Hereditary motor end-plate disease in the mouse: …Hereditary motorend-plate disease in the mouse:...

J. Neurol. Neurosurg. Psychiat., 1970, 33, 238-250

Hereditary motor end-plate disease in the mouse:light and electron microscopic studies

L. W. DUCHENFrom the Department of Neuropathology, Institute of Psychiatry and The Maudsley Hospital,

London S.E.5

With a genetic appendix by A. G. SearleFrom the M.R.C. Radiobiology Unit, Harwell, Berkshire

Hereditary neuromuscular disorders in mice provideuseful models for the study of the pathogenesis ofmany different disease processes and the reactionswhich they evoke in the nervous system. The clinicaland light microscopic features of a fatal hereditarydisease characterized by progressive muscular weak-ness were briefly described by Searle (1962) and byDuchen, Searle, and Strich (1967). The pathologicalchanges which were found suggested that theprimary abnormality in this disease was located inthe region of the motor nerve terminals, and thename 'motor end-plate disease' seemed appropriate.This paper presents the results of recent light andelectron microscopic studies of the disease which isnow known to be due to an abnormality in the func-tion of motor nerve terminals.

THE CLINICAL SYNDROME

This hereditary disease is transmitted by an auto-somal recessive gene (med). The heterozygous

carriers of the gene are clinically normal and noabnormalities are seen on histological examinationof their neuromuscular system. About 25% of theoffspring of matings of mice heterozygous for thegene are expected to be homozygous (med/med,henceforth called med mice) and show signs of thedisease (see genetic appendix by Searle). At birthand for the next eight to 10 days homozygous medmice cannot be distinguished from their normallittermates. The first signs of abnormality are un-steadiness of gait, the mouse swaying slightly fromside to side, with a nodding movement of the head.Within the next two to three days muscular weaknessbecomes increasingly severe and by the 12th to 14thday of age muscle wasting is apparent. The mousedrags the hind legs behind the trunk (Fig. la), theforelimbs cannot be extended and cannot properlysupport the body so that the animal tends to lie in aprone position with the head held low. Rightingreflexes are present but the mouse tends to fall overbecause of muscular weakness and takes longer

FIG. 1. An 18-day-old 'nied' mouse. (a) The hindlimbs are dragging because of weakness ofthe proximal thigh muscles.(b) The mouse has fallen over because ofmuscular weakness and can right itself though with difficulty.

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Hereditary motor end-plate disease in the mouse: light and electron microscopic studies

than normal to right itself (Fig. lb). The progressiveweakness affects especially the proximal muscles ofall limbs. The thigh can scarcely be flexed to thetrunk. By the 18th to 19th day the weakness is sosevere that the mouse cannot make effective move-ments. Most affected animals die at 19 to 23 days ofage. Occasionally they have survived until the 26thto 29th day, but some survive for only 15 days. Thecause of death is not entirely understood but clearlythe homozygotes have difficulty in obtainingsufficient milk, since they have to compete in suck-ling with their unaffected littermates and cannot eatsolid food or drink from the water-bottle availablein the cage. Sensory systems are not apparentlyaffected. Administration of anticholinesterase agentswas attempted in a few affected mice but did notproduce any significant effect on the muscularweakness.

MATERIAL AND METHODS

The studies described in this paper are the result ofhistological, histochemical, and electron microscopicexamination of more than 70 affected (med/med) mice.Some mice were killed at 10 days of age when there wasearly but definite clinical evidence of the disease. Thelongest surviving homozygous mouse examined was 29days old. For preparation of tissues for light microscopythe animals were killed with chloroform or with etherand, after removing the skin and opening the bodycavities, were fixed entire in formol-calcium (10%formalin in 1 % calcium acetate) or formol-alcohol (10%formalin in 60% alcohol). After decalcification in formic-citrate (90% formic acid-35 parts; 20% sodium citrate-65 parts) blocks were taken of all limbs, the entire trunkincluding spinal cord, and head including the brain andbrain-stem. Blocks of limbs were embedded in paraffinwax and sectioned in longitudinal and transverse planesand much use was made of the 'serial block' technique ofBeesley and Daniel (1956) in order to obtain transversesections at many levels of limbs, spinal cord, and brain.Paraffin sections were stained by various routine histo-logical methods including cresyl violet, with and withoutluxol fast blue; Palmgren's silver method; periodic acid-Schiff; and Sudan black. Silver impregnation of nervefibres by a modification of Schofield's method (1960)was carried out on serial frozen sections cut at 60tk fromblocks of limbs, trunks, and entire heads embedded ingelatin. Cholinesterase was demonstrated in serial 20Stlongitudinal frozen sections of entire limbs fixed byperfusion with cold (4°C) formol-calcium. The methodused was essentially that of Koelle and Friedenwald(1949) modified by Lewis (1961) and Henderson (1967).The substrates used were acetyl- or butyrylthiocholineiodide at a pH of either 5-2 or 5 6, with and withoutprior incubation for 30 minutes in tetra-isopropyl-pyrophosphoramide (iso-OMPA) in a concentration of3 x 10-M. The method of Namba, Nakamura, andGrob (1967) was used for silver impregnation of nerve

fibres in frozen sections after the completion of thecholinesterase reaction.The distribution in the muscles of the forelimb of two

enzymes, succinate dehydrogenase and phosphorylase,was studied in transverse cryostat sections of fresh tissuefrozen in iso-pentane cooled with liquid nitrogen.Succinate dehydrogenase was demonstrated by themethod of Nachlas et al. using Nitro-BT as described byPearse (1960). Phosphorylase was demonstrated by themethod of Lake (unpublished).

Electron microscopic studies were confined to m.biceps brachii after light microscopy had shown thismuscle to be severely abnormal in all med mice. Underether anaesthesia the skin over m. biceps brachii wasreflected laterally and the muscle covered by a pool ofcold (4°C) glutaraldehyde (25% in phosphate bufferpH 7-3). When the muscle was firm and yellow the bicepswas dissected out, pinned on cork board, and fixed forone to two hours in fresh cold glutaraldehyde. In morerecent work the animal was perfused through the leftventricle with cold buffered glutaraldehyde before thebiceps was dissected out and treated as described before.Blocks were taken from the region of motor innervationof biceps, washed overnight in buffer, post-fixed in 1 %osmium tetroxide, and dehydrated in graded alcohols.Blocks were stained in saturated alcoholic uranyl acetateand embedded in epoxy resin. In some cases blocks werewashed in cold buffer after fixation and then immersedin the substrate (acetylthiocholine iodide-copper sulphate-glycine) solution at 4°C andpH 5-2 for five to 15 minutesfor the demonstration of cholinesterase. No inhibitors ofpseudocholinesterase were used. The blocks were thenwashed and placed for a few minutes in cold 1 % sodiumsulphide solution in dilute acetic acid, washed overnightin cold buffer, post-fixed in osmium tetroxide, dehydrated,and embedded. Sections were stained with lead citrate.Tissues from many normal control animals, usuallylittermates of med mice but showing no clinical evidenceof disease, were treated for both light and electron micro-scopy in the same way as those from homozygous mice.

LIGHT MICROSCOPIC OBSERVATIONS

The characteristic histological abnormalities seenin this disease were atrophy of skeletal muscle fibresand a remarkable sprouting of the motor nervefibres innervating them. No changes were foundwhich suggested a primary myopathic disease. Nomorphological abnormalities were found in thebrain or brain-stem, in the ventral horn cells of thespinal cord, or in peripheral nerves proximal to themotor end-plates.

In the affected med mice aged 10 to 15 days therewere usually only mild abnormalities in the histo-logical appearance of skeletal muscles. In sections ofparaffin-embedded tissues the diameter of musclefibres was sometimes less than in normal littermates.At this stage the subneural apparatuses did notappear abnormal and no ultraterminal nerve fibreswere present. As the disease progressed it was found

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FIG. 2. Transverse section ofthe short head ofm. biceps brachii ofa normal 22-day-old mouse (a) and ofa 'med' mouseaged 24 days (b). There is severe atrophy ofalmost allfibres in the affected muscle. H and E, x 400.

that some muscles showed very much more markedabnormalities than others. The distribution of theseverely affected muscles was symmetrical and wasthe same in all the mice studied. The short head ofm. biceps brachii was uniformly very abnormalhistologically as were some other muscles of theshoulder girdle and also m. rectus femoris. Muscleswhich were less severely and less uniformly abnormalhistologically were the long head of the bicepsbrachii, triceps brachii, tibialis anterior, and gastro-cnemius muscles. In other muscles fewer fibres wereaffected but it seemed that no muscle was entirelynormal. In older med mice most muscle fibres ofm. biceps brachii were severely atrophied (Fig. 2)while in other muscles, such as the triceps (Fig. 3)atrophied fibres were found scattered throughoutthe muscle among fibres of larger diameter. Theatrophied fibres lay singly or in small groups. Therewas no fascicular arrangement of atrophied fibres.With longer survival the degree of atrophy and theproportion of muscle fibres affected seemed tobecome greater. The nuclei of the atrophied musclefibres were usually situated subjacent to the sarco-

lemma. No chains of nuclei were seen in longitudinalsections and no necrotic muscle fibres were found.

Silver impregnation of thick frozen sectionsshowed that there was marked sprouting of motornerve fibres at their terminals. Instead of the normalfairly simple terminal arborization (Fig. 4a) themotor nerve terminals were enlarged and complexand branching nerve fibres spread away from theregion of the motor end-plate along the muscle fibreand also across adjacent muscle fibres. Thesesprouting nerve fibres were of very irregular diam-eter, some fibres had a beaded appearance andlittle knobs were present at their tips. The pre-terminal motor nerve fibres did not appear to bebranched. No fragmentation or degeneration of pre-terminal axons was seen. In the mice surviving 19days or more the appearance of the innervation ofthe most severely affected muscleswasvery abnormal.The atrophy of muscle fibres resulted in markedcrowding together of nerve fibres and this, togetherwith the profuse nerve sprouting, caused the muscleto appear to contain motor nerves greatly in excessof normal (Fig. 4b).

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FIG. 3. M. triceps brachii of a 22-day-old 'med' mouse

shows many atrophied muscle fibres scattered among fibresofmore normal diameter. There is nofascicular distributionofatrophied fibres. H and E, x 400.

Marked abnormalities developed in the appear-ance of subneural apparatuses in sections stained todemonstrate cholinesterase (Fig. 5). The mostsevere abnormalities in cholinesterase preparationswere seen in those muscles with the most severe

- degree of atrophy and the most abundant motornerve sprouting. Instead of the normal regular

4 complex of gutters (Fig. 6a) the subneural ap-paratuses were elongated, irregular in size and shape,and had elongated branching projections. It seemedthat the distribution of cholinesterase followed thesprouting motor nerve fibres, and this was par-ticularly marked in the enzyme preparations in-cubated for 20 minutes in substrate (either acetyl-or butyrylthiocholine) at pH 5$6. The enzymeactivity along the newly formed motor nerve sprouts

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FIG. 4. Silver impregnation ofm. biceps brachii ofa normal mouse (a)and its 'med' littermate aged 19 days (b).In the normal muscle each motor nerve

fibre ends in a simple arborization.In the 'med' muscle the nerve fibres are

crowded together and there is sproutingfrom almost all nerve terminals so thatthe muscle contains more nerve fibresthan normal. Schofield, x 300.

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FIG. 5. Cholinesteraselocalization in m. bicepsbrachii ofa 'med' mouse aged19 days. The reaction productis seen in long brantchinglines extending from thegutters of the end-plates(compare with Fig. 6). Thereis no reaction product alongpreterminal axons. Acetyl-thiocholine substrate, pH 56,incubated 20 minutes. x 400.

was largely abolished by pre-incubation for 30minutes in iso-OMPA. Normally both true (acetyl)cholinesterase and non-specific cholinesterase arepresent at the end-plate, the reaction for the formerbeing much the stronger. In the muscles of the medmice it seemed that most of the enzyme associatedwith the motor nerve sprouts was non-specificcholinesterase. The sections stained to demonstratenerve fibres after the cholinesterase reaction showedthat the motor nerve sprouts arose from the terminals(Fig. 6b and c). The sprouts did not arise from pre-terminal myelinated axons. Cholinesterase activitycould be seen along the nerve sprouts in an irregularpatchy distribution.

DISTRIBUTION OF SUCCINATE DEHYDROGENASE ANDPHOSPHORYLASE The distribution of the enzymessuccinate dehydrogenase and phosphorylase inmuscle fibres of the forelimb was studied in 22 medmice aged 15 to 29 days and six normal mice (litter-mates of med) aged 15 to 19 days. In the muscles ofthe normal 15-day animal there was already differen-tiation of muscle fibres into two main classes, andthis differentiation was more clearly seen with in-creasing age and the intensity of the staining becamestronger. By the 19th day the histochemical differen-tiation of the muscle fibres and the distribution ofthe two main types was very like that of the adultmouse. In the superficial parts of m. biceps andtriceps brachii most of the muscle fibres gave astrongly positive reaction for phosphorylase, whilethe fibres rich in succinate dehydrogenase activitywere few in number (Fig. 7). In the deep parts ofthe muscles the majority of fibres appeared to berich in succinate dehydrogenase and fewer fibresgave a strong phosphorylase reaction. There wereconsistent changes in muscles of all the med micestudied histochemically (Fig. 8) biceps being affectedmore severely and uniformly than triceps, but inboth these muscles the changes were progressive

with longer survival. Succinate dehydrogenaseactivity was demonstrable in some muscle fibreseven in severely atrophied fibres of the biceps in thelongest surviving animal. The sequence of changesin the phosphorylase reaction was very differentfrom that of succinate dehydrogenase. In 16-day-oldmed mice there was a great reduction in the intensityof the colour reaction for phosphorylase in many ofthe muscle fibres of both biceps and triceps muscles.By the 19th day very few of the fibres in the shorthead of biceps, where the atrophy was uniformlysevere, showed any colour reaction and even thesewere weaker than normal and by the 23rd and 29thdays the phosphorylase reaction was almost absent.In triceps the abnormalities were always less severethan in biceps of the same animal and there wereusually some fibres which were not apparentlyatrophied and which gave a stronger phosphorylasereaction than the other smaller atrophied fibres.In the longest surviving mouse the colour reactionfor phosphorylase in triceps was weaker than inearlier stages of the disease. It seemed clear that theatrophy of the muscle fibres and the weakening ofthe phosphorylase reaction were both progressive.

MUSCLE SPINDLES AND OTHER SENSORY STRUCTURESIntrafusal muscle fibres of normal size and with astrong phosphorylase colour reaction were oftenseen among weakly reacting extrafusal fibres.Although the state of the motor innervation of theintrafusal fibres was not determined with certainty,the annulospiral sensory nerve endings appearednormal. The innervation of other sensory structuressuch as Pacinian corpuscles and vibrissae and ofskin and mucosae appeared normal.

ELECTRON MICROSCOPIC OBSERVATIONS

Electron microscopic examination of m. bicepsbrachii from normal mice showed the characteristicwell-known appearance of motor nerve terminals

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FIG. 6a

FIG. 6. Combined cholinesterase andsilver impregnation of m. biceps brachii ofnormal (a) and 'med' mice (b and c) 26days old. Each preterminal axon (arrowsto some) innervates one motor end-plate.Cholinesterase is localized to the subneuralapparatuses in the normal and there areno ultraterminal nerve fibres. In the 'mod'muscle nerve sprouts extenid beyond theend-plates and show patclhy cholinesteraseactivity (compare with Fig. 5). Acetyl-thiocholine substrate, pH 5 2, incubated15 minutes. Namba et al., a and b x 400;c x 600.

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and end-plates. The points which are relevant hereare that normally the end-plates are not easy tofind, that the motor nerve terminals lie in the guttersof sarcolemmal membrane, and that subneuraljunctional folds project into a region of sarcoplasmrich in cell organelles (Doyere's hillock) close to thecharacteristic sole-plate nuclei. Nerve terminals

normally always have subneural folds in theirimmediate vicinity. Cholinesterase can be demon-strated in the folds and in Schwann cell-axolemmaljunctions.

In m. biceps brachii of med mice aged 11 and 13days which showed definite clinical disease theappearances of the motor end-plates were normal

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FIG. 7. Succinate dehydrogenase (a) andphosphorylase (b) reactions in the superficial part of m. biceps brachii ofanormal mouse 19 days old. Most of the muscle fibres show a strong reaction for phosphorylase but few give a strongsuccinate dehydrogenase reaction. x 400.

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FIG. 8. M. biceps brachii ofa 'med' mouse aged 19 days. Succinate dehydrogenase (a) is demonstrable in most musclefibres although they are atrophied. The phosphorylase reaction (b) is very weak except in intrafusal fibres of musclespindles (arrows). x 400.

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Hereditary motor end-plate disease in the mouse: light and electron microscope studies

FIG. 9. Electron microscopic appearance ofpart ofa motor end-plate in m. biceps brachii ofa 'med' mouse aged 13 days.The appearance is like that of the normal. The terminal ofthe axon (A) contains abundant mitochondria, neurofilamentsand synaptic vesicles. The sarcolemmal membrane shows normal-looking subneuralfolds (arrows) and a sole-plate nucleus(N) lies close to the synaptic membrane. x 35,000.

(Fig. 9). The mice aged 18, 21, or 23 days showedabnormalities which were similar in all. Motornerve terminals were more numerous than normalthough their appearance was similar to the normalwith abundant vesicles, filaments, and mitochondria.The relationship between many of the nerveterminals and the muscle fibres was abnormal. Manynerve terminals were situated in regions wheresarcoplasmic organelles were not especially abundantand where no sole-plate nuclei were present-that is,they were outside the region of Doyere's hillock. Inrelation to some of these nerve terminals there w%reabnormally few or shallow junctional folds of thesarcolemmal membrane (Fig. 10). It seemed likelythat these were the terminals of the new nervesprouts. The nerve sprouts were always closelyenveloped by Schwann cells and their processes andcholinesterase reaction product was found inaxolemmal-sarcolemmal junctions and in axolemmal-Schwann cell junctions. Jnhibitors of pseudocholin-

esterase werenot used in electron microscopic studies.No denervated end-plates were seen in electronmicroscopic preparations. The appearance of axonsand myelin sheaths of intramuscular nerve fibreswas normal.

Within the muscle fibres the organization of themyofibrils was normal in the 11- and 13-day-oldmice. Abnormalities which were found to developin the muscle fibres were more conspicuous in theolder (21 and 23 day) mice than at earlier stages ofthe disease. In some randomly scattered musclefibres were dense bodies of varying size which wereusually rounded and occasionally contained vesiclesor membranes within them. The appearance of thedense bodies varied. They were either small andlying singly close to the Z line in the position wheremitochondria are normally situated or in aggregatesof larger dense masses which disorganized theinternal structure of some muscle fibres (Fig. 11).The most severely atrophied muscle fibres usually

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Hereditary motor end-plate disease in the mouse: light and electron microscopic studies2 c S *¢ .=, S i af~~~~~~~~L

FIG. 11. Electron microscopic appearance ofa muscle fibre ofbiceps brachii ofa 'med' nwuse 23 days old. Many densebodies (arrows to some) are present. Some appear to be membrane-bound and contain vesicles, while others are aggrega-tions of dense granules. The arrangement of the myofilaments is severely disorganized and dilatation of the sarcoplasmicreticulum is seen. x 27,000.

contained the most aggregations of such densebodies. Dilatation of the lateral cisternae of thetriads was found in some muscle fibres. These dilatedcisternae were few in number and scattered randomlyin young med mice but were found in greaternumbers in older mice. Lipid bodies were found inmuscle fibres of both normal and affected mice butappeared to be more numerous in the latter.

DISCUSSION

The clinical signs of this hereditary disease are dueto generalized muscular weakness. There are manyother hereditary neurological diseases in mice(Sidman, Green, and Appel, 1965) from which thesyndrome of the med mouse has to be distinguished.The progressive weakness leading to almost totalflaccid paralysis, the absence of inco-ordination orother evidence of cerebellar ataxia, and the shortsurvival time distinguish this disease from most ofthe mutants listed by Sidman et al. Progressivemuscular weakness is also found in the wobbler

mutant (Duchen, Falconer,and Strich, 1966; Duchenand Strich, 1968a) in which signs develop only atthe fourth week of age and which may survive formany months. Muscle weakness is also characteristicof hereditary dystrophia muscularis (Michelson,Russell, and Harman, 1955) but the clinical signsare recognizable only at three to four weeks of ageand survival is usually for many weeks (see alsogenetic appendix). However it is possible that amutant similar to med does exist elsewhere and mayhave been named and ailocated a gene symbol basedonly on the clinical syndrome.

Histological examination of the central nervoussystem ofmedmice failed to show any morphologicalabnormalities, particularly of the ventral horn cellsof the spinal cord, and no evidence of Walleriandegeneration was seen in ventral nerve roots or inperipheral nerves. The absence of such abnormalitiesin the lower motor neurone distinguished med fromwobbler in which vacuolation and degeneration ofmotor nerve cells was an early change found in thebrain-stem and spinal cord. In the skeletal muscles

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L. W. Duchen

of med mice the main abnormality found in themuscle fibres themselves was atrophy. The atrophyof muscle fibres was progressive in that, with longersurvival, fibres were smaller in the uniformlyseverely affected biceps, and in partially affectedmuscles such as triceps more fibres were atrophiedthan at earlier stages of the disease. There was noarrangement of atrophied fibres in groups or fasciclescharacteristic of partial motor denervation due tostructural loss of motor nerve fibres as in humanmotor system diseases or peripheral neuropathies(Adams, Denny-Brown, and Pearson, 1962) and inthe wobbler mouse. In none of the medmice examinedhistologically was necrosis of muscle fibres seen andthe appearances were vtry unlike the changes whichhave been described in hereditary dystrophiamuscularis in the mouse (Michelson et al., 1955;West and Murphy, 1960; Pearce and Walton, 1963;Banker, 1967) or in other primary myopathicdiseases (Adams et al., 1962).The changes in the innervation of skeletal muscle

fibres of med mice consisted of sprouting from motornerve terminals and the spread of demonstrablecholinesterase along the nerve sprouts. These changesare morphologically very like the terminal motornerve sprouting which occurs during the prolongedperiod of muscle paralysis induced by the localinjection of botulinum toxin in skeletal muscles ofthe mouse(Duchenand Strich, 1968b; Duchen, 1970).Botulinum toxin causes paralysis of skeletal musclefibres without causing degeneration of preterminalor terminal nerve fibres. The similarity between thechanges in muscles of the med mice and thoseoccurring as a consequence of the local action ofbotulinum toxin suggested that the paralysis ofmuscle in the hereditary disease may be due to afunctional interruption of neuromuscular trans-mission (Duchen and Strich, 1967; Duchenet al., 1967). In both conditions there developsan apparently paradoxical situation in which theparalysed and atrophied skeletal muscles contain anexcess of motor nerve fibres. The electron micro-scopic observations have shown that the motornerves and terminals of med mice contain a normal-looking complement of vesicles and mitochondria.No denervated motor end-plates were seen. In theearly stages of the disease the muscle fibres appearednormal and the abnormal multi-vesiculated bodiesand dense bodies within muscle fibres in the latestages of the disease are best interpreted as beiniglysosomes and lipochrome granules developingduring muscle fibre atrophy.The principal enzyme histochemical change con-

sisted of a progressive loss of phosphorylase activity,particularly in the superficial fibres of both bicepsand triceps muscles where, in normal muscles, fibres

show a strong phosphorylase reaction. Surgicaldenervation of muscle in the mouse causes a similarreduction of phosphorylase activity in muscle fibres(author's observations).The nerve sprouting in the muscles of med mice

begins from the intact motor nerve terminals. Therewas no apparent sprouting from preterminalmyelinated axons. In conditions in which it is knownthat motor nerve sprouting occurs after the structuralloss of lower motor neurones (Edds, 1950; Hoffman,1950; Wohlfart and Hoffman, 1956; Duchen andStrich, 1968a) the pattem of innervation is unlikethat seen in the present disease and in all these otherconditions the sprouting has been presumed tooriginate from surviving preterminal axons.There is thus evidence that in hereditary motor

end-plate disease there is a progressive loss of func-tional innervation of skeletal muscles which come tocontain an excess of motor nerve fibres and whichshow no positive evidence for a structural loss ofmotor axons. Positive evidence for structuraldenervation would have been degeneration of pre-terminal axons, a fascicular distribution of atrophiedmuscle fibres, preterminal sprouting from survivingaxons, or denervated motor end-plates on electronmicroscopy. None of these was found. The patho-logical findings suggest that the disease is due toprogressive failure of neuromuscular transmissionand progressive functional denervation of skeletalmuscle. Recent electrophysiological studies (Duchenand Stefani, in preparation) have shown that this isin fact the case.

It is not known whether a human counterpart ofhereditary motor end-plate disease exists. Possiblysome cases of rapidly progressive or congenitalhypotonia are due to a disorder of similar type as inthis disease and are clinically indistinguishable fromother forms of infantile hypotonia. In such casesnormal histological appearances of the spinal cordat necropsy should prompt study of the innervationof skeletal musculature.

SUMMARY

Clinical and pathological studies (including lightand electron microscopy) of 'hereditary motor end-plate disease' in the mouse are described. Thisdisease is caused by a fully penetrant and fullyrecessive autosomal gene med which is closelylinked to the gene 'caracul' Ca in linkage group VI.Clinically it is characterized by progressive weakness,of skeletal muscles beginning at about eight to 10days after birth and is fatal usually within 14 daysof onset. Light microscopic examination of thecentral nervous system failed to show any abnorm-alities and the peripheral nerves were normal

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proximal to the motor end-plates. In early stages ofthe disease the structure of the motor nerve terminalsand of skeletal muscle fibres appeared to be normalwith both light and electron microscopy. Withlonger survival there was progressive atrophy ofskeletal muscle fibres, particularly severe in proximallimb muscles. The histochemical reaction forphosphorylase became progressively weaker. Motornerves innervating the atrophied muscle fibresshowed marked terminal sprouting, the nerve sproutsgrowing beyond the normal confines of the motorend-plate. The sprouting nerve terminals containedabundant vesicles and mitochondria but the axolem-mal-sarcolemmal junctions of these sprouts weredeficient in subneural folds. The pathological find-ings suggest that this hereditary disease is due toprogressive failure of neuromuscular transmissioncausing progressive functional denervation ofskeletal muscle.

I should like to thank Mr. A. J. Davey, Mr. I. J. Stiff,and Mr. P. Marriott for the microscopic preparationsand Mr. P. M. Taylor for help with photography. Thiswork was supported by grants from the Research Fundof the Bethlem Royal and Maudsley Hospitals, theMuscular Dystrophy Associations of America, Inc., andthe National Fund for Research into Crippling Diseases.

ADDENDUM

Since this paper was written the results of an inves-tigation of med mice by Zacks, Sheff, Rhodes, andSaito (1969) have been published. These authorsreport that no abnormalities of the motor end-plateand no atrophy of muscle fibres were found. Theyconclude that the disease is primarily myopathic innature. The most likely reason for the failure ofZacks et al. to observe significant pathologicalchanges in the muscles of the med mouse may bethat they did not observe that different muscles areaffected with varying degrees of severity. Theirstudies were confined to intercostal and gastro-cnemius muscles, which are not uniformly severelyaffected in this disease.

REFERENCES

Adams, R. D., Denny-Brown, D., and Pearson, C. M. (1962). Diseasesof Muscle. 2nd ed., Henry Kimpton: London.

Banker, B. Q. (1967). A phase and electron microscopic study ofdystrophic muscle. I. The pathological changes in the two-week-old Bar Harbor 129 dystrophic mouse. J. Neuropath.exp. Neurol., 26, 259-275.

Beesley, R. A., and Daniel, P. M. (1956). A simple method for prepar-ing serial blocks of tissue. J. clin. Path., 9, 267-268.

Duchen, L. W. (1970). Changes in motor innervation and cholin-esterase localization induced by botulinum toxin in skeletalmuscle of the mouse: differences between fast and slowmuscles. J. Neurol. Neurosurg. Psychiat., 33, 40-54.Falconer, D. S., and Strich, S. J. (1966). Hereditary progressiveneurogenic muscular atrophy in the mouse. J. Physiol. (Lond.),183, 53-55P.

Searle, A. G., and Strich, S. J. (1967). An hereditary motor end-plate disease in the mouse. J. Physiol. (Lond.), 189, 4-6P.and Strich, S. J. (1967). Changes in the pattern of motor innerva-tion of skeletal muscle in the mouse after local injection ofClostridium botulinum toxin. J. Physiol. (Lond.), 189, 2-4P.- (1968a). An hereditary motor neurone disease withprogressive denervation of muscle in the mouse: the mutant'wobbler'. J. Neurol. Neurosurg. Psychiat., 31, 535-542.

(1968b). The effects of botulinum toxin on the pattern ofinnervation of skeletal muscle in the mouse. Quart. J. exp.Physiol., 53, 84-89.

Edds, M. V. Jr. (1950). Collateral regeneration of residual motoraxons in partially denervated muscles. J. exp. Zool., 113, 517-551.

Henderson, J. R. (1967). The use of silver for intensifying sulfidedeposits in the cholinesterase technique. Stain Tech., 42, 101-102.

Hoffman, H. (1950). Local re-innervation in partially denervatedmuscle: a histo-physiological study. Aust. J. exp. Biol. med. Sci.,28, 383-397.

Koelle, G. B., and Friedenwald, J. S. (1949). A histochemical methodfor localizing cholinesterase activity. Proc. Soc. exp. Biol. Med.,70, 617-622.

Lewis, P. R. (1961). The effect of varying the conditions in the Koelietechnique. Bibl. anat. (Basel), 2, 11-20.

Michelson, A., Russell, E. S., and Harman, P. J. (1955). Dystrophiamuscularis: a hereditary primary myopathy in the housemouse. Proc. nat. Acad. Sci. (Wash.), 41, 1079-1084.

Namba, T., Nakamura, T., and Grob, D. (1967). Staining for nervefiber and cholinesterase activity in fresh frozen sections. Amer.J. clin. Path., 47, 74-77.

Pearce, G. W., and Walton, J. N. (1963). A histological study ofmuscle from the Bar Harbor strain of dystrophic mice. J. Path.Bact., 86, 25-33.

Pearse, A. G. E. (1960). Histochemistry. Theoretical and Applied.2nd ed., Churchill: London.

Schofield, G. C. (1960). Experimental studies on the innervation ofthe mucous membrane of the gut. Brain, 83, 490-514.

Searle, A. G. (1962). Mouse News Lett., 27, 34.Sidman, R. L., Green, M. C., and Appel, S. H. (1965). Catalog of the

Neurological Mutants of the Mouse, Harvard Univ. Press:Cambridge, Mass.

West, W. T., and Murphy, E. D. (1960). Histopathology of hereditaryprogressive muscular dystrophy in inbred strain 129 mice.Anat. Rec., 137, 279-295.

Wohlfart, G., and Hoffman, H. (1956). Reinnervation of musclefibers in partially denervated muscles in Theiler's encephalo-myelitis of mice (mouse poliomyelitis). Acta psychiat. scand.,31, 345-365.

Zacks, S. I., Sheff, M. F., Rhodes, M., and Saito, A. (1969). MEDmyopathy. A new hereditary myopathy of mice. Lab. Invest.,21, 143-153.

GENETIC APPENDIX

A. G. SEARLE

The med gene arose by spontaneous mutation in astock homozygous for non-agouti (a), brown (b),chinchilla (Cch), pink-eyed dilute (p), dilute (d),6

short-ear (se), and piebald (s). A technician, MissDaphne Buck, noticed that amouse in litter CT/478.lborn 23 October 1958, was making wriggling seal-

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