Elis F. Stanley

Mechanisms of acetylcholine receptor loss in myasthenia gravis.

The fundamental abnormality affecting the neuromuscular junctions of myasthenic patients is a reduction of available AChRs, due to an autoimmune attack directed against the receptors. Antibodies to AChR are present in most patients, and there is evidence that they have a predominant pathogenic role in the disease, aided by complement. The mechanism of antibody action involves acceleration of the rate of degradation of AChRs, attributable to cross-linking of the receptors. In addition, antibodies may block AChRs, and may participate in producing destructive changes, perhaps in conjunction with complement. The possibility that cell-mediated mechanisms may play a role in the autoimmune responses of some myasthenic patients remains to be explored. Although the target of the autoimmune attack in myasthenic patients is probably always the acetylcholine receptors, it is not yet clear which of these immune mechanisms are most important. It is likely that the relative role of each mechanism varies from patient to patient. One of the goals of future research will be to identify the relative importance of each of these mechanisms in the individual patient, and to tailor specific immunotherapeutic measures to the abnormalities found.

Sensory and motor nerve conduction velocities and the latency of the H reflex during growth of the rat

Abstract The H reflex evoked in the plantar muscles of the rat hind limb was studied. The latency of the reflex and the conduction velocity of the sensory and motor nerves in the reflex arc were determined in rats of different sizes. Sensory conduction velocities were 38.8 ± ( sd )5.8 m/s in 100- to 150-g rats and 60 ± 5.9 m/s in 450- to 500-g rats compared with motor conduction velocities of 30.1 ± 2.8 m/s in 100- to 150-g and 55.0 ± 6.9 m/s in 450- to 500-g rats. Both motor and sensory velocities were directly related to rat size. However, the latency of the H reflex evoked at the ankle was relatively independent of rat size. It was 6.62 ± 0.36 m/s in 100- to 150-g rats and 6.05 ± 0.43 m/s in 450- to 500-g rats with a mean for 39 rats from 100 to 500 g of 6.44 ± 0.41 ms. This method of determining motor and sensory conduction velocities in the rat is simple, does not necessitate killing the animal, and should prove useful in the study of experimental neuropathies.

Canine Neuroaxonal Dystrophy

Canine neuroaxonal dystrophy, a newly recognized familial disorder in Rottweiler dogs, is characterized by progressive sensory ataxia. Two of four dogs studied clinically were autopsied and the cerebellum was mildly atrophic. Massive numbers of axonal spheroids were present in many regions of the neuraxis but were most prominent in the dorsal horn of the spinal cord and the nuclei gracilis and cuneatus. Ultrastructurally, spheroids appeared to be swellings of distal axons which were filled with accumulations of smooth membrane-bound vesicles, membranous lamellae, dense bodies, and other organdies. Neuropathological changes were similar to those identified in human neuroaxonal dystrophy.

Botulinum toxin blocks quantal but not non-quantal release of ACh at the neuromuscular junction

Botulinum (BOT) toxin is known to block quantal acetylcholine (ACh) release at the neuromuscular junction but little is known about its effect on non-quantal ACh release. We have examined the effect of BOT on non-quantal ACh release directly using a variant of the electrophysiological technique described by Katz and Miledi. This method is based on the observation that non-quantally released ACh results in a small, continual depolarization of the postsynaptic membrane, after inhibition of cholinesterase. This depolarization can be revealed by suddenly blocking ACh receptors with a pulse of curare, resulting in an abrupt hyperpolarization, the amplitude of which is presumed to be proportional to the rate of non-quantal ACh release. BOT treatment resulted in a marked decrease in quantal ACh release as shown by miniature endplate potential (m.e.p.p.) frequencies (control 0.65 +/- 0.33 m.e.p.p.s/s; BOT 0.03 +/- 0.03 m.e.p.p.s/s). However, non-quantal ACh release measured by the curare induced hyperpolarization, was not significantly different in control and BOT treated diaphragms (control 1.01 +/- 0.09 mV: BOT 1.03 +/- 0.11 mV). Our results show that BOT does not block non-quantal ACh release at a time when it has a profound effect on spontaneous quantal ACh release. This suggests that quantal and non-quantal ACh release take place through different release mechanisms.

Denervation accelerates the degradation of junctional acetylcholine receptors.

Abstract Acetylcholine receptors (AChRs) at innervated neuromuscular junctions are turned over at a far slower rate than the extrajunctional AChRs that appear along the muscle fiber after a period of denervation. We examined the effect of denervation on the degradation rate of junctional AChRs. Junctional AChRs were labeled in the mouse diaphram in vivo by injecting [ 125 I]α-bungarotoxin into the thoracic cavity. Two days later one hemidiaphragm was denervated. The radioactivity remaining bound to the denervated and innervated hemidiaphragms was measured in groups of mice at intervals of 1 to 21 days after denervation. Between 4 and 8 days after denervation the rate of loss of radioactivity from the denervated hemidiaphragms increased from the control rate of 7% per day (half-life = 9.6 days) to 11% per day (half-life = 5.6 days). Radioactivity was lost from labeled extrajunctional receptors at a much faster rate, more than 50% per day (half-life = 22 h). Thus the degradation rate of preexisting junctional AChRs increased by about 40% after denervation, but remained far slower than the degradation rate of extrajunctional AChRs. Because some junctional AChRs labeled after denervation may have much faster turnover rates, this suggests that there are at least two different populations of AChRs at denervated neuromuscular junctions.

Cholinergic transmission regulates extrajunctional acetylcholine receptors

To determine the role of ACh transmission in the regulation of extrajunctional ACh receptors, we compared the effect of postsynaptic cholinergic blockade with that of surgical denervation. Blockade of ACh transmission was produced in the soleus muscles of rats by continuous local infusion of α-bungarotoxin, delivered by implantable osmotic pumps. Extrajunctional ACh receptors were measured by an 125I-α-BuTx binding method. Our results showed an increase of extrajunctional ACh receptors quantitatively equivalent to that resulting from surgical denervation. This denervation-like effect is attributed to elimination of (i) impulse-dependent ACh transmission (which normally triggers muscle usage), and (ii) spontaneous quantal and nonquantal ACh transmission. The influence of the nerve in regulating extrajunctional ACh receptors appears to be due to the sum of these forms of ACh transmission.

Denervation and the time course of resting membrane potential changes in skeletal muscle in vivo

The effect of denervation on the skeletal muscle resting membrane potential (RMP) was studied in the soleus muscle of the intact rat. The RMP decreased much later than reported in previous in vitro studies. With a very short nerve stump (less than 2 mm), the RMP began to decrease 18 h after denervation and 4 h after miniature end-plate potentials ceased. With a longer nerve stump (39 to 43 mm) the decrease in RMP was delayed about 3 h. Our findings in vivo fail to confirm previous arguments against a role for acetylcholine in regulating the muscle RMP. The influence of usage and the possible effects of spontaneous quantal and nonquantal acetylcholine release or other hypothetical factors are discussed.

ANTIBODY‐MEDIATED MECHANISMS OF ACh RECEPTOR LOSS IN MYASTHENIA GRAVIS: CLINICAL RELEVACE*

The basic abnormality in myasthenia gravis (MG) is a decrease of available acetylcholine receptors ( AChRs) at neuromuscular junctions, due to an autoimmune attack. The receptor deficit was first identified by the use of 12iiI-aBungarotoxin ( 12+a-BuTx), a purified snake toxin that binds specifically and virtually irreversibly to AChRs to measure AChRs in muscle biopsies from myasthenic patients. In the initial studies, neuromuscular junctions of MG patients bound only 11% to 30% as much radioactivity as those of control individuals, indicating that they hed a markedly reduced number of AChR sites.l These observations have now been confirmed in several laboratories, by a varicty of techniques using a-BuTx binding,+: or electrophysiological measurements.G~ Indeed, our recent data, based on over 100 motor-point biopsies, suggests that the radiometric measurement of junctional AChRs in biopsy material may be clinically valuable as one of the most sensitive diagnostic tests for MG (Pestronk and Drachman, in preparation). The concept of an autoimmune mechanism involved in the pathogenesis of MG was first suggested by indirect evidence, including the association of MG with other autoimmune diseases,s the high incidence of thymic abnormalities in MG,” the reduction of complement levels in patients with MG lo and the development of an experimental animal model of MG produced by immunization with purified AChR from the electric organs of eels or rays.11 Based on the knowledge of a receptor deficit in MG, and the analogy to the experimental animal model, antibodies directed against AChRs were soon sought in sera of myasthenic patients. Anti-rcceptor antibodies were identificd by several different immunological methods, all of which depend on a-BuTx for their specificity.l?-lj With the most sensitive radioimmunoassay, circulating antibodies that bind to human AChR are detected in sera of nearly 90% of myasthenic patients,l* although the titer does not correspond closely with the patient’s clinical condition, The question of whether the circulating antibodies have a pathogenic role, or merely represent a sccondary response to AChR damage caused by some other agent, is critical in understanding the disease mechanisms in MG. At the last conference on MG sponsored by The New York Academy of Sciences, we reported initially results of passive transfer experiments that demonstrated

The effects of nerve section on the non-quantal release of ACh from the motor nerve terminal.

The spontaneous release of acetylcholine (ACh) from motor nerve terminals is now thought to occur by two mechanisms: (a) quantal release, giving rise to miniature endplate potentials; and (b) non-quantal release. In this study we have examined the effect of nerve section on spontaneous non-quantal ACh release, and have compared the time-course of cessation of non-quantal and quantal ACh release. Non-quantal ACh release, measured by an electrophysiological technique, declined 4 h after nerve section to approximately 50% of the control value. At 8-10 h it briefly rose again, then gradually declined to undetectable levels. Spontaneous quantal release (frequency of miniature endplate potentials) in the same muscle fibers remained close to control levels for 8 h after nerve section, and also increased prior to failure. Decline of non-quantal ACh release appears to be the earliest change in the nerve terminal following nerve transection; it may therefore be relevant in understanding the effects of denervation on the consequent changes in muscle properties.

Structure and function of urodele myelin lacking alpha-hydroxy fatty acid-containing galactosphingolipids: Slow nerve conduction and unusual myelin thickness

Myelin of several Caudata (Urodela) species appears to be unique in the fact that it lacks hydroxycerebrosides and hydroxysulfatides although it contains their non-hydroxy counterparts. Comparison of the nerve conduction velocities in the Urodeles Necturus (salamander) and Notophthalmus (newt) with that in a reptile, Anolis (chameleon) which contains hydroxycerebrosides and -sulfatides indicated that the values were significantly reduced in the urodeles. Furthermore, urodele myelin thickness remained uniformly the same regardless of the size of the nerve fiber. Despite these differences the myelins appeared structurally similar. Electron microscopic and X-ray diffraction studies did not disclose any structural difference between the two orders. A teased fiber technique established that the ratio of internodal distance and fiber diameter in urodele nerves was essentially similar to that in Anolis. These findings suggest that the absence of hydroxycerebroside and -sulfatide may be related to the reduction in nerve conduction velocity and unusual myelin thickness in the urodele nervous system.