Multiple innervation of rat pacinian corpuscles regenerated after neonatal axotomy

03Oa4522/Sl/OS1675-12102.00/o Pergamon Press Lid 0 1981 IBRO

Neuroscimce Vol. 6, No. 8. pp. 1675 to 1686. 1981 Printed in Great Britain

MULTIPLE INNERVATION OF RAT PACINIAN CORPUSCLES REGENERATED AFTER NEONATAL AXOTOMY J. ZELENA Institute of Physiology, Czechoslovak Academy of Sciences, Videiiska 1083, 142 20 Prague 4, Czechoslovakia Abstract-The regeneration of Pacinian corpuscles was studied on the crural interosseous membrane in rats in which the sciatic nerve has been crushed at birth. The original population of developing Pacinian corpuscles rapidly degenerated after neonatal axotomy. Subsequently, the regenerating axons induced the differentiation of corpuscles de nooo. The first regenerating Pacinian corpuscles were found on the interosseous membrane 19 days after neonatal axotomy. Each was, as a rule, supplied by several regenerating axons and contained a rudimentary inner core enclosing the largest axon terminal. By day 40 postnatal, the regenerated corpuscles usually contained several axon terminals enclosed by 2-7 inner cores compressed within a common outer capsule. The majority of corpuscles remained polyaxonal and contained multiple inner cores up to at least 11 months. This is in contrast to normal corpuscles that have one terminal enclosed in an inner core and capsule. The mean number of regenerated Pacinian corpuscles was 13.2 + 1.3 (*SE.; n = 5) at one to four months after axotomy, i.e. 27.5% of the mean number 48.0 k 1.3 (n = 5) corpuscles found on normal interosseous membranes. The number of regenerated axons of the interosseous nerve supplying the corpuscles was decreased to about 40% of the normal number. The regenerated corpuscles were small and the diameters of the regenerated axons were much reduced. The permanent polyaxonality of newly formed Pacinian corpuscles together with their small size and number is an example of an aberrant course of regeneiation in the rat after axotomy performed during the critical perinatal period of development.

PACINIAN corpuscles

are mechanoreceptors with a highly regular structure (Fig. 1), consisting of a single axon terminal enclosed in a lamellar inner core and an outer capsule (LOEWENSTEIN, 1971; HUNT, 1974). In the rat, Pacinian corpuscles begin to develop shortly before birth and their differentiation is complete by the end of the second postnatal week (ZELENA, 1978). Their development is dependent upon an intact nerve supply. Section of the sciatic nerve in new-born or l-day-old rats causes the Pacinian corpuscles on the crural interosseous membrane to degenerate and to disintegrate within 5 days after neurotomy (ZELEN.~, SOBOTKOVA& ZELENA,1978; ZELENA,1980). Indeed, the denervated extremity becomes devoid of most encapsulated receptors (ZELENA,1976). It appeared of interest to learn whether and how Pacinian corpuscles would differentiate anew, if the crushed nerve was allowed to regenerate. Newlyformed Pacinian corpuscles have been previously observed under pathological conditions in men (KR~~cKE,1974) and under experimental conditions in adult cats (SCHIFF& L~EWENSTEIN,1972; CHALIXNA & ILYINSKY,1976). In very young rats, however, nerve regeneration is deficient (BUEKER& MEYERS,1951; ZELENA & HN~K, 19636) which may adversely affect the differentiation of new corpuscles. In the present study, the formation of new Pacinian corpuscles on the crural interosseous membrane was investigated after crushing the sciatic nerve in new-born rats. The 1675

time course of regeneration and the ultrastructure of regenerated Pacinian corpuscles are described in this paper. EXPERIMENTAL

PROCEDURES

The experiments were performed on Wistar rats of both sexes. The right sciafic nerve was crushed in new-born rats 6-8 h after birth. The crush was made with a forceps that had oblong parallel tips 0.2 mm wide and a stop controlling the space between the closed tips. Electron microscopy

Tissue from twenty-nine animals was prepared for electron microscopy 12-330 days after the operation. The rats were decapitated and both hind limbs were injected, in the region of the interosseous membrane, with fixative containing 1% glutaraldehyde and 1% paraformaldehyde in 0.12 M phosphate buffer at pH 7.3. After 5-15 min of fixation in situ, the membranes were dissected out together with the bones and put into fresh fixative for 2-4 h. After rinsing in a 7.5% sucrose solution, the interosseous nerves and membranes were excised, cut into small tissue blocks, fixed for 2 h with 2% osmium tetroxide in phosphate buffer, dehydrated and embedded in Durcupan. Semi-thin sections 0.5-l pm thick were stained with toluidine blue and examined for nerve branches and Pacinian corpuscles. Ultrathin sections were cut from selected areas on a Reichert OMU 3 ultramicrotome, stained with uranyl acetate and lead citrate and examined in a JEM 100 B electron microscope.

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J. ZELENP;

Light microscopy In 10 animals, the interosseous nerves and membranes of the denervated and the normal limbs were dissected out under ether anaesthesia 35-124 days after the crush. They were fixed, processed and stained for cholinesterase (SILVER,1963). The whole-mounts of nerves with their branches and attached corpuscles were examined under the light-microscope to determine the number of Pacinian corpuscles supplied by regenerated and normal interosseous nerves. In five animals, pieces of pre-fixed interosseous nerves were removed from the crural interosseous membrane 175 days after the operation and processed as for electron microscopy. Transverse sections 0.5 pm thick were stained with toluidine blue and all myelinated fibres were counted in each nerve, using magnification x 1000. To determine the number of myelinated axons entering the corpuscles, seven tissue blocks were cut in series of semi-thin sections, stained with toluidine blue and examined. RESULTS The onset of regeneration

Regenerating axons were already found in the interosseous nerve 12 days after neonatal nerve crush. Pacinian corpuscles, however, were first detected on the interosseous membrane at 19 days, i.e. about 2 weeks after the disintegration of the original population of corpuscles and their removal by macrophages following nerve crush (ZELENA,1980). Normal Pacinian corpuscles of control rats are well differentiated at 3 weeks of age (Fig. 1) and measure about 150~ in diameter. Regenerating CorpusCles found on day 19 (Figs 2-4) resembled developing corpuscles of foetal rats about 2 days before birth (LY. ZELEN.~,1978) and measured only 6-14~ in diameter. The morphogenetic process started within a thin perineurial capsule which Contained several regenerated axons (Figs 3a and 4). One of the axons formed an enlarged cylindrical terminal about 2 crm in diameter which contained numerous mitochondria (Figs 2-4) and sent off axonal proCesses into the Clefts between the Schwann cells of the rudimentary core (Fig. 2). The Schwann cells of the diirarentiating core had dense bulky cytoplasm (Fig. 2) and began to form the first inner Core lamellae around the axonal surface (Figs 3a and 4). Tiny axons that accompanied tbe main axon terminal were localizd in an adjacent Schwann cell band and at the outer aspect of the differentiating cells; some of the axonal pro&s were filled with prominent dense core vesides SGloo-nm in diameter (Figs 3a and b). Regeneration 40 days ajkr nerve crush The differentiation of the corpuscles was markedly advanced by day 40. The corpuscles had multiple axon tuminals surrounded by multipb hmn&u inner cores. Their average diameter wII) about 4ojan. Usually they were supplied by more than one axon. In a typical corpuscle at this stage (Figs 5, 7 and 8),

two myelinated and several unmyelinated axons were found in the lumen of the corpuscle at its proximal pole and an inner core was already formed around a nerve terminal (Fig. 5). The number of inner cores enclosing axon terminals increased to two further distally (Fig. 7), rose up to five in the middle part of the corpuscle (Fig. 8) and again declined towards the distal end. The inner cores comprised maximally 10 concentric lamellae and had no distinct radial clefts. The number of axon terminals exceeded that of the inner cores, since often additional terminal profiles were found in the center of or between the inner core lamellae (Fig. 8). The nerve terminals were usually small and of irregular form. Occasionally, a well developed terminal was found which contained numerous mitochondria, some axially oriented neurofilaments and microtubules and a number of vesicles in lateral processes (Fig. 11). The axolemma appeared thickened and undercoated in limited areas of some terminals. Sometimes a coated pit was found in the axolemma. Numerous collagen fibrils were found in the interstices and in the space beneath the capsule (Figs 5 and 7). The capsule was underdeveloped, for it was formed of only 3-5 lamellar layers of capsular cells. Basal laminae covered both the capsular and inner core lamellae.

One of the five regenerated corpuscles examined at this stage was monoaxonal; the diameter of its axially localized axon terminal was less than 1 m (Fig. 6). Its inner core which consisted of about 12 concentric lamellae was approximately 8 p in diameter; the radial clefts were absent. The outer capsule comprised seven lamellar layers divided by interstices containing numerous collagen fibrils. The diameter of the whole corpuscle measured only 12 pm. Regenerated corpuscles crush

3 to 11 months after nerve

With age, the regenerated Corpuscles increased in size, but their maximal diameter did not exceed 70 jnn which is less than 20”/, of the mean diameter of mature rat Pacinian corpuscles (&LENA, 1978). The regenerated corpuscles remained polyaxonal throughout the period of study (Figs 9 and 10); only three of 18 corpus&s studied during this period were small, monoaxonal and monoterminal as described above (Fig. 6). One corpuscle was monoaxonal, with multiple terminals and inner cores. In polyaxonal corpuscles, 2-6 myelinated axons about $3~ thick were found to enter the outer capsule; the mean number of myelinated axons in 10 corpuscles examined in serial sections at 4 and 11 months was 3.5 + 0.45 (~-SE.).The maximal number of axon terminals endosed by inner cores varied 5-8 in the equatorial region and declined towards both poles. The axon terminals localized eccentricany in relation to the inner cores (Figs 8 and 9) were gradaally eliminated and were absent at late stages of regeneration (Fig. 10). The terminals were roundish or ellipsoid in ~XOSS-~tion, about l--3 pm wide, with simple or branched axond processes at one or both poles (Figs 13 and 14). At earlier

FIG. 1. A normal Pacinian corpuscle in transverse section; t, axon terminal; ic, inner core; c, capsule. Arrows indicate radial clefts. From a 20-day-old rat. x 6000. Ail scale markers in this and subsequent figures indicate 1 pm. FIGS 2-4. Initial stages of corpuscles regeneration 19 days after neonatal axotomy. FIG. 2. A sensory terminal (t) in transverse section, filled with mitochondria and flanked by Schwann cells (S) which contain, among other organelles, several conspicuous dense bodies (d). Lateral axonal processes are indicated with arrows. Axon terminal and Schwann cells are covered with a common basal lamina (1); c, capsule. x 17,000. FIG. 3a. A differentiating corpuscle which contains within one capsule (c) a large axon terminal (t) surrounded by lamellar processes of a rudimentary inner core and small axonal profiles with prominent dense-core vesicles (box and arrows). The axons are accompanied by Schwann cells (S) and their processes. x 15,000. FIG. 3b. The boxed area from Fig. 3a, enlarged, shows dense core vesicles within an axon. x 40,000. FIG. 4. An oblique section of an axon terminal (t) enclosed by Schwann cells (S) forming the first inner core lamellae (arrow); c, capsule x 15,000. FIGS. 5-8. Regenerated corpuscles 40 days after axotomy.

FIG. 5. A polyaxonal corpuscle containing, at the proximal pole, one myelinated axon (ma), several unmyelinated axons (a) and an inner core enclosing a nerve terminal (t); c, capsule; S, Schwann cells; col, collagen fibriis. x 12,OCQ FIG. 6. A small monoaxonai corpuscle regenerated 40 days after axotomy. x 4.500. FIG. 7. The same corpuscle as in Fig. 5, further distally; 2 inner cores are formed around two axon terminals (t). One additional terminal (t) and two axonal profiles (a) are seen within the capsule (c). x 12,ooo. FIG. 8. At the middle part, the corpuscle contains five inner cores and about 10 axonal (a) and terminal (t) profiles within a multilayered capsule (c). x 6000. FIG. 9. Transverse section of a regenerated Pacinian corpuscle four months after axotomy. Distal half of the corpuscle. The lameliae of severat inner cores are pressed together in a common shape. Four terminals (l-4) are found among the lamellae at this level. The multilayered capsule (c) has about 10 additional outer lamellae with wide interspaces which are not included in the figure. x 6000. FIG. 10. A regenerated corpuscle 11 months after axotomy; mid-level in an oblique section. Well-defined multiple inner cores encircling eight axon terminals (l-8) are condensed in a common ovoid structure with a smooth outline. The capsule (c) consists of tightly packed lamellae; the spaced-out external lamellae are missing. x 5300. FIG. 11. An axon terminal from a regenerated corpuscle 40 days after axotomy is oval in transverse section, filled mainly with mitochondria (m) and mostly covered by inner core Iamellae (ic). Blunt lateral processes (arrows) contain a number of vesicles (v); 1, basal lamina. x 40,000. FIG. 12. An axon terminal from a regenerated corpuscle four months after axotomy. The major part of the axolemma is covered by basal lamina (1). An inner core lamella approaching the axon is fixed to it by an attachment plaque (arrow); v, vesicles; 1, basal lamina; col, collagen fibrils; p, pinocytotic vesicles in the inner core lamellae. x 34,000. FIG. 13. Part of an axon terminal with a branched lateral process containing vesicles (v), 11 months after axotomy. Axolemma enclosing a collagen pocket at the base of the branched process has a dense undercoating (arrows). x 50,000. FIG. 14. Part of an axon terminal with a lateral process of irregular form, 11 months after axotomy. Numerous mitochondria (m) are arranged at the axonal circumference. Axonal process has a dense membrane undercoating (arrows) at its base and contains vesicles (v), some of them with dense core (dv); ap, attachment plaque; p, pinocytotic vesicles; 1, basal lamina. x 60,000.

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Multiple innervation of regenerated Pacinian corpuscles stages of regeneration, some of the axonal profiles were covered mostly by basal lamina (Fig. 12). Later on, the major part of the axolemma became covered by the innermost lamellae of the core, and the basal lamina became restricted to small areas of exposed axolemma (Fig. 14). Clear vesicles about 60nm in diameter and occasional dense-core vesicles were concentrated in the axonal processes (Figs 13 and 14). A thin layer of dense undercoating (cf. SPENCER & SCHAUMBURG, 1973) appeared about 20 nm beneath the axolemma at the base of axonal processes (Figs 13 and 14). Attachment plaques were often found at the lateral processes, connecting the lamellar tips with the axolemma (Figs 12 and 14). The inner cores were not arrayed in parallel but had a random arrangement and were cut transversely, obliquely or longitudinally in one cross-section (Fig 10). The number of lamellae in an individual core remained restricted to 5-10. Radial clefts were absent or incomplete; the subdivision into hemilamellae was usually found in the innermost layers of the core, so that lateral processes could protrude into the clefts. The surfaces of the lamellae were indented by numerous pinocytotic pits and vesicles (Figs 12 and 14). Basal lamina covered the lamellar surface at places exposed to wider clefts, but disappeared from stacked lamellae separated by clefts about 30nm wide. All of the cores in a corpuscle were compressed together (Figs 9 and 10); inner-core nuclei with perinuclear cytoplasm sometimes filled out the angular spaces between the cores. Layers of collagen fibrils appeared in occasional clefts between inner-core lamellae and in the capsule.

Two forms of capsule were found. In some recep tom (Fig. 9), the capsule resembled that of normal Pacinian corpuscles, with S-6 lamellar layers of capsular cells divided by narrow clefts, and 7-12 outer lamellae with wide spacing. Other regenerated corpuscles had a thin capsule consisting of 6-12 concentric layers separated by very narrow interspaces (Fig. 10). The number of regenerated corpuscles and axons

The total number of regenerated corpuscles found on the interosseous membrane was small (Table 1). In the whole-mounts of the interosseous nerve with the attached corpuscles, the mean number of the latter was 13.2 & 1.3 (~s.E.) in five rats investigated 35-124 days after neonatal nerve crush. This is about 27.5% of the mean number of 48.0 f 1.3 corpuscles found in the group of five control rats (Table 1). The regeneration of the interosseous nerve was also deficient. In a group of five animals investigated 175 days after axotomy, the mean number of all myelinated axons in the regenerated nerves was reduced to 31.4 f 2.8 which is about 40% of the mean number of 79.2 f 4.7 myelmated axons found in contralateral control nerves. Umnyelinated axons contained in the interosseous nerves were not counted, since Pacinian sensory terminals of both normal and regenerated mature corpuscles are exclusively formed by myelinated axons or their branches. In normal interosseous nerves, most myelinated fibres had diameters 8-12~; this large-diameter group comprised 68.0 f 5.2 axons. Small thinly myel

TABLE 1. THE NUMRER

OP PACINIAN

CORPUSCLES ON THE

CRURAL INTERGSSEOUS MEMBRANE AFTER REGENERATION AND IN NORMAL CONTROL RATS

Control

Regenerated Rat No.

Age in days

1 2 3 4 5

35 56 70 124 124

Mean number f 13.2 f 1.3

Number of PC 13 10 11 15 17 S.E.

Rat No.

Age in days

35 1 35 2 3 90 4 90 100 5 Mean number k 48.0 f 1.3

Number of PC 51 49 50 45 45 S.E.

PC, Pacinian corpuscles

linated axons present in normal nerves do not innervate corpuscular receptor organs. In regenerated nerves, the maximal fibre diameter was 6~. The

group of myelinated fibres with diameters 4-6pm comprised about 60% of the mean number of myelinated fibres in the nerve, but smaller myelinated axons were also occasionally found to participate in the innervation of regenerated Pacinian corpuscles. In three rats, no interosseous nerves and no Pacinian corpuscles were detected in their usual territory in re-innervated hind-limbs 5 months after nerve crush at birth. It could not be established, whether in these instances regenerated nerve fibres were deviated to another target area or whether nerve regeneration had been, for unknown reasons, seriously impaired.

DISCUSSION Permanent and transient polyaxonality The present study demonstrates that rat Pacinian corpuscles will regenerate again when the original population of developing corpuscles has degenerated after neonatal denervation. Most regenerated corpuscles become atypical: they are supplied with two or more myelinated axons that form multiple axon terminals enclosed by multiple inner’cores. The polyterminal innervation of the regenerated corpuscles is permanent, unlike the transient polyaxonal and polyneuronal innervation of developing motor end-plates (REDFERN, 1970; BENNBIT & PETI’IGUW, 1974; BROWN, JANSEN & VAN ESSEN, 1976; RG~ENTHAL&

TARA~KEVICH, 1977; JANSEN, ‘THOMPSON& KUFFLER, 1978; O’BRIEN, &TBFXG & VRBOVA, 1978; ZELJZNA,

Vvs~oEn. & Jm~A~ov.4,1979) and some other synaptic sites during ontogenesis (RONNEVI & CGNRADI, 1974; CIWPEL, MARUNI 8~ DEI_IWYE-BOUCKWD, 1976; LICHTMANN,1977) where the redundant axons retract, in the rat, during the first 2-3 weeks after birth. Polyaxonal innervation has not yet been described in developing mechanoreceptors. During ontogenesis, only a single terminal and a single inner core differentiate in each Pacinian corpuscle, but other tiny axonal

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J. ZELENA

profiles are found at the outer aspect of the nascent core in foetal rats; these additional axons are no longer detected in postnatal rats (ZELENA,1978). In contrast, in regenerating corpuscles additional terminals and cores are soon formed beside the first differentiating ending and are maintained throughout further life. It has been suggested that the transient polyaxonal innervation observed during ontogenesis is a phenomenon preceding and ensuring the establishment of selective connectivity by competitive neurons (cf. JANSENet al., 1978; CHANGEUX & MIKOSHIBA,1978). On the other hand, the persisting polyterminal innervation of regenerated Pacinian corpuscles may be regarded as an attempt to compensate for an insufficient sensory nerve supply of the given peripheral field by increased peripheral branching. Deficient nerve regeneration and its consequences

The number and diameters of myelinated axons in the regenerated interosseous nerve were reduced, 5 months after crushing the sciatic nerve at birth, by more than 50%; this exceeds the reduction found in sensory fibres of a rat muscle nerve after the same operation (ZELENA& HN~K, 19636). The decreased number of regenerated axons is presumably associated with the loss of neurons after early axotomy and the decreased axonal diameters are probably due to a permanent deleterious effect of injury upon surviving neurons. It is known that retrograde reaction of neurons to injury is particularly severe in immature organisms (KINGSLEY,COLL~S 8c CONVERSE,1970; LIEBERMAN,1974; HUGHFB& CARR, 1978; RISLING, REMAHL,HILDEBRAND & ALDSKOGNS,1980). An insufficient nerve regeneration after nerve crush in new-born rats has its consequences in the periphery. In rat muscles re-innervated after neonatal axotomy, muscle spindles and Golgi tendon organs eliminated due to denervation do not regenerate at all or differentiate in an atypical form (ZELENA& H&K, 1963a,b). The numbers of motor axons and the size of motor units are reduced to about one half after regeneration, but the surviving muscle fibres become hypertrophic in compensation (HELENA & HNIK, 19636). In the case of the regenerated interosseous nerves, a reduced number of sensory fibres supplies a still further decreased number of Pacinian corpuscles. This reduction is, however, counterbalanced by increased terminal branching, so that the calculated total number of Pacinian terminals and inner cores becomes roughly equal on the regenerated and control side. Under normal conditions, the ratio between axons and Pacinian corpuscles is, presumably, 1: 1 in the rat, as it appears to be in the cat (ICOO, 1976). A direct correlation between the number of myelinated axons and the number of Pacinian corpuscles supplied by the interosseous nerve is not possible, since the nerve also contains axons terminating elsewhere. After regeneration, direct counts of myelinated axonal pro-

files entering Pacinian capsules show a ratio 2.8 axons per corpuscle (the calculated ratio is 2.4). This indicates that either all myelinated fibres of the regenerated nerve enter Pacinian corpuscles unbranched, or the actual number of fibres associated with corpuscles is smaller and the difference is compensated by preterminal branching. These considerations suggest that most regenerated corpuscles are supplied by more than one neuron, on condition that all myelinated fibres in the regenerated nerve are unbranched peripheral processes of primary sensory neurons. It would be of interest to learn how effective these polyaxonal corpuscles are physiologically and how much they differ from normal Pacinian corpuscles. The specific morphology of regenerated corpuscles indicates that discrimination of adequate mechanical stimuli will be decreased and conduction velocity of sensory fibres diminished because of their reduced diameter. Regenerated corpuscles with a thin outer capsule are likely to be slowly adapting. Neoformation and re-innervation of corpuscles in adults

In adult cats, new formation of Pacinian corpuscles was achieved after transplantation of a cutaneous nerve to the mesentery (SCHIFF& L~EWENSTEIN, 1972; ILYINSKY,CHALISOVA& KUZNE~~OV,1973; CHALISQVA, CHUMASOV& ILYINSKY, 1980). The newly formed corpuscles induced by the foreign nerve were monoaxonal and had a surprisingly normal ultrastructure (ILYINSKY et al., 1973). In the re-innervated territory, however, several bead-like Pacinian corpuscles were often found in succession on a single axon, and twin corpuscles supplied by one axon were also common (CHALISOVA & ILYINSKY,1976). The regenerated axons apparently had a tendency to produce an increased number of corpuscles per axon. A transient polyaxonal innervation was observed in avian Herbst corpuscles during re-innervation after nerve crush (CHOUCHKOV, 1978). Multiple axons were found within a single inner bulb 4-8 weeks after axotomy, but re-innervated corpuscles became again monoaxonal about four months after nerve crush (CHOUCHKOV,1978). It was mentioned that a similar process occurred in re-innervated Pacinian corpuscles of adult cats (CHOUCHKOV, 1978). This transient polyterminal innervation of re-innervated corpuscular receptors deserves further study, especially with regard to factors initiating transition to a monoaxonal and monoterminal supply. Morphogenetic mechanisms

The question arises as to which factors are responsible for the differentiation and maintenance of polyaxonal corpuscles described in this study. Normal development of rat Pacinian corpuscles (-4 1978) and of analogous Herbst corpuscles in birds (SUCOD,1978)‘is brought about by m-tic interactions of sensory terminals and the hmervated tissue. The onset of morphogenesis is only possible when the target tissue has attained a certain stage of

Multiple innervation of regenerated Pacinian corpuscles

histogenesis, as has been proved by heterochronic transplantation experiments (SAXOD, 1978); when the development is triggered, sensory terminals induce the differentiation of the non-nervous components of the receptor (SAXOD, 1978; ZELENL et al., 1978; ZEJXNL, 1980). The basic mechanisms underlying the neuronal induction and the reverse effect of innervated tissue upon nerve terminals are not known. The neuronal intluence may be mediated by contact induction or by an inductive substance (PARKER, 1932) presumably contained in dense-core vesicles and released from the terminals (LENIZ, 1967; ZELENA,1978). In view of the latter assumption it is surprising to find, at the initial stage of corpuscle regeneration, the accumulation of dense substance not in the differentiating terminal itself, but in small diameter axons which have not yet formed a true Pacinian ending. As regards the effect of the peripheral environment upon sensory terminals, one may speculate that it is exerted by some regulatory macromolecules taken up by endocytosis into the nerve ending (JJRMA-

1685

NOVA& ZELEN~ 1980). It has been surmised that the target tissue manufactures a substance that stimulates the nerves to sprout and regulates the density of sensory nerve endings at the target; the substance is normally neutralized by neuronally transported factors (DIAMOND,CXIOPER,TURNER & MACINTYRE, 1976). If the balance is disturbed the nerves sprout until the nerve terminals can release enough of the neutralizing factors to restore the equilibrium (DIAMOND et al., 1976). It remains to be elucidated which of these hypothetical mechanisms actually operate in enhancing terminal branching of regenerating Pacinian axons and which factors are responsible for the aberrant corpuscle regeneration.

Acknowledgements---I wish to thank Mrs M. SoeonrovA

for her expert technical assistance and Mrs M KRUPKOV~ Mr H. KUNZ and ING. V. POKORN+for their skilful techni-

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