are responsive to nerve augmentation. The effectiveness of implanted ganglia to provide neurotrophic stimulation was demonstrated in denervated blastemas. Blastemas implanted with 2 dorsal root ganglia and simultaneously denervated 14 days after amputation exhibited control levels of cell cycle activity 6 days later, as measured by 3H-thymidine pulse labeling. Denervated blastemas that were sham-operated or implanted with pituitary glands exhibited cell-cycle declines similar to those of denervated blastemas without implanted ganglia. Thus, 2 implanted ganglia provide neurotrophic stimulation equivalent to that of the normal nerve supply. Dorsal root ganglia implanted into normally innervated blastemas, which should effectively double neurotrophic activity to the blastema, had no effect on cell-cycle activity, innervated blastemas implanted with ganglia for 6 days exhibited pulse labeling indices similar to those of normally innervated blastemas. These data indicate that neurotrophic stimulation is not normally limiting in innervated limbs, and that some other factor, whether extrinsic or intrinsic to blastema cells, accounts for the relatively low level of active cell cycling in the adult newt blastema.     

Functional Recovery of Denervated Skeletal Muscle with Sensory or Mixed Nerve Protection: A Pilot Study

Functional recovery is usually poor following peripheral nerve injury when reinnervation is delayed. Early innervation by sensory nerve has been indicated to prevent atrophy of the denervated muscle. It is hypothesized that early protection with sensory axons is adequate to improve functional recovery of skeletal muscle following prolonged denervation of mixed nerve injury. In this study, four groups of rats received surgical denervation of the tibial nerve. The proximal and distal stumps of the tibial nerve were ligated in all animals except for those in the immediate repair group. The experimental groups underwent denervation with nerve protection of peroneal nerve (mixed protection) or sural nerve (sensory protection). The experimental and unprotected groups had a stage II surgery in which the trimmed proximal and distal tibial nerve stumps were sutured together. After 3 months of recovery, electrophysiological, histological and morphometric parameters were assessed. It was detected that the significant muscle atrophy and a good preserved structure of the muscle were observed in the unprotected and protective experimental groups, respectively. Significantly fewer numbers of regenerated myelinated axons were observed in the sensory-protected group. Enhanced recovery in the mixed protection group was indicated by the results of the muscle contraction force tests, regenerated myelinated fiber, and the results of the histological analysis. Our results suggest that early axons protection by mixed nerve may complement sensory axons which are required for promoting functional recovery of the denervated muscle natively innervated by mixed nerve.     

Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses

To elucidate the nature of signals that control the level and spatial distribution of mRNAs encoding acetylcholine receptor (AChR), alpha-, beta-, gamma-, delta- and epsilon-subunits in muscle fibers chronic paralysis was induced in rat leg muscles either by surgical denervation or by different neurotoxins that cause disuse of the muscle or selectively block neuromuscular transmission pre- or postsynaptically and cause an increase of AChRs in muscle membrane. After paralysis, the levels and the spatial distributions of the different subunit-specific mRNAs change discoordinately and seem to follow one of three different patterns depending on the subunit mRNA examined. The level of epsilon-subunit mRNA and its accumulation at the end-plate are largely independent on the presence of the nerve or electrical muscle activity. In contrast, the gamma-subunit mRNA level is tightly coupled to innervation. It is undetectable or low in innervated normally active muscle and in innervated but disused muscle, whereas it is abundant along the whole fiber length in denervated muscle or in muscle in which the neuromuscular contact is intact but the release of transmitter is blocked. The alpha-, beta-, and delta-subunit mRNA levels show a different pattern. Highest amounts are always found at end-plate nuclei irrespective of whether the muscle is innervated, denervated, active, or inactive, whereas in extrasynaptic regions they are tightly controlled by innervation partially through electrical muscle activity. The changes in the levels and distribution of gamma- and epsilon-subunit-specific mRNAs in toxin-paralyzed muscle correlate well with the spatial appearance of functional fetal and adult AChR channel subtypes along the muscle fiber. The results suggest that the focal accumulation at the synaptic region of mRNAs encoding the alpha-, beta-, delta-, and epsilon-subunits, which constitute the adult type end-plate channel, is largely determined by at least two different neural factors that act on AChR subunit gene expression of subsynaptic nuclei.     

Possible functional role of substance P on the mammalian motor nerve terminals

The effect of substance P (sP) on mammalian skeletal myoneural transmission was studied employing innervated and denervated isolated rat diaphragm preparations. sP at a concentration of 3.7 nM facilitated the indirect twitch responses of the rat diaphragm and antagonised the paralytic effect of d-tubocurarine (d-Tc). sP failed to affect the direct twitch responses as well as the contractures induced by acetylcholine (ACh) and potassium chloride (KCl) in the denervated diaphragm. The amount of ACh released into the bathing medium in response to tetanic stimulation of the phrenic nerve was doubled in presence of sP. The study illustrates a presynaptic facilitatory involvement of sP on mammalian myoneural transmission.     

Acetylcholine receptor alpha-, beta-, gamma-, and delta-subunit mRNA levels are regulated by muscle activity

Denervation of adult skeletal muscle results in increased sensitivity to acetylcholine in extrajunctional regions of the muscle fiber. This increase in acetylcholine sensitivity is accompanied by a large increase in the level of mRNAs coding for the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. To determine whether muscle activity is sufficient to regulate expression of extrajunctional acetylcholine receptor mRNA levels, denervated muscles were stimulated with extracellular electrodes. Direct stimulation of denervated muscle suppresses both the increase in extrajunctional acetylcholine sensitivity and the expression of mRNA encoding the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. These results show that muscle activity regulates the level of extrajunctional acetylcholine receptors by regulating the expression of their mRNAs.     

The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture

This paper describes the physiological and pharmacological parameters of the response of mature muscle fibers that develop from myoblasts in vitro to iontophoretically applied acetylcholine (ACh) and the distribution of ACh sensitivity over fibers innervated in vitro by spinal cord cells and uninnervated (control) fibers. Peaks of sensitivity were detected near nerve terminals on functionally innervated fibers, but the “extrasynaptic” chemosensitivity remained high. The distribution of chemosensitivity over uninnervated fibers is not uniform: peaks or “hot spots” were detected over most fibers. Autoradiography of cultures exposed to ¹²⁵I-α-bungarotoxin is consistent with the uneven distribution detected by iontophoresis. Sensitivity peaks were usually located in the immediate vicinity of obvious muscle nuclei and conversely the membrane near most nuclei was more sensitive than that over other regions along the same cell. The relation between innervation and distribution of ACh sensitivity is discussed.