reflex and behaviour patterns elicited from implanted ...peripheral connexion is thas t of local...

Reflex and Behaviour Patterns Elicited fromImplanted Supernumerary Limbs in the Chick

by GEORGE SZEKELY and JOHN SZENTAGOTHAI1

From the Department of Anatomy, University of Pecs

WITH TWO PLATES

INTRODUCTION

EXPERIMENTAL disarrangement of orderly nervous connexions has for manyyears been used as an important tool to test the capacity of the nervous systemto re-adapt itself, i.e. to re-arrange either by purely functional means or with theaid of actual changes in the neuronal connexions of the original, more or lesspurposive, functional pattern of the system. By allowing us to introduce at will(by the transplantation of organs or even parts of the central nervous system)gross changes with certain completely new types of connexions, experimentalembryology contributed much to the development of our understanding of thebasic phenomena that underlie the orderly development of nervous connexionsand the elaboration of the whole complicated fabric and function of the nervoussystem. One of the fundamental concepts that has emerged from this line ofinvestigation and thinking is that of neurone specificity (Sperry, 1951; Weiss,1955), a term introduced to label the capacity of neurones to establish function-ally effective connexions selectively only with a certain definite set or sets ofother neurones. Another related concept concerned with neurones which haveperipheral connexions is that of local sign specificity (Sperry & Miner, 1949;Miner, 1951 a, b), a particular case of the more general concept of Weiss (1936,1942) of specific modulation, according to which tissues or organs transplantedheterotopically exert a specific modulatory influence upon the neurones thathave innervated them and provide them with a supposedly biochemical specificcharacter that enables them to establish in the centre synaptic connexionscharacteristic of the normal nerve supply of the tissue or organ. Similar mechan-ism might be at work also in the normal development of nervous connexions.

Most of the experimental work leading to these concepts having been doneon amphibia and fishes, it might be worth while to extend this investigation also tohigher vertebrates in order to obtain information on the validity and working ofthese phenomena of neurone specificity in warm-blooded animals. Additionally,

1 Author's address: Department of Anatomy, University Medical School, Dischka u 5, Pecs,Hungary.[J. Embryol. exp. Morph. Vol. 10, Part 2, pp. 140-51, June 1962]

G. SZEKELY AND J. SZENTAGOTHAI—SUPERNUMERARY LIMBS 141

the higher vertebrates (both birds and mammals) offer some important ad-vantages: (1) they have in general better differentiated and more specificreflexes—both with respect to reflexogenic sites and localization and individ-ualization of the response—compared with the often somewhat vague anddiffuse reflexes and behavioural patterns of the lower vertebrates and especiallyof the urodeles; and (2) because of their considerably restricted regenerativecapacity, especially of the central nervous tissues, the formation of completelynew nervous connexions absent in normal animals is less likely to distort thefunctional results of the disarrangement intended by the experimenter, especiallywhen the new connexions are over long distances.

Since from the higher vertebrates only the embryos of birds can be handledexperimentally in the early stages needed in these investigations, our experimentshave been performed in the chick. As a first step in a longer series of investiga-tions now under progress in our laboratory, we repeated an experiment per-formed in the frog by Miner (19516) in transplanting homoplastically anadditional limb-bud into a heterotopic site of the host in order to let it becomeinnervated by segments of the cord which do not normally innervate limbs.Attention in this experiment was primarily focused on reflex and behaviourpatterns elicited by stimulation of these heterotopically innervated super-numerary limbs, and an account of this is presented in this paper.

MATERIAL AND METHOD

Inbred strains of two local varieties of fowl (Hungarian Yellow and Speckled)were used for the experiments. Limb-primordia, both legs and wings, wereexcised from embryos on the 3rd day of incubation (stages 19-21 of Hamburger& Hamilton, 1951) and implanted into embryos of similar age and of the samestrain, usually in the region of the 19th to 23rd somites. For implantation of thegraft three types of operation were performed. (1) The implant was fixed into alongitudinal slit between the ectoderm and the somatopleur immediatelyventral to the somites (Hamburger, 1947). (2) The medullary tube was splitlongitudinally exactly in the midline over the length of three segments and theexcised limb primordium was simply placed upon the opening (Sandor, 1958).(3) Three somites were split with a paramedian incision penetrating into thesomite cavity and the limb primordium was fixed into the incision. In spite ofthe haemorrhage occurring during operation this latter procedure has provedthe best of the three methods in respect of successful innervation of the graft andundisturbed development of the host. Most of our successful experiments weretherefore performed with the third method. From about 120 operated embryos11 living chicks were hatched, of which 5 had wing grafts and 6 leg grafts. Oneof them had been operated upon by the first, 2 by the second, and 8 by the thirdmethod. After hatching the chicks were kept alive for 3-10 weeks. During thisperiod the reflexes and behaviour of the grafted limbs in response to mechanicalstimuli were frequently investigated and the most interesting cases were recorded

142 G. SZEKELY AND J. SZENTAGOTHAI—SUPERNUMERARY LIMBS

by cinephotography. The sacrificed animals were fixed either in formol (4 percent, for subsequent treatment by Bielschowsky, and 10 per cent, in other cases)or in a mixture of formol, ethanol, and glacial acetic acid. After careful ana-tomical dissection to ascertain the segmental source of innervation of thesupernumerary limbs, the respective parts of the cord were investigated inserial sections impregnated according to Bielschowsky or to Bodian or stainedwith Nissl's method.

RESULTS

General form and behaviour of the animals

Form and behaviour of the animals were generally not appreciably changed,save for the supernumerary limb on their backs or flanks. They seemed to takeno notice of it and the graft usually did not interfere with normal movements.The supernumerary limbs remained perfectly motionless and contained nomuscles at all. Degeneration of muscles, however well innervated, started on the10th day of incubation and by the 13th day practically all muscle tissue wasundergoing degeneration (Strazniczky, 1959). At hatching the joints were alreadyankylotic. The covering skin and feathers were normal and sensitive to mechani-cal stimulation. Only two chicks displayed serious difficulty in movement. Oneof them had been operated upon according to the first method, and the graft layfairly ventrad, quite near to the normal leg. Probably because of simple mechani-cal interference by the graft, the hip joint of the host's own ipsilateral leg hadbecome luxated and its walking was affected in consequence. The other embryooperated upon by the second method developed, after hatching, a defectivemovement of both legs and in consequence the chick soon learned to performall its locomotion by a rapid fluttering of the wings. The wings became largerand the feathers longer than is usual in normal chicks of the same age. Havingan additional grafted wing, this chick exhibited the remarkable phenomenon ofdeveloping on the immobile graft feathers as long as those on the hypertrophiednormal wings. None of the chicks operated upon by the third method showedany abnormality of movement or body posture, and their grafts were generallywell innervated. The growth rate of the grafted limbs was considerably reduced,being only about one-third of that of the normal limbs.

All transplanted limbs survived until the hosts were killed, except for a singlecase in which the animal was kept alive for a longer time; on the 60th day thegrafted wing became swollen and inflamed, and within a few days gangrene setin. The chick was killed on the 73rd day, before the complete destruction of thegraft, which still maintained some sensitivity and blood circulation. Perhaps theimmunological tolerance induced by the limb transplantation at an earlyembryonic age lasted only for a relatively short period, i.e. 60 days, but sincewe let only this single case survive we have no information whether this is theusual period under such circumstances.

G. SZfiKELY AND J. SZENTAGOTHAI—SUPERNUMERARY LIMBS 143

Reflex responses from the graft

Immediately after hatching mechanical stimulation of the supernumerarylimbs usually failed to provoke any specific responses of the host. The chicksproduced loud chirping and general escaping movements indicating pain. Fromthese general reactions the specific responses gradually emerged during the first3 days. To slight pressure applied to the transplanted limb with forceps nearlyall animals yielded a flexion reflex of the normal leg on the side from which thegrafts received their nerves. Only three of our cases did not show any specificresponse; one of them had a well-developed though perfectly insensitive leggraft, another with a wing graft indicated exclusively pain, and the third diedwithin 2 days of hatching. Irrespective of whether a wing or a leg had beentransplanted, the chick always responded with the limb nearest the graft. Thegrafts being situated mostly at the level of the lower thoracic segments, hind-limb flexion was the response obtained most frequently. Simultaneously with theflexion reflex of the leg an extension of the ipsilateral wing propped against theground as support could often be observed. The same supporting movementof the wing was also observed in normal chicks if painful stimulation causedelevation of one leg. But this reflex was never observed normally if the chick washeld in the hand of the experimenter during stimulation of the leg, whereassimultaneous wing extension downwards upon stimulation of the supernumerarylimb occurred both when the chick was freely standing on the ground and whenit was kept firmly in hand. Two cases which had leg grafts quite near to thebrachial segments (nos. 3 and 8) exhibited wing reflexes proper. Upon stimula-tion of the graft the ipsilateral wing was elevated.

It is most significant that such 'limb-specific' reflexes could be elicited withouta single exception from the graft only. Even the strongest stimuli applied to theskin in the close vicinity of the graft yielded only general escaping movements,struggling and loud chirping, but never 'limb-specific' reflexes. On the graftitself we could localize districts of different sensitivity for evoking the limbreflexes. There were some small regions of high sensitivity, while other zonesproved to be less sensitive or even ineffective. Of the six animals with a leggraft, the distal part covered with scales was sensitive to pain in only a singlecase (no. 8). At the same time it produced 'limb-specific' reflexes, while threeother chicks yielded the limb reflexes only from the perfectly painless scaled skin(or underlying tissues) of the transplanted legs. Animals with a wing graft didnot show such dissociation of sensation except the above-mentioned case withpain sensation only.

In one of the chicks operated upon according to the second procedure thegraft underwent duplication and developed a normal leg on the right side of theback and a single digit on the left. Both parts yielded a clear flexion response onthe left side, and an extension reflex on the right side which was most easilyproduced by stimulation of the complete leg. Anatomical investigation showed


later that both parts of the graft were innervated from the left side of the cord,so that the animal had the usual flexion reflex on the ipsilateral normal leg anda crossed extension reflex; this has not been seen—or at least not been clearlyseen—in other animals.

Behavioural responses from the graftsWe collected under the term of behavioural responses all more complex re-

action patterns elicited by stimulation of the graft obviously involving thefunction of higher centres. The simplest and most common form of this sort ofresponse was that after stronger pressure on the transplanted limb the chickstarted to walk lamely, frequently elevating its own leg on the graft's side andstanding on the contralateral leg for a while as if feeling strong pain in theelevated leg. Even more impressive were the characteristic cleaning and dis-entangling movements made with the beak as if to remove something unpleasantor painful attached to the normal ipsilateral leg. These responses could be sus-tained by applying a steel clamp to the graft thus making a prolonged painfulstimulation on the otherwise free animal (Plate 1, figs. A-D). The chick triedagain and again—for as long as the clamp was on—to clean or disentangle its legfrom the apparent painful stimulus. In one of our chicks stimuli applied to awing graft, though apparently localized on the ipsilateral normal leg, were noteffective in eliciting appropriate cleaning behaviour. However, a piece of threadtied on to one of the toes of the ipsilateral leg caused the chick to bend down andtry to tear it away; but when the clamp was removed from the graft, the chickignored the thread. In other cases, whenever by chance the clamp made contactwith normal skin or was caught sight of, the chicks localized its place correctly(Plate l, figs. E-H). In these cases we frequently witnessed (and recorded onfilm) the comic scene of the animal's hesitation at not being able to 'decide'whether to start cleaning where it saw the clamp or where it felt the pain to belocalized. One of the chicks (no. 8) with a transplanted leg in the neck regionexhibited a still more complicated behaviour, the graft being constantly in itsvisual field. As it was described above, slight mechanical stimuli elicited theelevation of the wing. Applying the clamp to the graft, the chick first localized iton its own wing. The erroneous localization lasted, however, no longer than theinitial phase of development of this behavioural pattern, and within a few daysit 'learned' that the clamp was placed somewhere on the graft. There was,nevertheless, no exact localization on the graft; the chick simply started cleaningthe grafted foot beginning with its nearest place (Plate 2, fig. I), and the clamp,if attached in such a way as to be hidden from sight by feathers, was foundgenerally by chance. Correct localization was, however, always experiencedwhen the clamp was visible to the animal. A few days later a new erroneouslocalization appeared in the same case when the chick occasionally looked forthe clamp on its own foot (Plate 2, fig. K). Projection of the unpleasant feelingcaused by the clamp on the [cervically innervated] graft to the ipsilateral host


leg was even more obvious when it occasionally 'tried to get rid' of somethingunpleasant by repeated quick jerky movements of the foot—the characteristicdisentangling behaviour of chicks.

TABLE 1

Exp.no.

1

2

3

4

5

6

7

8

9

10

11

Natureof graft

Leg

Leg(doubled)

Leg

Wing

Wing

Wing

Wing

Leg

Leg

Wing

Leg

Responses to stimulation of the grafts

Limb reflexes Behavioural patterns elicited Innervation of the grafts

Ipsilateral flexion reflexof the host's leg. Nopain sensitivity fromthe scaled skin of thegraft

Flexion of the contra-lateral extension of theipsilateral leg. No painsensitivity from thescaled skin of the graft

Elevation of the ipsi-lateral wing

Flexion reflex of the ipsi-lateral leg




Elevation of thelateral wing

lpsi-

Perfectly senseless

Sensitive only to pain

Looks at right foot andtries to clean it withbeak

After stronger pressure ofthe graft lame walking;cleaning the right footwhen clamp applied tograft (Plate 1, figs.A-D)

Same behaviour as inno. 6

Pressing the graft by for-ceps or application ofclamp the chick startscleaning either its ownwing or part of the graftlying in visual field;occasionally, especiallywith advancing age,projection of pain uponipsilateral own leg(Plate 2, figs. I-K)

Branches from the last n. inter-costalis and a cutaneousbranch from the lumbarplexus

Branches from the left spinalnerve of the 22nd segment

Branches from the 17th rightspinal nerve

A single nerve formed by theroots of the 23rd and 24thsegments for the innervationof the graft which gives a side-branch to the lumbar plexus

Strong plexus formed by thenerves of the 18th and 19thsegmental nerves, 5 branchesof which penetrate into thegraft

Plexus formed by two strongbranches from the 20th and21st segments, as well as bysmaller nerves from the 19thand 22nd segments

A small branch from the 20thand a large one from the 21stspinal nerves formed plexusfor innervation of the graft(Plate 2, fig. L)

Not investigated, presumablyfrom the 12th—13th segments

Not investigated

Strong branch from the 18thspinal nerve, smallerbranches from the 16th and17th nerves to the upper andlower rim of the graft's base

Died on 2nd day


These complex behavioural responses developed even later than the simplereflexes. It was not until the 5th day that the first response of this type appeared,and the 'kicking off' response just mentioned appeared only in the 3rd weekafter hatching. Limb reflexes could be evoked in 8 out of our 11 successfullyoperated cases, but only 4 chicks exhibited these behaviour patterns. Differencesin ' reflexogenic capacity' of various sites of the graft for evoking such behaviouralresponses were still more pronounced than for that of simple reflexes. As a rule,the former ones could be provoked only from a few particular points, whilst thechick was ready to yield limb reflexes from nearly the entire surface of the graft.These sensitive points showed some 'extinction' after long-lasting repeatedstimulation, and we had to wait for a few hours or even days till the behaviouralresponses appeared again.

Dissection and histological findingsAfter having been killed the animals were submitted to painstaking examina-

tion under the dissecting microscope in order to determine the exact segmentsfrom which the grafts had derived their nerve supply. We were unable to deter-mine the exact segmental origin of nerves supplying the graft in animal no. 1,which had been operated upon by inserting a leg-bud into the coelom and whichhad received its innervation from secondary branches of the lumbal plexus.Nor could we unfortunately investigate the innervation of the graft in case no. 8which, whilst being kept in the garden of our animal farm, probably fell victimto a stray cat. Presumably the transplanted leg was innervated by segments11-13. Innervation of the normal wings is furnished from segments 13 to 16,and of the legs from segments 23 to 29. In the majority of cases the nerves of thegraft emerged from one or two out of the segments 17-22, i.e. from thoracicsegments which under normal circumstances convey neither wing nor legsignals. Detailed data on segmental innervation of the grafts in the several casesare given in Table 1 with a short summary of the main functional observations.

Histological examination of spinal ganglia and spinal segments innervatingsupernumerary limbs in serial sections (Bielschowsky and Bodian methods) didnot reveal anything of interest beyond that described earlier by Detwiler (1936)on the newt and Hamburger (1939) and Hamburger & Levi-Montalcini (1949)on the chick, i.e. a hyperplasia of the respective spinal ganglia.

DISCUSSION

The results presented substantiate in every respect the findings of Miner(19516) and also our own in the newt indicating that those general mechanismsof nervous tissue development which Weiss (1924) first demonstrated and triedto explain, and which Sperry (1951), analysed more closely, must undoubtedlybe at work also in higher vertebrates. It is, however, quite another questionwhether one can agree with the hypothesis already briefly touched upon in theintroduction. The existence of specific differences not only between different sets

G. SZEKELY AND J. SZENTAGOTHAI—SUPERNUMERARY LIMBS 147

of neurones but even those between neurones of the same pool, or group, havingdifferent local positions may be considered as an established fact. It would alsobe difficult to account for such specific differences without assuming some minutebiochemical individuality, perhaps of their surface membrane, rendering theestablishment of some connexions between certain types of neurones possibleand others impossible. One can hardly fail to recognize the ingenuity of themodulation concept which shifts the primary cause—at least in the case of someneurones—to the periphery, i.e. to the tissue innervated, where it is much morelogical to assume fundamental differences in biochemical character according tospecificity of the tissue and location in different parts of the body. No objectionwhatever could be raised against the assumption of specific modulation ofneurones by the individual character of peripheral tissue with which they haveestablished connexion. Additional evidence has been produced recently byEccles, Eccles, & Magni (1960) for changes that are at least quantitative inmonosynaptic connexions of motor neurones in the kitten, when nerves werebrought into connexion with foreign muscles by cross-suturing. Thus the changeof synaptic connexion patterns, at least in motor neurones brought about bymodulation from the periphery, seems to be established experimentally.

This explanation of the observed phenomena appears to us, however, not tobe entirely satisfactory for the following reasons. (1) There is no correlationwhatever between the type of the graft—whether wing or leg—and the evokedresponses, since wing responses were elicited from leg grafts (nos. 3 and 8) andleg responses from wing grafts (nos. 4, 5, 6, and 7). (2) Impulses arising from theimplanted limb have a tendency to spread (or to be projected) in a cranio-caudal direction downward and to evoke responses in the framework of thegeneral structural organization and functional patterns of the spinal cord, i.e.ipsilateral flexion and contralateral extension reflex of the leg, occasionally witha supporting reaction in the ipsilateral wing. (3) The delayed appearance of the'limb-specific' reflexes, and even more the retarded development of 'limb-specific' behaviour, both only gradually emerging from rather unspecificgeneral responses to stimulation of the grafts. (4) Independence between painsensitivity and specific reflexogenic capacity of the whole graft or different partsof it. On the other hand, 'limb-specific' behaviour localized erroneously on thehost's own limbs is generally experienced only upon painful stimulation of pain-sensitive parts of the transplants. (5) 'Limb-specific' reflexes may be elicited bygrafts supplied by a single segmental nerve (nos. 1, 2, 3, and 4), whereas morecomplex behaviour reactions have exclusively been experienced in grafts withmultisegmental innervation (nos. 5, 6, 7, and 8) and some plexus formation be-tween the several nerves supplying the grafts (Plate 2, fig. L). (6) Interactionbetween information reaching the c.n.s. of the host through normal (e.g. visual)and the inappropriate (nerves supplying the grafts) channels, with the resulteither of reinforcing or suppressing the inadequatedly projected 'limb-specific'behaviour.

5584.10 L


These facts at least show that there is probably no simple, or no single,mechanism responsible for the strange phenomena observed. In particular theobservations summarized under the first four of the above points are not infavour of the assumption that new specific central pathways have been de-veloped by the thoracic nerves that happen to innervate the grafted limb andlead directly to the motor apparatus of one or the other limbs of the host. Itlooks much more as if the c.n.s. deals with information received from the graftswithin the framework of its general inherent functional patterns for the move-ment and use of the limbs. The observation mentioned under points (3) and (6)suggest a purely functional mechanism—resembling somewhat the process in-volved in learning—to be at work rather than actual establishment of newanatomical connexions. The anatomical observation summarized in point (5)also underlines the importance of quantitative—therefore more probablyfunctional—rather than qualitative factors (i.e. changed connexions). The dis-crepancies indicated in point (4) and especially our case no. 8, in which 'limb-specific' reflexes have been projected exclusively to the wing and 'limb-specific'behaviour shifted from the wing later to the leg, clearly show that ' limb-specific'behaviour cannot be simply a consequence of the established reflex broughtabout by some secondary feed-back mechanism.

In agreement with findings reported earlier (Szekely, 1959) on corneal reflexesin the newt elicited from the regeneration blastema of a limb that has beentransplanted into the region of, and has become innervated by, the vagus,evidence on the whole suggests analysis and 'recognition' of afferent impulsepattern flowing in from the graft. As an alternative hypothesis to the modula-tion concept, the possibility must therefore be considered that the c.n.s. mighthave the capacity to recognize information on the basis of certain cues, e.g. onthe basis of space-time patterns, independent of the channels through whichthey have been received, and to direct them to more or less adequate' addresses'.The importance of the periphery should not be overrated in the elaboration ofcentral mechanisms, as we shall soon be able to demonstrate in the forthcomingreports on the functional results of exchange of different parts of the spinal cordby transplantation of medullary tube both in the newt and the chick.

SUMMARY

1. Limb primordia, both leg and wing, have been transplanted heterotopicallyin chick embryos on the 3rd day of incubation. Of 11 chicks hatched, 1 had a limbgraft situated in the neck, 9 in the thoracic, and 1 in the lumbar region. Thesesupernumerary limbs were supplied with nerves from the respective segments.

2. Muscle tissues underwent degeneration and the joints were ankylotic. Thecovering skin and feathers were normal and sensitive to mechanical stimuli.Responses of the hosts to stimulation of the supernumerary limbs have beenstudied.


3. The most common reflex response to light mechanical stimulation of thegraft was a flexion reflex of the host's ipsilateral leg. If the graft was situated inclose vicinity of the wing, elevation of the host's own wing could be elicited.These 'limb-specific' reflexes could be exclusively evoked from the grafts, butnever from their neighbourhood. Four cases yielded more complex behaviourreactions always referred to one of the host's own limbs, i.e. lame walking afterstronger stimulation of the graft and cleaning of the host's foot with the beakwhen a permanent, painful, stimulus was applied to the supernumerary limb.

4. There was no correlation whatever between the type of the graft and theevoked 'limb-specific' responses which appeared only some days after hatching.Pain sensitivity and specific reflexogenic capacity were not strictly associated,since 'limb-specific' responses could be elicited from painless parts of the graftsand the reverse.

5. According to anatomical investigation most of the grafts were suppliedexclusively by intercostal nerves which never under normal circumstances con-vey limb signals and stimulation of which normally never evokes 'limb-specific'responses. Grafts producing only simple reflexes were generally supplied by asingle segmental nerve, whereas complex behaviour reactions could be ex-clusively evoked from grafts with multisegmental inervation and some plexusformation among the supplying nerves.

6. It is difficult to explain the observations made on supernumerary limbs ofchicks on the basis of the Weiss-Sperry concept of 'specific modulation'. Asalternative hypothesis the capacity of 'analysis' and 'recognition' of specificafferent impulse patterns by the c.n.s. is suggested.

RESUME

Types de reflexes et de comportements obtenus a partir de membres surnumerairesimplant es, chez le poulet

1. Les ebauches de membres d'embryons de poulets, a la fois alaires etposterieures, ont ete transplantees en position heterotopique le troisieme jourde l'incubation. Sur onze poulets eclos, Tun avait un membre greffe situe surle cou, neuf l'avaient dans la region thoracique, et le dernier dans la regionlombaire. Ces membres surnumeraires etaient pourvus de nerfs provenant deleurs segments respectifs.

2. Les tissus musculaires avaient subi une degenerescence et les articulationsetaient ankylosees. Le revetement cutane et les plumes etaient normaux et sen-sibles a des stimuli mecaniques. On a etudie les reponses des notes a la stimula-tion des membres surnumeraires.

3. La reaction la plus repandue a une legere stimulation mecanique du greffonetait un reflexe de flexion de la patte ipsilaterale de l'hote. Si le greffon etaitplace tres pres de l'aile, on pouvait provoquer l'elevation de l'aile meme del'hote. Ces reflexes 'specifiques du membre' ont pu etre provoques exclusive-ment a partir des greflfons, mais jamais a partir de leur voisinage. Quatre cas ont


fourni des reactions de comportement plus complexes, se rapportant toujoursa un des propres membres de l'hote: claudication apres stimulation assez fortedu greffon et nettoyage de la patte de l'hote avec le bee quand un stimuluspermanent, douloureux, etait applique au membre surnumeraire.

4. II n'y a pas de correlation entre le type de greffon et les reponses' specifiquesdes membres' provoquees, qui sont apparues quelques jours seulement apresPeclosion. La sensibilite a la douleur et la capacite reflexogene specifiquen'etaient pas etroitement associees, puisque des reactions 'specifiques desmembres' ont pu etre obtenues a partir de regions indolores des greffons etinversement.

5. D'apres les examens anatomiques, la plupart des greffons etaient pourvusexclusivement de nerfs intercostaux qui ne transmettent jamais de signaux auxmembres dans les conditions normales et dont la stimulation ne provoquejamais, normalement, de reponses 'specifiques des membres'. Les greffons quipresentaient seulement des reflexes simples etaient generalement pourvus d'unseul nerf segmentaire, tandis que les reactions des comportements complexespOUVaient etre provoquees exclusivement a partir de greffons a innervationplurisegmentaire avec ebauche d'un plexus des nerfs interesses.

6. II est difficile d'expliquer les observations faites sur les membres sur-numeraires des poulets, sur la base du concept de 'modulation specifique'de Weiss-Sperry. Comme hypothese de remplacement, on suggere l'existenced'une capacite d"analyse' et de 'reconnaissance' des ensembles d'impulsionsspecifiques afferentes, par le systeme nerveux central.

REFERENCES

DETWILER, R. S. (1936). Neuroembryology: An Experimental Study. New York: The Macmillan Co.ECCLES, J. C , ECCLES, R. M., & MAGNI, F. (1960). Monosynaptic excitatory action on motorneurones

regenerated to antagonistic muscles. / . Physiol. 154, 68-88.HAMBURGER, V. (1939). Motor and sensory hyperplasia following limb-bud transplantation in chick

embryos. Physiol. Zool. 12, 268-84.(1947). A Manual of Experimental Embryology. Chicago: Univ. Chicago Press.& HAMILTON, H. (1951). A series of normal stages in the development of the chick embryo.

/ . Morph. 88, 49-92.& LEVI-MONTALCINI, R. (1949). Proliferation, differentiation and degeneration in the spinal

ganglia of the chick embryo under normal and experimental conditions. / . exp. Zool. I l l ,457-502.

MINER, N. M. (1951a). Cutaneous localization following 180 degree rotation of skin grafts. Anat.Rec. 109, 326-27.(19516). Integumental Specification of Sensory Neurons in the Genesis of Cutaneous Local Sign.

Ph.D. Thesis, University of Chicago.SANDOR, ST. (1958). Personal communication.SPERRY, R. W. (1944). Optic nerve regeneration with return of vision in Anurans. / . Neurophysiol. 7,

57-69.(1951). Regulative factors in the orderly growth of central circuits. Growth Symposium, 10,

63-87.& MINER, N. M. (1949). Formation within sensory nucleus V of synaptic associations mediating

cutaneous localization. / . comp. Neurol. 90, 403-24.STRAZNICZKY, CH. (1959). Postembryonic growth of supernumerary limbs grafted into chick embryos.

Acta Biol. hung. 10, Suppl. 3, 49.SZEKELY, G. (1959). The apparent'corneal specificity'of sensory neurones. J. Embryol. exp. Morph.

7, 375-9.

/ . Embryol. exp. Morph. Vol. 10, Part 2

G. SZEKELY and i. SZENTAGOTHAT

Plate 1

/ . Embryol. exp. Morph. Vol. 10, Part 2

G. SZEKELY am/J. SZENTAGOTHAI

Plate 2


WEISS, P. (1924). Die Funktion transplantierter Amphibienextremitaten. Aufstellung einer Reso-nanztheorie der motorischen Nerventatigkeit auf Grund abgestimmter Endorgane. Roux Arch.EntwMech. Organ. 102, 635-72.(1936). Selectivity controlling the central-peripheral relations in the nervous system. Biol. Rev.

11,494-531.(1942). Lid-closure reflex from eyes transplanted to atypical locations in Triturus torosus :

evidence of a peripheral origin of sensory specificity. / . comp. Neurol. 11, 131-69.(1955). Nervous System (neurogenesis). In Analysis of Development, ed. B. H. Willier, P. A.

Weiss, & V. Hamburger, pp. 346-401. Philadelphia and London: W. B. Saunders.

EXPLANATION OF PLATES

PLATE 1

Frames from a cinematographic film. The chick has a wing graft (white protruding below ownwing) stimulated by application of a steel clamp indicated by arrows (animal no. 6). Insert of B showsthe clamp somewhat magnified.

FIGS. A-D. Pain caused by the clamp is referred to the ipsilateral own leg; the chick tries to get ridof unpleasant feeling by cleaning the foot with the beak.

FIGS. E-H. After catching sight of the clamp its place is localized correctly.

PLATE 2

FIGS. I and K are taken from a cinematographic film. The chick holds a leg graft in the neck region(animal no. 8).

FIG. I. The chick has learned that the painful stimulus (clamp indicated by arrow) is on the graftand cleans the graft's part lying in its visual field.

FIG. K. Pain caused by the clamp is referred to the chick's own leg.FIG. L. Dissection finding of animal no. 7. The graft is innervated by dorsal branches of the 20th

and 21st spinal nerves which form a plexus (indicated by white arrow). Intercostal nerves of thesesegments are covered by underlying strip of black paper. Graft has been removed. Lumbar plexusto the left.

(Manuscript received 31 : vii: 61)

reflex and behaviour patterns elicited from implanted ...peripheral connexion is thas t of local...

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