Slowly adapting cutaneous mechanoreceptors (displacement detectors) have. Hensel, 1959; Hunt and McIntyre, 1960; Boman and Hensel, 1960; Fjallbrant

Transcription

1 Quarterly Journal of Experimental Physsology (1972) 57, THE STRUCTURE AND FUNCTION OF THE SLOWLY ADAPTING TYPE II MECHANORECEPTOR IN HAIRY SKIN. By MARGARET R. CHAMBERS, K. H. ANDRES, MONIKA v. DUERING and A. IGGO. From the Department of Veterinary Physiology, University of Edinburgh, Edinburgh, EH9 1QH, U. K. and Lehrstuhl fur Anatomie II, Riihr-Universitat, 463 Bochum, Westdeutschland. (Received for publication 15th March 1972) The slowly adapting type II mechanoreceptor in hairy skin of the cat was studied in detail using histological and neurophysiological methods. The end organ (Ruffini ending) was lightly encapsulated, situated in the dermis and supplied by one axon which bifurcated within the capsule to form a terminal arborisation. A serial diagrammatic reconstruction was made of one ending. The afferent units almost invariably carried a resting discharge and responded to vertical displacement of the skin over an area of 2-5 sq. cm and to directional stretching of the skin over an area up to 25 sq. cm. For each unit there was a single small region of maximum sensitivity (less than 2 sq. mm). The response to vertical displacement consisted of a dynamic and static phase, linked by a period of adaptation. These three phases were subjected to quantitative tests, using displacements of different amplitude, velocity and duration. The units were directionally sensitive to skin stretch and this response was of a similar pattern to that caused by vertical displacement. The units were temperature sensitive over a range from C and the resting discharge was increased by a fall in skin temperature and temporarily silenced by an increase. Slowly adapting cutaneous mechanoreceptors (displacement detectors) have frequently been reported since they were first described electrophysiologically by Adrian and Zotterman in 1926 [Adrian, 1931; Zotterman, 1939; Fitzgerald, 1940; Frankenhaeuser, 1949; Maruhashi, Mizuguchi and Tasaki, 1952; Witt and Hensel, 1959; Hunt and McIntyre, 1960; Boman and Hensel, 1960; Fjallbrant and Iggo, 1961; Tapper, 1965]. We reported previously that there are two physiologically distinctive groups of slowly adapting mechanoreceptors in hairy skin in the cat [Iggo, 1961] and monkey [Iggo, 1963]; the two groups were originally called 'touch spots' and 'touch fields', but later 'slowly adapting types I and II' (SA I and SA II) respectively [Chambers and Iggo, 1967]; the morphology of one group in the cat has been described [Iggo, 1961], and a detailed account of type SA I has been given [Iggo and Muir, 1969]. Previous descriptions of the SA II units have been given by Witt and Hensel [1959] with special reference to their thermal sensitivity, by Fjallbrant and Iggo [1961] who examined their responses to intra-arterially injected chemicals, by Iggo [1963] and Perl [1968] in surveys of primate cutaneous afferent units, by Werner and Mountcastle [1965] in a detailed examination of the stimulusresponse characteristics of a sample of units which included both kinds of slowly adapting units and by Brown and Iggo [1967] and Burgess, Petit and Warren [1968] in surveys of cat myelinated units. The subdivision of slowly 417

2 418 Chambers, Andres, Duering and Iggo adapting mechanoreceptors into two classes in monkey skin was re-affirmed by Merzenich and Harrington [1969] and Harrington and Merzenich [1970] and has now been extended to human cutaneous receptors by Knibestbl and Vallbo [1970], and reptilian cutaneous receptors by Kenton, Kruger and Woo [1971]. All the results taken together indicate that the two categories are a general feature of the slowly-adapting skin mechanoreceptors in hairy skin and perhaps of non-mammalian skin also. The extent to which the same two categories are also characteristic of glabrous skin is uncertain, although there is some evidence that they may be [Iggo, 1963; Talbot, Darian-Smith, Kornhuber and Mountcastle, 1968; Knibestbl and Vallbo, 1970]. The present paper correlates the functional characteristics of the SA II units with a specific intradermal receptor (Ruffini ending) and describes a quantitative examination of the stimulus-response properties of the units to mechanical stimuli. The SA II units can now be distinguished from SA I mechanoreceptors on both morphological and functional grounds and a tabulated summary of the major differences is given. Preliminary reports based on the results described in the present paper have been published [Chambers and Iggo, 1967, 1968; Iggo, 1968]. A description of the ultrastructure of the Ruffini corpuscle from a limb joint has been published by Goglia and Sklenska [1969]. METHODS Thirty domestic cats, male and female, weighing between 1-5 and 3.0 kg were used. Anaesthesia was maintained with an intravenous injection of chloralose, 80 mg/kg body-weight, following induction with ethylchloride and ether, and with subsequent injections of pentobarbitone sodium as required, through a cannula in the right brachial vein. The right saphenous nerve was exposed in the thigh and prepared for dissection in a pool of paraffin. The rectal temperature was maintained at 37 C using a thermostatically controlled electric blanket but the temperature of the skin of the right hind leg was not otherwise controlled except when the thermal sensitivity of receptors was examined. Details of the preparation of the hind leg and the dissection of the fibre strands are given by Brown and Iggo [1967]. Each strand usually contained a small number (8-12) of active axons, but by careful restriction of the peripheral stimulus, type II units could be excited selectively. When selective excitation could not be achieved, either because (a) there were too many active axons in the strand or (b) more than one unit carried a resting discharge, then the strand was divided using a fine needle and fine forceps, once or repeatedly until a satisfactory preparation was isolated. The single action potentials were ,uv in amplitude. Controlled displacement of the 8kin. The mechanical stimulator consisted of a perspexcapped steel probe attached to the core of a vibration generator. Details of this instrument are given by Brown and Iggo [1967] under the heading Moving Coil Transducers-the second version. The movement of the probe was detected by a displacement transducer, the output of which was used (1) for negative feed-back control of the displacement, (2) display on the oscilloscope screen and, (3) display on a digital voltmeter, calibrated to read directly the displacement of the probe to the nearest micrometre (,um). The system was designed to maintain a constant displacement and not constant force, so that any movement or release of tension in the skin or underlying tissues during maintained displacement did not result in an adjustment of the probe position. Three parameters of the vertical displacement could be varied

3 I: I a~~~~~~~~~~amud'tse SA II Cutaneous Mechanoreceptor 419 independently:-the velocity ( pum/msec), the amplitude (0-500,m) land the duration (100 msec-10 sec.) The positioning of the probe over the receptor was considered to be a very important part of the experimental procedure. The aim was to place the probe normal to the surface of skin containing the receptor. Type II units are not accompanied by any evident surface feature such as a hair or a touch corpuscle, and so initial location of the most sensitive spot was achieved by examination using a von Frey hair and binocular microscope (magnifications times) while recording electrically from a unit. The hairs around the sensitive spot were then cut very close to the skin or removed using a depilatory. Then, while continuing to view the skin through the microscope, the probe tip was brought down onto the receptive spot. In order to facilitate this manoeuvre the mechanical stimulator was mounted on a frame giving controlled movement in three planes. By a combination of movements of the various parts of the frame, the probe was positioned quite accurately relative to the receptor and then locked into position. Final adjustments in the vertical plane could be made electrically. The position of the probe was not altered during a recording session, unless the relative sensitivities of different parts of the field were being explored. Recording system (Fig. 1). A Devices digitimer was used to trigger the probe, oscilloscope, relay unit and Biomac synchronously or sequentially, so that selected phases of the discharge elicited could be recorded either as analogue signals on an F.M. tape recorder or photographically from the oscilloscope screen, and also analysed on-line as interval or time-dwell histograms by a Biomac 500 (Data Laboratories Ltd, London). To ensure secure triggering of the Biomac the action potentials l DiM _ timer Probe \\\\ IIW o, ~~~~~~~~~~~~~voltmeter Preparation Oscillosc Film \>\~~~~~~pif ierrx A A omtrelay 500mac W Ta A FIG. 1. The recording system. The cross hatching indicates the system used for the initial experiments, to which the Biomac 500 and its supplementary equipment was added in later experiments. were converted to uniform pulses using a Schmitt trigger circuit. The data was stored by the Biomac as interspike intervals; the resolution of the intervals was variable (possible range 256 points/msec to 256 msec/point). For this work sufficient separation of the intervals was achieved by msec/point. The contents of the stores was displayed on an oscilloscope screen in the Biomac but, for permanent data storage, the store contents were punched out on paper tape in binary code using a

4 420 Chambers, Andres, Duering and Iggo Data Dynamics 110 paper tape punch. The interspike intervals were retrieved and further analyses performed on a remote Atlas Autocode Computer (KDF 9 Edinburgh), using programs written in Atlas Autocode, Edinburgh. A fuller explanation of these methods is available (Chambers, 1969). Histological Methods Light microscopy. Two methods were used to mark the receptor during physiological recording. The receptive field of a SA II receptor was marked with Indian Ink or silver nitrate solution and the piece of skin then dissected from the anaesthetized animal and fixed in a buffered formalin solution [Iggo and Muir, 1969]. After 30 min in the fixative, it was post-osmicated in osmium and finally dehydrated and embedded in araldite (Ciba). Semi-thin sections (0 5 pm) were cut from the tissue embedded in araldite and for light microscopy the sections were stained with toluidine blue/ thiamine. In some of the later experiments the location of a receptor in the skin was marked by inserting two fine stainless steel wires (diameter 100 ltm), one on each side of the most sensitive part of the receptor field. This technique was introduced by Andres and Hensel [1971]. At the end of the electrophysiological experiment, the cat was perfused with a formalin/glutaraldehyde mixture. The marked pieces of fixed tissue were then dissected out, post-osmicated and processed as described above, to yield semi-thin sections of araldite-embedded skin. Electron microscopy. The skin of the cat was perfused with glutaraldehyde in situ, with successive fixation with osmium tetroxide and embedding in araldite [Andres, 1966]. Serial sections were cut, 0 5,um thick for the light microscopy and 50 nm thick for the electron microscopy. The semithin serial sections supplemented by ultrathin sections were used for a total reconstruction of a Ruffini corpuscle. RESULTS Forty-five slowly adapting type II units were examined as single units and were found in fascicles supplying skin over the knee, inner aspect of the leg and the ankle. Each unit survived for many hours, the longest recording period being twenty hours. A minimum of 4 hr on each unit was necessary to allow a full analysis of the discharge produced by repeated mechanical stimuli, which were separated by rest periods long enough to permit the receptor and the surrounding skin to recover their original positions after each stimulus. The recovery period, i.e. the time taken for the resting discharge to return depended on the amplitude and duration of the stimulus; for a standard stimulus of 500,um amplitude and 8 sec duration, the recovery period was 20 sec. With this precaution taken, the units responded without fatigue to repeated stimuli for several hours. Fig. 2 shows the typical discharge pattern of a SA II unit to vertical displacement of the skin. Characteristics of the receptive field No distinctive surface feature of the skin could be correlated with the presence of an underlying receptor. SA II units were never found in association with touch corpuscles or any particular type of hair, in contrast to the reports by Burgess et al. [1968] that a few type II units were associated with structures indistinguishable from 'touch corpuscles', and by Werner and Mountcastle

5 SA II Cutaneous Mechanoreceptor 421 A unit sec Final displacement (pm) / ~~~ _~2ESiL_ 432 B 310 unit 2358 Igiio-1 0.l 600k Y,-- -C aq} Q C 200.~~~~~~~~~~.4..ioo r., ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~. a b c d e Time - secs Io. 2. A, Sing(le recor(ls of the typical response of type II unit (Ino ) to vertical displaceealt of the skin. The Unlit fires rapidly durilg the dy-namnic phase of the stimulus anid then adapts to a steady discharge, the mean interspike iinterval of wshich is related to the mpillplitulde of displacement (see text). The stimitilus velocity durinig the ramp remaiined 10 tti-n/msec, aind the imaxiurntttisplacemeint in /nm is constant throughotut the series at inidicated for each response. The timre marks are set at 1 sec. B, Graph showing all six niamiecd phases of the response of a type II mechanoreceptor. Each poinit is the iimeain of six recordlings aind the vertical bars iindicate the standard error of the meani. The upper linie inidicates the stinmullus. The six )hases are: (a) restinlg (lischarge; (b) dynamile discharge; (c) aclapting discharge; (d) adapted clischarge: (e) silent period waudi (f) recoxvery of the resting dlischarlge f

6 422 Chambers, Andres, Duering and Iggo [1965], that the typical type II response could be recorded from 'touch corpuscles'. When the skin was displaced laterally, the focus of the unit shifted by the same amount, confirming that the unit was cutaneous. The histological preparations established that the terminals are situated intradermally. Each axon invariably innervated a single focus of sensibility, again in contrast to the report by Burgess et al. [1968]. For example, the diameter of the receptive field of one unit for vertical displacement, tested with 300 um displacement, was 2-5 mm and the central point of this area was the most sensitive (i.e. producing the smallest intervals during the dynamic phase of the stimulus). The smallest interspike interval recorded from a type II unit in response to all kinds of deformation stimuli was 1-3 msec (frequency 800 Hz), a property that distinguishes the type II from the type I (touch corpuscle), as the latter invariably produces a discharge with intervals <1 0 msec (> 1000 Hz) in response to a light brush across the dome [Iggo and Muir, 1969]. The response to vertical displacement varied at different points in the receptive field, despite its small size, so that there was an evident effect of the size of the probe tip and the probe position on the pattern of discharge. Fig. 3 shows the dynamic response at five A Probe position 'I I I I I ~Guard hair SAII unit D,(,o) Position 1 D 1 mm 2 0> FIG Displacement - pm A, a diagrammatic cross section of the skin showing the presumed relative positions of the probe and receptor terminal. The probe was moved in steps of 0-2 mm using a micrometer calibrated to 0 01 mm. B, unit No Graph to show the variation in dynamic response to the same stimulus (250 tum displacement and 0-31,um/msec velocity) at five different points in the receptive field as shown diagrammatically in A. The vertical bar indicates the standard deviation of the mean interspike interval of the resting discharge (RD).

7 FIGc. 4. Type 11 slmv lx a(lal)ting eutaneous iiechanoreceptor repr(senlted 1)y ai encal)psulate( ltiltfilli (o1p)lsele ini the haliry skill of the eat fonle g(,,. The CI(e1Ile(1 i) coitains terminal amnificationis of the axoil. Th( Cole is siirroiliit (li <1 liilt conilective t issue space, plmor in struct ure (es). The perinieural capsule sheath (c) (coisisting (f four thinl cell aii(i colnective tissue lavers) seplarates thei rece)tor organ fromn the dense eorim1ni rich iii (c011agen fibres (kf); k1, p erfuised capiillaries: lif, lia ir follile. Selmithin sect imin (1-5 sl1). M\Jagl`iifiatoli ( x.(().

8 Fi(*... Two sections froi tihe equnat orial regmit 01 of a Rtnffiii endloig in lair skin of thei cats forele-g(. (oi). Eio(Iformat in()of' the Im lin sheath (ins) with (llarlgedl i()()t ()f the axoni (ax); tb), termwill 1 nlnllifieatims1 (If tina\(i, tarnx arrv s) ill th( 1il1(1 (.I t he coijpuele( i) (e npare wsithl 'ig (8). (1). ITrminal ra inifiaetion (t )) w ith. aee'c( pan villog SP ills of Sehiw ann eel Is in the Oili(ddle allea of till eapsile ((e) (at Iarrow). T1h. airea, ()f' t i ramnifieat ioll is (Ilelosedl hix tedllilir (colliutgeo)lis fihrils (ldf ) which( arll clear I lec rllisblile in liem'inelr(l eocn in tilis lighttl-liero)se es 1pi(ctulre. e, pierilitillall caipslll(; is, Ili( Ilieliral ealipsile sliaee; ec, (e1(- nlelural cell. Selllithlill sectioi (0-5.in). ( Idlt)).

9 FI('. 6. Sectionl through the p)oiala r(gion of a Bltuffini ending, in cat's skin. The til) of the s dil(le- shaped colrp)lisce contains at its (cenl ie tw o coliagenouis finres (arow ) which w ele f(w ined by the( oliagenotis fibtilis of the i1111' co( v. The perineural capsule (1)(&) (dissolves 110or uthil a 1d)or at the p lie; kf. xltrtac ip)sular collagenouis fires. Secmiithin sec(tmml (0>-5 ttiii). (>,' 2),000>).

10 PNM: r-1., Fi(:. 7. I'l((trnInleirwr( Id r I of )(8I-t o t he 11111OF(1 W(d1 a1( IIlfIilli ( 1II,i-1 of the (ctt S.skiII. thi(e te IIioII l rdt1iii(f 1 ilulls o,f t hoe aoi II, (t h). Imssess touloo,i r p)woo00sso8. (1'), NviIei olile palri I (OXV01('1 hyx Schimxi 0ll ( lls (S( ). Thiov 0010(1tl make l (iloolt 0001t01(1t withi thle (c11l10( XiV ti ll)s Space \vi t Io I tiii l r t 11 10i(0,1 theo I:se1m10oIi1 10I,1h0, Ilw (I.100 i- 11'' r)). hf, c(d 1o1-el0lls filwlilso fit', IIOioloofihilils; -1,01OI1l,I1l(o Xej-bls (S1. i;:i Illor ii ill 1t h0( pl)s1ll;1 (,, t hl te'r-limull I1r 10fitoi 1 0i0s (tl). ( 30,040 ).

11 SA II Cutaneous Mechanoreceptor points in the receptive field over a track of 1 mm. Although each component of the response was altered at each new point of stimulation, the effect on the dynamic response was the most striking (see section, Dynamic Response, for details). Normally our intention was to place the probe over the most sensitive spot in the receptive field as described in 'Resting Discharge', but to find this in the absence of a surface feature was often very difficult and failure to do this was reflected in the variation of the response from unit to unit. Consequently the complexity of the analysis of the stimulus-response patterns for the whole sample was increased. The size of the receptive field to stretch was very difficult to define. Most units responded if the tension of the skin in the lower leg was altered, for example, during flexion and extension of the ankle joint. Presumably this was due to tension transmitted through the skin. Morphology of receptors Receptors were located during electrophysiological experiments for subsequent histological examination either by marking the epidermal surface at the most sensitive spot with a small drop of silver nitrate solution or by inserting a pair of fine wires through the skin. The first technique proved to be unreliable, probably because the receptors could be deeply situated in the corium. In the later experiments in which fine stainless steel wires (100,m diameter) were inserted into the skin on either side of the most sensitive spot in the receptive field, the typical receptors were found lying between the holes made by the wires. Six receptors were examined histologically after marking their location in electrophysiological experiments. The detailed structural results reported in this paper were obtained from a similar receptor in the skin of the fore-limb of a cat. In low power photomicrographs of semithin sections, the receptor lies between the two holes made by the marking wirepieces which were drawn out of the specimen after fixation and dehydration. Such receptors show the structure typical of a Ruffini ending: a thinly lamellated capsule, like a perineural sheath, surrounding a 'fluid-filled' space at the centre of which is the core (called the 'supporting spindle' by Ruffini) composed of nerve-terminals, connective tissue and cells giving rise to membranes that divide the space surrounding the core into compartments (Fig. 4). The receptors have a similar appearance in formalin-fixed tissue, stained with the Holmes' silver technique, except that the detail is not so well preserved. The receptors were examined by both light- and electron-microscopy. They were situated in the corium, and had a spindle-shaped form, oriented parallel to the long axis of the limb. The receptors examined ranged from 2-0 to 0'5 mm in length, with largest diameter amounting to 150,um in the equatorial area. The polar region had a diameter of 30 to 40,um. The capsule arose from the perineural sheath of the afferent nerve fibre and had four to five layers of perineural cells (thus corresponding exactly with Ruffini's original description). The interior of the capsule contained endoneural connective tissue that was separated into an inner core and an outer envelope. The space between the core and the envelope was filled with fluid substances (Fig. 5). At the poles of the VOL. LVII, NO

12 424 Chambers, Andres, Duering and Iggo ~. 3a -. 3b 100pm,4 FIG. 8. / 1:1. "' I J] Reconstruction of a Ruffini ending of the cat's skin from a series of semithin sections and from electron-microscope step cuts. The picture shows two-thirds of the 05 mm long Ruffini organ. Lettering as in Figs 5 to 7. Nerve fibre (nf). The drawn lines indicate the levels of Figs 3 and 4.

13 SA II Cutaneous Mechanoreceptor 425 receptor the collagenous fibrils formed collagenous fibres which entered the fibre network of the corium (Fig. 6). The receptor was supplied by a myelinated nerve fibre which entered at the equatorial area of the organ in the receptor illustrated in Fig. 8, but in other receptors it entered at one pole. After fixation and embedding the diameter of the fibre was 7-12,um in different receptors. The myelin sheath could be traced up to the inner core of the receptor (Fig. 5a) when serial sections were examined. There a club-like enlargement of the axoplasm was located from which numerous fine branches emerged. On each side a group of these fine branches of 200,um length ran up to each pole. The terminal ramifications (Fig. 5b) of the nerve fibres had a meandering course. They were accompanied by spurs of the endoneural cells and also had a direct contact with the collagenous fibrils of the inner core (Fig. 7). At the surface of these terminal ramifications there were thornlike or tubular processes of about 0-2 to 1 um in diameter, which did not contain the usual cell organelles. Instead they included thin filaments and, at their base, vesicles which occasionally were granulated. A diagrammatic reconstruction of this particular Ruffini ending, prepared from a series of semithin and electron microscope sections is shown in Fig. 8. The general structure has some similarities with muscle spindle receptors which also have a fluid-filled capsule space. They are quite distinct from Merkel cells. PHYSIOLOGICAL RESULTS Resting discharge The resting discharge was defined as the discharge of a unit in the absence of any stimulus intentionally applied by the experimenter. Forty one units carried a resting discharge with mean interspike intervals ranging from 61-35±15*17 msec (SD) to msec (SD). The interspike intervals were very regular (Fig. 9), the coefficients of variation for different units ranging from The regularity and length of the interspike interval of the resting discharge were characteristic for the type II units, and the term 'ticker' applied originally to corneal units by Tower (1940) aptly describes them. The regularity of the resting discharge often became more pronounced after initial mechanical stimulation of the unit, possibly due to persistent tension changes in the skin or receptor caused by this initial identation. Two other types of mechanoreceptor unit may carry a resting discharge: these are the type D hair follicle units [Brown and Iggo, 1967] which are sometimes stimulated by the arterial pulse in the skin, and SA I units which sometimes carry an irregular low frequency discharge. Such a resting discharge is not usual and can easily be distinguished from type II units by its failure to respond vigorously to stretching of the surrounding skin. After gentle mechanical stimulation the resting discharge of type II units sometimes increased slightly in frequency as well as in regularity, and was maintained at the new higher level even when the stimulus was removed. Excessive lateral or vertical deformation of the skin occasionally caused both permanent disappearance of the resting discharge, and a decrease in discharge caused by deformation stimuli, effects attributed to receptor damage. Such units were excluded from the sample analysed in detail in this paper.

14 426 Chambers, Andres, Duering and Iggo Resting discharge unit c r 0-02 n: 3208 c Mean isi 87±5(SD) u) Frequency 11 5 i/sec o8 'a> Coeft variation 0o06 cc 0 ~~~~~~~~~~~~~C 0 0 C 32,c o S OC 0 o.~~~~~~~~~~~~~~~~~0 E z c c FIG. 9. cc I,I oil 0 N Time - msecs Unit No Interval histogram of the resting discharge of a type II unit collected on the Biomac 500 over 7 min, S1. The restinlg discharge of this unit was the most regular of all the units encountered, with a coefficient of variation of 0e06. Four units were silent when they were first isolated, but a resting discharge appeared after a period of gentle mechanical stimulation. There was also some indication that heating of the skin by the microscope lamp may have aroused the resting discharge in these units, but this was not considered to be a simple thermal response, because when the source of heat was removed the resting discharge persisted. Burgess et al. [1968] reported that many of their type II units did not carry a resting discharge and it is possible that the present sample may be biased in favour of units carrying a resting discharge, as they were more readily found. It was usual, however, to identify all the slowly adapting units even in 'silent' strands; hence the discovery of only 4 silent units suggests that they were uncommon in skin innervated by the saphenous nerve. The resting discharge could be changed by (1) vertical deformation immediately in the vicinity of the receptor, (2) stretching the skin over a wide area and (3) heating or cooling the skin. The responses to each of these stimuli are described under separate headings in. the following pages. The discharge usually increased in response to deformation stimuli but it could be inhibited when the skin was stretched in a certain direction for each unit and also when vertical deformation was applied at certain points in the receptive field (see later sections). The resting discharge invariably returned on release of the stretch or removal of the vertical deformation and the pattern of the returning discharge was an inversion of the normal dynamic and adapting response (Fig. 2B).

15 SA II Cutaneous Mechanoreceptor 427 Quantitative mechanical stimulation SA II receptors were stimulated by vertical deformation and by lateral stretching of the skin and a detailed systematic examination of the response to vertical deformation was made, as well as a less systematic examination of the response to skin stretching. The response to vertical deformation had a basic characteristic pattern and was divided for analysis into several components (a-f) (Fig. 2B). Dynamic response Type II units were subjected to indentation stimuli with linear velocities from 0-1 to 6*25,um/msec and maximum displacement of 350,um, repeated every 20 or 30 sec depending on the amplitude and duration of the stimulus. The dynamic responses for 9 units were analysed in detail by calculating the mean interspike interval for the 1st, 2nd, 3rd... nth interval of ten consecutive responses and plotting these against the mean displacement at which the 2nd, 3rd, 4th... (n+ Ith) action potential occurred. Three kinds of dynamic response were recorded (Figs. 10 and 11). (i) (5 units) Consecutive interspike intervals decreased in length up to maximum displacement. This was regarded as the 'standard' response because Dynamic response B A unit * A Stimulus velocty t b 320 A 012 )Om/msec C A A.0:24 o AA a A c ~~~~~A E 20 N~~~ A~ A A, < 60 _ * c *Es > w A 40 A 70 to * 20[-L-_ b A A - _ FIG DispIacement - um The 'standard' dynamic response of a type II unit (No ) at 5 velocities of displacement plotted on (A) linear, (B) semilogarithmic and (C) logarithmic co-ordinates. Each point is the mean for 10 repetitions of the stimulus. The regression lines were calculated by the Method of Least Squares. The first interval (i.e. from the beginning of the stimulus to the first spike) is not plotted. This kind of dynamic response was recorded and analysed in detail for the 5 units in group i (see text). The maximum displacement throughout was 225,um. For B and C only one regression line is inserted (0-12,um/msec slope).

16 428 Chambers, Andres, Duering and Iggo Dynamic response A 60o - 0 unit Stimulus velocity A 0-25 rm/msec * b E ~~~~~~~~~~~~~~~20 - O~ ' 30 -too -c ~~~~~~~~~~~~~~~~~~~~ A Displacement - jum FIG. 11. The dynamic response of this unit (468-2) was typical for the 3 units in group ii (see, text). The stimulus response relationship plotted on linear co-ordinates was curvilinear- (A), but linear on semilogarithmic (B), and logarithmic (C), co-ordinates. This difference from the 'standard' dynamic response (see Fig. 10 and text) is thought to be due to the location of the stimulus probe remote from the most sensitive spot in the receptive field. Each point is the mean ISI calculated from not less than 10 records, and the lines were calculated by the Method of Least Squares in B and C. it occurred when the most sensitive part of the receptive field was stimulated (Fig. 10). (ii) (3 units) The interspike interval decreased up to maximum displacement, but the rate of the decrease itself fell, producing a curvilinear relationshipbetween response and stimulus (Fig. 11). (iii) (1 unit) After a decrease of the first two or three interspike intervals the interspike interval remained more or less the same. The last two kinds of response were attributed to stimulation at varying remoteness from the central point in the receptive field. These groupings therefore, reflect variability of the discharge in response to stimulation at different parts of the receptive field, rather than a further sub-division of the type II (refer to Fig. 3). The variability may be attributable to directional sensitivity (p. 436). Relation between velocity of displacement and afferent discharge Regardless of the pattern of discharge during displacement, the mean frequency of discharge increased as the velocity of displacement was increased

17 IT>> <XmSB ooo ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~OZ < < e>xo o > o : _ b > ~~~cq SA II Cutaneous Mechanoreceptor,,, ;~~~~a oe * ~ _l~~ ~ ~ e 11~~~~~~~~~~~~~~~~4 ell *4 *0 a _~~~~~~~~~~~~~~e _- *0 0s r o >bx: X_ O XbOO 00 >- M.t~~~~~~~~~~~~~~~~~~~~~~~~~~~~. 429 *.e~~~~~~~~~~~~~~b ~~~~~~~~~~~~~~~~~~~~~ o * 4 m O Ci e * * s~~ >= * 8~~~~~~~~0 * co *- > cc s t- e O < GO > > CiU4 U~~~

18 430 Chambers, Andres, Duering and Iggo (Figs. 10 and 11) and so the final interspike interval was proportional to the velocity of displacement even when, as in Fig. 11, the stimulus-response for a given velocity was curvilinear. Therefore, the type II receptors, like the type I [Iggo and Muir, 1969] and the hair follicle units [Brown and Iggo, 1967] are sensitive to the velocity of displacement. In addition, the decrease in ISI during displacement indicates that the type II is displacement sensitive also, so that like the type I the dynamic discharge is determined by both the amount and rate of displacement. Regression equations (y=a+bx) have been calculated for the regression of ISI (y) on amount of displacement (x) at different velocities and results for two units and two velocities are given in Table I. Iggo and Muir [1969] suggested that in such a representation the value of the ordinate intercept 'a' is related to the velocity component alone, and that the slope of the line 'b' is related to the displacement component of the response. If this is so 'b' should remain constant at all velocities of displacement. For the type II, 'b' was found to decrease as the velocity of displacement increased, up to a velocity of 6 um/msec, and this suggests that the velocity component influences the value of the slope, 'b', as well as that of the intercept, 'a'. A similar pattern in the values of 'b' at different velocities for the type I can be seen in Fig. 5A of Iggo and Muir [1969]. An attempt was made to relate these results to the psychophysical relations adduced by Fechner and by Stevens. Fechner's relation is satisfied if a straight line is obtained when the response is plotted against the logarithm of the stimulus (a logarithmic relationship) and Stevens' is represented graphically by plotting the logarithm of the response against the logarithm of the stimulus and is satisfied if a straight line is obtained (power function relationship). The results were plotted on the appropriate axes (Figs. 10 and 1 1 B and C), regression lines were calculated by the method of Least Squares and their significance tested using Fisher's Variance Ratio test (Table I). The three kinds of dynamic discharge were treated separately and each unit was analysed separately: (i) (5 units) No statistical distinction could be made between the three scalings, so that linear, logarithmic and power function relationships are equally valid (Fig. 10). (ii) (3 units) Again no statistical distinction could be made between the straight lines produced on the semilogarithmic and logarithmic scalings, whereas on linear axes the relationship was distinctly curvilinear (Fig. 11). (iii) (1 unit) This unit was not subjected to this analysis. The dynamic response of the type II can therefore be regarded as satisfying both the Fechner and Stevens Laws. No statistically valid distinction could be established by the statistical methods used, especially for the five units in section (i) which we consider to have been stimulated over the most sensitive spot and which also satisfied a direct linear relationship. Relation between velocity of displacement and terminal ISI All the units were included in a similar analysis of the relation between the terminal ISI at maximum displacement and the velocity of displacement, and

19 SA II Cutaneous Mechanoreceptor the results plotted on three scales. These three forms of scaling resulted in (1) a curvilinear relationship on linear axes, (2) a straight line relationship over the whole velocity range on linear-log axes and (3) a straight line relationship up to 5,um/msec velocity on log-log axes. This result indicated that the relation between stimulus velocity and ISI for the type II best satisfies Fechner's Law (2), a result that may depend on the relative strengths of the velocity and displacement components of the stimulus in determining in the ISI. This result is applicable to all three kinds of dynamic response, despite the fact that the probe was positioned at points of differing sensitivity for each of them. Adaptation In order to use the analysis of time constants (T=k, where y=e- t, y=variable declining exponentially, t =time for particular value of y, and k slope of the line, log y against t) the dependent variable, i.e. the response expressed in ISI, had to be represented as a declining variable. Therefore, the adapting response was plotted as the difference between the instantaneous ISI and the mean ISI of the resting discharge. The semilogarithmic plot, Fig. 12, revealed three time constants during the first 8 sec of the response with average values of 077 sec, 7-27 sec and 3002 sec for unit at four displacements (Table II). A fourth time constant which became evident after 8 sec, was measured for only one unit and was min. The adapting response of the cockroach mechanoreceptor [Pringle and Wilson, 1952] and of the frog muscle- spindle [Roberts, Boyd and Cairnie, 1956; Davey, 1959] has also been resolved into three components, similar to the components of the first 8 sec adaptation of the type II. Preliminary experiments to eliminate the elastic and compressive properties of the underlying tissues have shown that these contribute significantly to adaptation. When a small metal plate was placed under a type I receptor, the amount of adaptation during maintained stimulation with constant displacement was greatly reduced. Attempts to repeat this for the type II unit were unsuccessful, probably due to the inaccessibility of the terminal in the dermis, which prevented accurate positioning of the metal plate. Static response The response during the phase of maintained constant displacement has been termed the 'static response'. In this present work the static response is defined as the discharge (expressed as the mean ISI) during the 4th to 8th sec of a maintained 8 sec stimulus. During this part of the response the time constants of adaptation are long, sec. This defined period lies between the 'early' and 'late' steady states of Werner and Mountcastle [1965]. During the 'static response' the unit fires regularly and the coefficient of variation of ISI is always less than 0-25, in contrast to the variability of the static response of the type I, (Coeff. var. > 0.5), as shown in Figs. 9 and 10 by Iggo and Muir [1969]. The mean ISI of the static discharge was linearly related to displacement over the range ,um displacement. However, when the straight lines of this relationship were extrapolated, they did not cut the 431

20 432 4Chambers, Andres, Duering and Iggo Adaptation End of rising unit :~ phase of stimulus 80- Maximum displacement r T Q 80 J 100pm E 60 - \ 0 U E ) _T 20L' I 0 o ~~~~~O [ L% S Pm FIG pum _ Time - secs Graphs to show the process of adaptation during displacements at four different amplitudes. The response was plotted as the difference between the interspike interval and the resting interspike interval discharge in order to produce an expontially decreasing variable for the analysis of time constants. The time constants (7) are in seconds, and are not corrected for interactions between them. ordinate at the resting discharge (i.e. at zero displacement) and experimental interspike intervals below indentations of 100,um and above 350,m tended to be smaller than the extrapolated values, resulting in a curvilinear relationship. The regular resting discharge was a very distinctive feature of these units and was used as the more-or-less fixed base-line from which the 'static' response was measured. The response of each unit in Fig. 13 was therefore corrected for its resting discharge by subtracting the resting discharge from each interspike interval. This data, including points below 100,um and above 350,m displacement for 8 units was normalized, pooled (see legend Fig. 13 for details) and plotted on linear, semilogarithmic and logarithmic co-ordinates (Fig. 13) in order to test the correspondence of this relationship to the psychophysical laws. Analysis of the fit of the lines B and C, Fig. 13, by the Method of Least Squares and Fisher's Analysis of Variance showed that no statistical distinction could be made between the significance of the logarithmic and power function

21 SA II Cutaneous Mechanoreceptor 433 a0, d 0 0,o m to = o C) O= oo 0 o 0. c eb *4 0 0, 0lRC~ CO = N- P- N- N- C~cic 6< A 10 N 0- O-- O - Cs bq :z 0,IIq l g CA) *0; 0U0 't 0,-4 8.0, o 004 4, r00 _N 6 roo N N NC0 al0 c r O * -* td =cs; r- w **.*.~~~~~CB** - 10~~~~~~~~~~~~~~~~~~~~~~~~~4 ~ Q ,) o0 o$ 4.4 0, 10~~~~~~~~~~~C bu: Cslg 10 o o CON10 4- O o 0 : co 0 r CO +X- :~0 14Q, 0 xo xo (D ed 4 1* 0-0 S 0 P- 0H 0 - c, a. 1 1to 0 0 C> ) 1OC1C1O 0 to lf C)e 0 e _0 CoC C-oC00 X _ ~~oho H) H)C 0 C, 0: fpi04._c 0, N I 0 CO N K t < to t~~~~a C*b 14

22 434 Chambers, Andres, Duering and Iggo 0 0 n- ut 0 b- ns'. Static reesponse Final displacement units c9 [.E Z; loo -x c co sc 6c jum posn posn w~-6 A... A 0 A U c *e C C B C - ~~~~A * 0 j- 10 A ' U Q *,0 0oo 40 " 't loo 1 Stimulus (as % of maximum stimulus) FIG. 13. Graphs to show the analysis of the static response. The data consists of the static stimulus response relationships from 7 units. The mean interspike intervals of each unit were corrected for the resting discharge of the particular unit and then calculated as percentages of the response to maximum stimulus. The results are shown plotted on (A) linear, (B) semilogarithmic and (C) logarithmic axes. The curve shown in (A) was not calculated but the straight lines in (B) and (C) were calculated by the Method of Least Squares and their significance tested by Fisher's F Test. The regression equations for the lines are: B. Semilogarithmic co-ordinates R = log S (r = 0-89, F = (1,57 d. of f.)) C. Logarithmic co-ordinates Log R = log S R = xs0o36- (r = 0-85, F = (1,57 d. of f.)) Where S = displacement, 1? = response, r = correlation coefficient of regression relationship (see Fig. legend), but that on linear co-ordinates the fit was unsatisfactory. This result is contrary to the report by Werner and Mountcastle [1965] that, for a mixed population of type I and type II units [see Iggo and Muir, 1969], the logarithmic relationship can be rejected and that only the power function is significant. Werner and Mountcastle however did note that for six of their eighteen fibres the logarithmic relationship was also statistically valid and these fibres may be the type II units of their sample. The stimulusresponse relationship of the type I has been reported to obey a power function by Tapper [1965] and Werner and Mountcastle [1965]. The fact that the type II satisfies both the logarithmic and power function relationship may indicate a real difference between the type I and type II slowly adapting receptors in their transducer/spike interaction mechanisms. Recovery of the resting discharge on withdrawal of a stimulus (silent period) When a steady stimulus was removed from a SA II unit a silent period was observed followed by an exponential decrease in successive ISI as the resting

23 SA II Cutaneous Mechanoreceptor 435 discharge gradually reappeared (Fig. 7). The duration of the silent period was proportional to the duration of the stimulus, as observed in the frog muscle spindle [Adrian, 1926]. The time course of the recovery of the resting discharge could, like adaptation, be described by three time constants; the first time constant was very short, secs, the subsequent two being longer (Fig. 14 and Table II). A similar pattern of recovery was observed when the skin was released from maintained stretch t Stimulus oft Recovery unit \ Velocity of displacement 500-6pm/ms 0bo E -a L._ (I) 0, C c: lv0 1000: *~~ "D I I * I* * I I 500- \1pm/ms : 500-\ jim/ms *vv 1tv.~ I,I,,',I I ',II I I I Time - secs FIG. 14. The recovery of the resting discharge after the return to sero from a 350.Lm displacement applied at three different velocities. The duration of the stimulus was 1-0 sec. The recovery consisted of a silent period (in this case < 1 0 see) followed by an exponential decrease in interspike interval. In this figure the logarithm of the interspike interval is plotted against the time at which the interval finished. The time constants (X) are in seconds and are not corrected for succeeding ones. The time for recovery is directly proportional to the duration and amplitude of the stimulus. Sensitivity to lateral stretching and directional sensitivity The type II units were the only slowly adapting cutaneous receptor units in the skin of the inner thigh and leg which were excited by lightly stretching the skin surrounding the receptive field. This characteristic was so well defined that it was used for the initial identification of a unit. The time dimensions and patterns of the response to controlled stretch were similar to those produced by vertical displacement.

24 436 Chambers, Andres, Duering and Iggo The receptor units were directionally senaitive to lateral stretching of the skin, as has also been reported by Knibestol and Vailbo [1970]. For each unit the resting discharge was increased by stretching along one axis of the receptive field and was silenced by stretching the skin at right angles to this axis. Twelve units were tested for directional sensitivity-the resting discharge of nine of them was increased by stretch along the proximal-distal axis of the leg. If a unit was excited by stretch along one axis and then stretch was applied at right angles while still maintaining the original stretch, the discharge was not abolished but was reduced, and was restored when the inhibitory stretch was released. When the skin was released from stretch along either axis the discharge changed. If the stretch had excited the unit, then on release the discharge ceased for a while followed by a recovery of the resting discharge, whereas if the stretch had inhibited the unit, release caused a sharp increase in the discharge, similar to a dynamic discharge, followed by a period of adaptation to the resting 330 unit j '~~~~~~~~~~~~~~i I'I i1iflhnlinii I,IiI ''I,'i I11 111, 11,I,III,IllIIIIIllllllll IIIlIIIIuulIull'I 220 unit FIG oo msec The typical response of the type II unit to temperature change. Unit is excited by a drop in temperature and unit is inhibited by a rise in temperature. After both temperature changes there is adaptation to a new discharge appropriate to the new temperature. In these examples there is an increase in the discharge because the new temperatures (i.e. 280C and 360C) are near the peak temperature for each of these particular units. Both time marks are at 100 msec intervals. discharge. Extreme maintained stretch abolished the resting discharge permanently and more quickly than extreme vertical displacement. It was observed that the unit remained sensitive to vertical displacement even if the resting discharge had been abolished by extreme stretch. This fact may account for the large number of silent units found by Burgess et al. [1968], for if they held the leg in such a position that the skin was tightly stretched few units would carry a resting discharge but all would remain responsive to vertical deformation. Thermal sensitivity The type II, like the type I, is excited by a fall in temperature and the resting discharge is temporarily depressed when the skin temperature rises (Fig. 15). The thermal sensitivity of the type II was tested by placing a metal thermode

25 SA II Cutaneous Mechanoreceptor 437 Hz 7 Static temperature sensitivity curve 6_ unit , Temperature--C FIG. 16. Unit The static temperature curve of a type II. The discharge was measured when the unit had been held at each given temperature for at least 3 min. The peak discharge was recorded at 270C. on the skin and adjusting its pressure to produce a continuous adapted discharge, at a neutral temperature of about 33 C. The adapted discharge which was temperature dependent was measured when a given temperature had been held steady for at least 3 min. The peak discharge was found at 270C for the unit in Fig. 16. It is this property of the SA II units which suggests that the population of units with a spontaneous discharge described by Witt and Hensel [1959] at least includes, if it is not composed exclusively of, slowly adapting type II mechanoreceptors. DIscUSSION Comparison with earlier descriptions [Ruffini, 1891, 1894, 1905; Dogiel, 1903] leads to the conclusion that the receptor investigated by us corresponds to the Ruffini corpuscle. There are several features of exact correspondence: (1) spindle-shape, (2) capsule composed of 4 or 5 layers of perineural tissue, (3) an inner core composed of nerve terminals and other tissue surrounded by (4) capsule space filled with fluid, (5) innervation by a single large myelinated axon, (6) located in deeper layers of the skin and (7) size. The structural results reveal a number of criteria already known from previous electron-microscopical investigations of peripheral receptors. The

26 438 Chambers, Andres, Duering and Iggo close contact of axoplasmatic endings and their tubular processes had been demonstrated in the lanceolate terminals of afferent fibres in the sinus hair [Andres, 1966] and of the Golgi tendon organ [Andres, 1971]. Thus the investigated terminal of the cat does not belong to those mechanoreceptors which possess an inner lamellation (e.g. Pacinian corpuscles, Golgi-Mazzoni corpuscles, Herbst bodies [Andres, 1966, 1971]. The microstructure of the capsule and the inner capsule space corresponds in some degree to the situation known from the Pacinian corpuscle and the muscle spindle. The special suspension of the inner core by the collagenous fibres emerging from the poles of the terminal support the suggestion that the Ruffini ending is a stretch receptor. The central area or core may be sensitised to deformation in a special direction by the water-cushion effect of the capsule space, as is reported in the physiological section of this paper. The Ruffini-ending therefore, unlike the Merkel cell, does not possess any specialised presumed 'transducer' cell. The neurone itself must function as the mechano-electric transducer and this function probably resides in the thornlike or tubular processes 02,um-1l0,m in diameter that are carried on the terminal ramifications of the nerve throughout the core. Transmission of the mechanical deformation to these spurs is presumably via the collagenous fibrils that run throughout the core to emerge at the poles as collagen fibres that attach the receptor to the connective tissue of the corium. This arrangement provides another explanation for directional sensitivity since stretch of the skin in the long axis of the Ruffini ending would be very effectively transmitted via the collagen fibres and fibrils to the nerve terminals. The very regular discharge of these afferent units implies a single site for spike initiation which summates depolarisation arising from all the active transducer sites scattered over the core of the receptor. This spike initiation site may be the club-like enlargement of axoplasm seen in Fig. 3a. A recent description of Ruffini-like endings in joints [Goglia and Sklenska, 1969] agrees in general with the fine structural details reported here. Joint afferent units with a slowly-adapting response have been identified as innervating Ruffini-like endings [see Boyd and Roberts, 1953; Skoglund, 1956] found in joint capsules. Their myelinated afferent fibres are 5 to 12,um diameter, thus corresponding closely to the SA II afferent fibre diameter (5 to 15,um-[Brown and Iggo, 1967]). As with SA II units, the joint capsule Ruffini-like afferent units are also excited by stretch and carry a discharge with regular interspike intervals. A Ruffini ending in the skin differs characteristically in location from a Merkel touch corpuscle; it lies in the dermis or corium and is connected by its polar collagen fibres with the connective tissue of the corium, and is therefore readily affected by tension transmitted through this tissue. The Merkel cells, in contrast, are invariably found at or close to the basal layer of the epidermis as a cluster innervated by a single myelinated axon. The receptor cells are firmly embedded in the dermis, and protected, by the collagen core of the receptor and annular binding of the circumferential epidermal cells to subjacent collagen, from all except stimuli delivered to the epidermal surface of the receptor [Iggo

27 SA II Cutaneous Mechanoreceptor and Muir, 1969]. In Merkel's original descriptions [1875, 1880], especially in the frog epidermis, the Merkel cells lie at the base of a distinctive dome or plate of thickened epidermis. This structure has been re-discovered since then by many investigators and there is now a profusion of names for it (Pinkus Haarscheibe; touch spot; tactile pad; touch corpuscle; Iggo or Iggo-Pinkus domes; etc.) and the confusion might best be resolved by attaching to it the original discoverer's name and using his functional classification, i.e. Merkel tastflecken or touch spots. A physiological classification of the afferent units, of which the Merkel tastflecken form the receptor apparatus, is used in the present and previous papers from this laboratory, and they are termed SA I units-slowly adapting type I cutaneous mechanoreceptors. The functional characteristics of the type II receptor appear to depend on both the structure of the terminal and its position in the skin. The lower frequency of discharge of these units compared to the type I slowly adapting units is likely to be due at least in part to the damping of the stimulus by the overlying epidermis and dermis, whereas the characteristic regularity of discharge of the type II units can be attributed to the morphology of the receptor itself. In contrast to the type I receptor in which the afferent fibre branches into many terminals, each ending in a Merkel cell, the axon of the SA II enters the Ruffini ending without dividing and the terminal arborisation is confined to the intracapsular part of the receptor. Depending on the site of the initiation of impulses these structural differences provide a simple basis for the regular sustained discharge in the type II unit and the irregular sustained discharge in the type I, if in the former there is only a single or dominant spike initiation region (as in the crustacean stretch receptor) and in the latter many relatively independent sites of spike initiation. A single site would be expected to respond to the mean generator potential and, if the latter were constant, the spike discharge would be at a rate determined by the recovery time of the membrane at that place-possibly a node of Ranvier, a situation analogous to the crustacean stretch receptor [Edwards and Ottoson, 1958]. The variability in resting or static discharge would be an index of variability of the membrane (random fluctuations in excitability) at this site and/or the stability of the generator potential. Multiple sites on the other hand, if they were entirely independent of each other, would give rise to a Poisson distribution of spike intervals [Iggo and Muir, 1969] with a dead-time determined by the refractory period of the afferent nerve fibre. Because of antidromic invasion of other terminals and spike generation sites in the touch corpuscle ending, however, the dead-time would be longer than that determined solely by refractoriness. This would arise from antidromic invasion causing a 're-setting' of the spike train rhythm at the invaded sites [Matthews, 1931; Iggo, 1958]. Depending on the site of impulse origin there may be another contributory factor. Katz [1950] and Ottoson and Shepherd [1970] have reported abortive spike potentials in the non-myelinated terminal arborisation of frog muscle spindle receptors and a similar phenomenon may occur in the touch corpuscle. The latter, however, differs from frog muscle spindles in an important respect. The terminal arborisation is myelinated to within less than VOL. LVII, No

28 440 Chambers, Andres, Duering and Iggo 1 uxm from each Merkel disc (the presumed source of generator potential) and by analogy with Pacinian corpuscles it would be expected that action potentials could arise at the first or second node of Ranvier. It remains a possibility, however, that such spikes may fail to propagate orthodromically because of low safety factors at regions of axonal branching. If this happens then the generation of orthodromically conducted spikes would depend on the near-synchronous arrival of terminal branch spikes at the branch junctions. Since individual terminal branch spikes would be independent in origin this could be a further factor leading to random interspike interval lengths. On either of these models, or a combination of them, strong stimulation of all the Merkel cells would give rise to large individual generator potentials. In such circumstances the branch junctions more remote from the Merkel cells may be depolarised by an amount sufficient to move the spike generation site proximally along the axon, thus reducing the number of independent generators, and could give rise to a more regular train of spikes, especially if the inter-spike intervals were short enough at one site for it to dominate all others by antidromic invasion. This could also happen if any spike generating site developed an enhanced sensitivity. Another difference between type I and II units also finds a morphological explanation. Type I in the skin of the leg are characteristically insensitive to stretching of the skin, unless it is severe, in contrast to type II. A careful examination of their structure shows that the Merkel cells in type I receptors (Merkel tastflecken) in the cat, i.e. the presumed transducers, are insulated from, mechanical changes in adjacent skin by the dense collagen that fills the core of the corpuscle and by the strong attachment of the perimeter of the receptor to the underlying dense collagen [Iggo and Muir, 1969]. The Merkel cells therefore, would be expected to be relatively insensitive to all except direct displacement of the overlying epidermal surface. The type II receptors (Ruffini ending) in contrast, lie more or less free in the dermis and their capsular organisation makes them more easily influenced by displacements transmitted from a distance. The directional sensitivity to stretching is probably determined by the structural geometry of the ovoid terminal structure (Ruffini ending), the bilateral symmetry of the receptor resulting in two axes with different excitability patterns. The Ruffini ending is anchored to adjacent tissue by collagen fibres which emerge from the poles of the receptor (Fig. 8). The collagen fibres form continuous fibrils that run through the core of the receptor and make intimate contact with the nerve terminals. Longitudinal extension of the receptor is likely, therefore, to be detected more effectively than transverse extension, which would tend to unload the receptor. The variation in the response to perpendicular displacement at different points in the receptive field may reflect either the differential sensitivity of the terminal membrane or the effect of the topography of the skin in each particular receptive field. All these suggestions, however, cannot be tested satisfactorily without recourse to an isolated receptor. The discharge of the type II in response to mechanical stimulus has been divided into six parts (a-f, Fig. 2) and for each phase the stimulus/response

29 SA II Cutaneous Mechanoreceptor relationship has been investigated. How many of these relationships actually transmit information to the central nervous system is difficult to judge and their importance will only be established when it is discovered what the discharge pattern is after one or two synapses. Some evidence has accumulated to suggest that the discharge patterns are retained in identified cutaneous spinocervical tract units [Brown and Franz, 1969]. If, as Harrington and Merzenich [1970] claim, the type II is a pressure receptor then the response to static displacement is of primary importance, and the response to the velocity of the displacement of little relevance. Similarly it is difficult to decide for which effective stimulus-skin stretch, vertical displacement and/or temperature change-these units transmit information centrally. Casey and Hahn [1970] have recently reported that a change in temperature alters the mechanical sensitivity of the type I. The thermal sensitivity of the type I and II has led to their description as 'spurious' thermoreceptors [Iggo, 1969] but they could be regarded as simultaneously transmitting information to the CNS regarding both temperature and mechanical displacement; it is supposed that this information would be re-sorted centrally by correlation with information from other adjacent receptors of different properties. It has also been suggested that the type II units may function as proprioceptors [Chambers, 1969; Knibest6l and Vallbo, 1970] on account of their directional sensitivity to stretching. However, adjacent units do not always respond in the same way to a particular movement of the leg and this property militates against a proprioceptive function. The type II units are unique among the cutaneous mechanoreceptor units so far described in hairy skin of the trunk and limbs, in that a single axon innervates only one receptive focus and this may aid two point discrimination. The low density of these units on the inner thigh and leg of the cat corresponds with the relatively poor two point discrimination in this region in man, although their high sensitivity to skin stretch may militate against this role. This type of unit may be more abundant in the extremities and in the forelimbs in the cat, where more detailed tactile input is presumably required. The units described by Mountcastle, Talbot and Kornhuber [1966], in the hand of the monkey are closely similar functionally to the type II receptors and their fibres appeared to be abundant in the median nerve as has also been suggested for similar units in man by Knibest6l and Vallbo (1970). Harrington and Merzenich [1970] have identified type II receptors electrophysiologically, in both glabrous and hairy skin of primates and ascribe to them without reservation the function of transmitting information regarding pressure. At the same time on questionable grounds Harrington and Merzenich implied that type I units have no sensory function. Neural codes A receptor can be classified as a particular type by qualitative and relatively simple quantitative methods [Adrian and Zotterman, 1926]. Delicately controlled quantitative stimulation and recording, as described in this present paper, is necessary to establish, in addition, details of the processes by which information is transmitted in the primary afferent fibre. This has recently been 441

30 442 Chambers, Andres, Duering and Iggo presented as a search for a neural code. When only simpler quantitative methods were available the frequency code was generally accepted as the neural code, but with new methods a more exacting scrutiny becomes possible. The exact details of the stimulus-response relationship of the frequency code seemed difficult to establish but early attempts were made by Matthews [1931] and Hartline and Graham [1932] for the muscle spindle and Limulus eye receptor respectively. For these two units, the frequency of the discharge was related to the logarithm of the stimulus, in agreement with the psychophysical law of Fechner [1860]. However, in 1957, Stevens demonstrated that sensation and stimulus may be related by a power function and since then many reports have been made that the stimulus-response relationship in the primary afferent fibre can be described by a power function. The velocity components of the response of the types D, G and T hair follicle units [Brown and Iggo, 1967], and of the SA I units [Iggo and Muir, 1969] as well as the static components of the SA type I [Tapper, 1965; Werner and Mountcastle, 1965] and the carpal hairs [Nillson, 1969] have all been found to be related to the stimulus by a power law (R=KSn; where R=response, S=stimulus, K a factor of proportionality and n is the exponent [Stevens, 1957]). Using the interspike interval rather than the frequency as the basic unit, this paper has presented evidence to show that the events in the primary afferent fibre of type II units in relation to the stimulus can be described equally validly by a logarithmic (i.e. Fechner) or power function (i.e. Stevens) relationship. Since it has been established by Iggo and Muir [1969] that Werner and Mountcastle presented results from a mixed population of type I and type II units, the seven fibres for which they found that the logarithmic and power function were equally valid may have been type II units. The response to stretch may follow more closely a power law relationship and if so this would suggest that the function of the type II is to transmit information regarding tension in the skin. This has not been established during this investigation. As a result of these various combined electrophysiological studies, histological experiments and independent electron-microscopical studies, the type II slowly-adapting cutaneous mechanoreceptor has become established as a distinctive and well-defined afferent unit. Independent electro-physiological experiments (cited above) amply confirm the functional properties reported in this paper and indicate a widespread occurrence of this class of receptor in the skin of mammalian species (Table III). The sinus hairs in the face of the cat have also been reported to have slowly-adapting mechanoreceptors with the general characteristics of the SA I and SA II units [Gottschaldt, Iggo and Young, 1972] and it has been proposed that the functional differences have a morphological basis-the type I response arising from Merkel cell receptors and the type II from lanceolate terminals as described electromicroscopically by Andres [1966]. The major distinction drawn between SA I and SA II receptors in the present paper has been sustained and extended in a recent, thorough, description of slowly-adapting receptors in reptilian skin by Kenton et al. [1971].

31 SA II Cutaneous Mechanoreceptor TABLE III. Comparison of the properties of slowly adapting type I and II cutaneous mechanoreceptors. Similarities 1. Slowly adapting. The discharge to maintained mechanical stimulus lasts for several minutes, at least. 2. Respond to vertical displacement of the skin with dynamic and static components. 3. The dynamic response has both velocity and displacement components. 4. Found in cat, rabbit (Tapper, 1965; Brown and Iggo, 1967; Burgess et al., 1968), the monkey (Iggo, 1963; Perl, 1968; Harrington and Merzenich, 1970) and units with similar properties in man (Boman and Hensel, 1960; Vallbo and Hagbarth, 1968; Knibestol and Vallbo, 1970). 5. Innervated by myelinated axons, conduction velocities ranging from 20 to 100 m/sec (mean 57 and 54 in cat, 47 and 31 in rabbit (Brown and Iggo, 1967)). Differences 443 SA I SA II 1. Irregular discharges to maintained stim- Regular discharge to maintained stimulus ulus Coefficient of variation > 0.50 Coefficient of variation < Does not respond to stretching, unless Responds readily to stretching of the severe and prolonged skin 3. Capable of high frequency discharge to Generally low frequency response to all all effective stimuli effective stimuli Smallest ISI < 10 msec ( > 1000 Hz) Smallest ISI 1-3 msecs (800 Hz) 4. Resting discharge unusual Usually has a resting discharge 5. Distinct dome on surface of the skin (cat) No evident surface feature 6. From 1-5 receptors per axon. These may One receptor per axon be scattered over an area of 25 sq. cm. 7. Receptor terminals are Merkel discs Receptor is Ruffini ending associated with Merkel cells. * SA I units in the skin covering the dorsum of the hand in monkey (Iggo and Ogawa, unpublished) are more readily excited by stretching the skin than those described previously. ACKNOWLEDGMENTS We are grateful for financial assistance from the Wellcome Trust, the Science Research Council and Deutsche Forshungsgemeinschaft. REFERENCES ADRIAN, E. D. (1926). The impulses produced by sensory nerve endings. Journal of Physiology, 61, ADRIAN, E. D. (1931). The messages in sensory nerve fibres and their interpretation. Proceedings of the Royal Society, Section B, 109, ADRIAN, E. D. and ZOTTERMAN, Y. (1926). The impulses produced by sensory nerve endings. Pt. III. Impulses set up by touch and pressure. Journal of Physiology, 61, ANDRES, K. H. (196-6). tber die Feinstruktur der Rezeptoren an Sinushaaren. Zeitschrift fiur Zellforschung und mikroskopische Anatomie, 75, ANDRES, K. H. (1971). Structure of cutaneous receptors. Proceedings of the International Union of Physiological Sciences, 8, ANDRES, K. H. and HENSEL, H. (1971). Personal communication. BOMAN, K. K. A. and HENSEL, H. (1960). Afferent impulses in cutaneous sensory nerves in conscious human subjects. Journal of Neurophysiology, 23, BOYD, I. A. and ROBERTS, T. D. M. (1953). Proprioceptive discharges from stretchreceptors in the knee-joint of the cat. Journal of Physiology, 122,

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