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90 J. Physiol. (1962), 164, pp. 90- With (^9) text-figures Printed in Great^ Britain
THE EFFECTS OF^ SUBLIMINAL STIMULATION^ ON^ THE EXCITABILITY OF^ FROG^ SKIN^ TACTILE^ RECEPTORS
BY W. T. CATTON
From the Department of Physiology, Medical School, King's College,
University of Durham, Newcastle-upon-Tyne, 1
(Received 11 April 1962)
After discharging a spike in its sensory axon the frog tactile receptor shows a^ refractoriness lasting^ from^ 500 to^ 2000 msec^ (Catton,^ 1960,^1961 b). In the present paper the excitability changes accompanying subliminal mechanical and electrical stimulation are described. Some of the results have already been reported (Catton, 1961a). As^ in^ the^ previous work, single receptor endings were excited by careful localization of the stimulus.
METHODS These were chiefly as described in an earlier paper (Catton, 1961 b), with two main differences; first, in some experiments dorsal-skin-nerve preparations were used instead of the calf-skin-nerve preparation; secondly, in order to obtain an improved wave form with prolonged stylus displacements a moving coil drive system was devised. This consisted of the moving-coil and magnet assembly of a transistor radio loudspeaker (coil impedance 80 Qi), on which was mounted the stimulating stylus, an ordinary pin blunted at the tip. Stylus movements were monitored, as with the earlier crystal stimulator, by means of a variable- capacity device which gave a record on the second beam of the oscilloscope. In order to reduce overshoot at the 'on' and 'off' deflexions of a long pulse, and to damp mechanical resonance, the moving coil was fed from a power transistor, giving an output impedance of about 1 Q as compared to the 500 Ql output of the main stimulator. The circuit includes provision for algebraic addition of two stimulus pulses (Fig. 1). In spite of being fed from a low impedance there was still some overshoot and resonance, but this could mainly be removed by suitable damping material. The p6rformance of the moving-coil stimulator was as follows: maximum displacement, 50 ,; rise time, 0 7 msec; minimum duration, 2-0 msec; maximum duration, 150 msec. Electrical pulses were applied through an isolating trans- former to the metal stimulating stylus in contact with the outer skin surface. Spike dis- charges in the skin nerve could be obtained at many points, and it^ was^ difficult^ to^ find situations where a single response could be maintained over a reasonably wide range^ of stimulus strengths; the appearance of additional 'recruited' responses, when^ strength had^ to be (^) increased as the threshold of the originally chosen receptor rose, made measurement difficult. Neither shielding of the stylus by plastic nor the^ application^ of^ an^ insulating layer of (^) liquid paraffin to the preparation gave much improvement, the spread of current being presumably largely within the skin itself. Nevertheless, some favourable sites were found from which reliable measurements could be made. The criteria by which excitation of the sensory terminal by an electrical pulse could be distinguished from direct excitation of the sensory axon were as follows: (i) response obtained at low-threshold strength and long latency, (ii) response fatigued rapidly when stimulation frequency was increased beyond
STIMULATION OF FROG SKIN TACTILE RECEPTORS 91
about (^) 20/sec. Direct nerve excitation was accompanied by very short (^) latency (< 1 msec) and high threshold. All stimuli were applied to the outer surface of the skin-nerve (^) preparation mounted in (^) a moist chamber, impulses being recorded from the skin-nerve branch. The stylus was (^) placed in light contact with the skin before the stimuli were applied.
Pi Pll
I (^) 0-6 V
T R 3 TRs
R, (^) R2 (^) N~~~~~~~~~~~~-
stages TR3 and TR4 (^) provide a (^) very low output impedance to feed thecoil (^) a. Adjust- ment of feedback control VR1 enables emitter of TR4 to be set to -2 V, when zero current flows through the coil, as indicated on (^) meter M. This is the condition in the absence of drive pulses. I?1 and R2 together with base input resistance of TR1 con- stitute an 'adding circuit' giving algebraic addition of (^) pulse Pl and (^) PII delivered from the main stimulator. R1, (^) R2, 100kQ;RI, 3 R9k(;I4, 68kQ; I?,2c2kQ; R*, 4.7 kQ.; 1R, 39 Qt; Vri1, 0orkQi.C, (^) moving coil astembly (^) of transistor radio speaker, impedance 80t. M, 100mA full scale deflexion. (^) Te1,TRc , Mullard 0071;ofR3, 0C72; (^) tlR4,0036.
RESIJLTS
Studies were made on about 80 receptors from 27 dtifferent skin pre-
parations, mechanical or electrical (^) pulses of various durations and in
different combinations being used. Electrical pulses were alternatively
cathodal or anodal and varied from (^0) 5 to 25 msec. Throughout this paper a 'short' mechanical pulse (^) is one of (^) 2 msec, the shortest practicable; a 'long' stimulus is one of 20-40 msec. The pulse pairs were presented at (^) a rate of one pair (^) every 2 sec.
Two short subliminal mechanical stimUli. With the strength of the con-
ditioning pulse (PI) held constant and just subliminal, a subliminal test
pulse (PII) was applied at different times before, during, and after PI.
STIMULATION OF FROG SKIN TACTILE RECEPTORS 93
the initial sharp phase of threshold fall and the later phase of threshold rise were enhanced by increasing the strength of the subliminal conditioning pulse, until a spike was discharged over a certain summation interval which was difficult to measure exactly but was of the order of 05-2-0 msec. A special feature of these experiments was a tendency for the phase of threshold depression to become less marked with repetition of the pulse pairs until in some cases the effect ultimately became hard to detect; this did not always occur and the results relate to receptors which did not show this effect. On the other hand, the initial phase of threshold fall did not diminish with repetition.
a d
b e
c 500 cls
1*
1~~~~~ ~P subimna ,0.79 _
If 1. 1 I 1 I _ 0 5 10 15 20 25 30 40 Time (msec) Fig. 3. Excitability changes accompanying a long subliminal mechanical pulse. Upper trace, mechanogram of stylus displacement (moving coil unit); lower trace, spike response in skin nerve. Graph shows time course ofexcitability changes during a 16 msec pulse. In the oscillograms (a-e) the long pulse is 30 msec, short test pulse is 2 msec, and adds algebraically. Thresholds were adjusted so that a spike response occurred only at or near the 'on' deflexion (b), (c), and 'off' deflexion (d), corre- sponding to the peaks of threshold depression seen in graph, which was^ taken from a different experiment. Spikes in^ b, c, d retouched.
Long, subliminal mechanical conditioning pulse. When the^ duration^ of the subliminal (^) pulse was increased to values between 16 and 40 msec the general result was as shown^ in^ Fig. 3.^ There^ was^ a^ sharp fall of threshold
at the 'on' deflexion, similar to that described for a short pulse; but in- stead of being followed by a period of subnormal excitability the threshold now remained below normal during the (^) long pulse, though gradually rising. At the 'off' deflexion there was usually another sharp fall of threshold, which was^ followed by a^ subnormal^ period of greater extent than that which followed a short subliminal pulse. The fall of threshold at the (^) 'off' deflexion was (^) usually smaller than that at the 'on' (^) deflexion. With stimuli supr&liminal for an 'on' response it was found that the 'off' threshold was correspondingly higher than the 'on' threshold (Fig. 4, oscillograms).
a_ c
~~~~~~~~500 c/s
2- 26 224
212 E4)1 -6 off ~1 4
1-0 -
- 1020 40 60 80 100 150 On Time (msec) Fig. 4. 'On' and 'off' thresholds shown by progressively increasing the amplitude of a (^) single 30 msec mechanical displacement (moving coil unit). b, 'on' threshold; c, 'off' threshold; a, subliminal for 'on' response. Graph shows effect of pulse duration on the 'off' threshold (0); 'on' threshold (0) remains constant for pulse durations exceeding 2 msec, the shortest pulse available.
'On' (^) only responses. Some receptors showed a behaviour different from that described above, in that instead of a threshold fall at the 'off' deflexion (^) there was a threshold rise. These receptors failed to give an 'off'
spike even at maximum strength (about four times the 'on' threshold).
Figure 6 illustrates the behaviour of such a receptor, for a pulse duration of
40 msec.
'On' and 'off' thresholds in relation to pulse length. 'On' and 'off'
thresholds were measured for a single pulse of duration varying from 5 to
94 W. T.^ CATTON
Discharge of the spike at 'on' was followed by the refractory phase which characteristically follows a spike (Lindblom, 1958; Catton, 1961 b), so that the static phase of supernormality was abolished; the rise of threshold at 'off' was then superimposed on the existing subnormality (Fig. 6 (iv)). Similar behaviour^ was^ observed^ in^ 'on'-'off'^ units,^ with the difference that the threshold fall at 'off' was of sufficient magnitude to cause firing of an 'off' spike. The plateau phase of excitability change was often small, though always detectable, and onily in certain units could it be measured with reasonable accuracy. The unit illustrated in Fig. 6 was particularly favourable for measurement.
1-6 _ (i) _(ii)
12-OnOffon spik
04
Fig. 6. Behaviour of an 'on' unit. Graphs (i)-(iv) showeffect ofincreasingstrength of a 40 msec subliminal mechanical deflexion on excitability as tested by a 2 msec mechanical pulse added algebraically. Stimulus strengths were (i) 0 7, (ii) (^) 0-85, (iii) 0 95 and (iv) liminal, for the 'on' response. In (iv) an 'on' spike was discharged (f) and was followed by the characteristic refractory phase. In this receptor there was a decrease, not an increase of excitability at the 'off' defiexion, which persisted even after a spike was fired at 'on' (iv). No 'off' response could be obtained at maxrimum strength. Note in (i) only the plateau (static) phase is present; the 'on' and 'ofE' (dynamic) phases appear in (ii), and are larger in (iii).
Short subliminalmechanical pulse with cathodal or anodal test^ pulse. With
a cathodal test pulse the (^) excitability changes were similar to those with a mechanical test (^) pulse, except that the initial period of enhanced excita- bility was^ prolonged up to^ 5 msec. With anodal test pulses there^ was no phase of enhanced (^) excitability.
Lonr subliminal cathodal or anodal pulse with cathodal test pulse.
Long cathodal pulse. The results obtained (Fig. 7, graph) differed from those reported above (Fig. 3) for the long subliminal mechanical pulse, in^ that
96 W. T. CATTON
STIMULATION OF FROG SKIN TACTILE RECEPTORS 97
there was a sharp rise instead (^) of a fall in threshold at 'off', although the time courses were otherwise similar. Summation, with spike firing, occurred at 'on' (Fig. 7, oscillogram), but not at 'off'. With increasing strength of a long cathodal pulse presented in isolation, it was found that a spike was ultimately fired at 'on'. Long anodal pulse. The result (Fig. 8, graph) was the mirror image of that for a^ long cathodal pulse, there being a threshold rise at 'on', a main- tained elevation of threshold during the pulse, and a sharp fall below normal at 'off'. Summation, with spike firing, occurred at 'off' (Fig. 8, oscillogram), but not at 'on'. With increasing strength of a long anodal pulse presented in isolation, a spike was ultimately fired only at 'off'.
a c
b d
20 msec 1-4_
7: 1 04 t
(^0 7) Sublim inal
i (^) FPi E (^) -yeve r 5 10 1 5 20 25 30 35 40 On Time^ (msec) Fig. 7. Graph shows excitability changes accompanying an 18 msec cathodal pulse, tested by a 05 msec cathodal test pulse. In the oscillograms deflexions X and Y show beginning and end of long pulse. S shows artifact of testing pulse. A spike is discharged at 'on' (b), but not during the pulse (c), or at 'off' (d).
Long cathodal or anodal subliminal pulse with mechanical test pulse. The
results (Fig. 9) were similar to those obtained with short cathodal test
stimuli as reported above, i.e. a short mechanical test pulse behaved in
the same way as a cathodal test stimulus.
Repetitive subliminal pulses. Short subliminal mechanical pulses were
presented repetitively at frequencies between 1/sec and 100/sec for periods
7 Physiol. 164
STIMULATION OF FROG SKIN TACTILE RECEPTORS 99
of several minutes. There was a small rise of threshold not exceeding about (^10) % of the resting value. The same result was obtained with subliminal cathodal stimuli. DISCUSSION Detailed studies of the effects of subliminal stimulation of tactile receptors are not numerous in the literature. Lindblom (1958) described a brief period of refractoriness which followed a subliminal stimulus applied to the toad tactile receptor, but gave no indication of a preceding phase of enhanced excitability. The following interpretation of the results of the present experiments will be based on the assumption that, as in the case of all receptors so far studied electrophysiologically, a depolarizing receptor potential accom- panies the natural stimulus and is the essential precursor of spike initia- tion by the receptor. Such a receptor potential has not yet been demon- strated in a cutaneous free nerve ending, such as the amphibian tactile receptor. It is, however, possible to see whether the excitability changes observed may reasonably be correlated with the known characteristics of the receptor potential of other mechanoreceptors. Of these the most thoroughly investigated is the Pacinian corpuscle (e.g. Alvarez-Buylla & Ramirez de (^) Arellano, 1953; Gray & (^) Sato, 1953). The characteristics of the receptor potential of this structure which will be considered are as follows. (^) First, the amplitude is (^) proportional to stimulus (^) strength, and receptor potentials are evoked by subliminal stimuli, which can sum over a short interval. Secondly, a period of depression follows a given receptor potential so that a succeeding potential is smaller than the first. Thirdly, a spike is discharged when the receptor potential reaches a level of critical depolarization (= threshold). Fourthly, with a^ prolonged mechanical deflexion there are separate receptor potentials at 'on' and 'off', of the same sign. The Pacinian corpuscle is a rapidly adapting receptor, and like the frog tactile receptor seldom discharges more than a single spike, either with a short stimulus, or at^ the 'on'^ and^ 'off' deflexions of a long stimulus. Taking the case of a short subliminal mechanical^ conditioning pulse, the observations on the frog receptor were a sharp transient rise of excita- bility at about the peak of the pulse, followed by a^ more prolonged period of subnormality. The most probable explanation of this sequence of events is that the rise of excitability is due to summation of the receptor potentials of the conditioning and test pulses, and^ the subsequent sub- normal period is due to the period of depression following the receptor potential of the^ conditioning pulse. This^ period of^ depression lasts^ 10- msec in the Pacinian corpuscle, comparable with the subnormal period of the tactile receptor.
7-
When both pulses are subliminal, what is measured is the summation interval, which is the period of overlap between the two receptor potentials within which the critical depolarization may be achieved, and it is clearly dependent on both the amplitude and the duration of the individual receptor potentials. It was difficult to measure summation interval in the present work, owing to the considerable overlapping of the stimulus pulses (each of 2 msec duration), and their rounded shape. Values between 0- and 2 msec were regarded as rough estimates only. For the Pacinian corpuscle Alvarez-Buylla & Ramirez de Arellano (1953) give a summation interval of about 2-2 msec, the receptor (^) potential duration being about
6 msec. If we take the summation interval (0.5-2 msec) for the frog tactile
receptor, in conjunction with the transient sharp rise of excitability (2-3 msec) (^) revealed by the testing pulse method, it may be concluded that the receptor potential of the frog tactile receptor, in (^) response to a (^2) msec deflexion, is brief, though no precise value can be suggested. The results with long (^) pulses will (^) now be (^) considered, since they appear to throw another light on the nature of the receptor process. With long- duration subliminal mechanical pulses two (^) distinct types of behaviour were seen, which differed, however, only in respect of the changes at the ' off' deflexion. Thus with both types there was a (^) sharp fall (^) of threshold at the (^) 'on' deflexion, similar in duration to that which occurred with short conditioning pulses; but instead of this being followed by a (^) period of sub- normality, there succeeded a low plateau of increased excitability which declined slowly during the pulse. At (^) the 'off' (^) deflexion there was a rise of excitability for the first and more common type, a fall for the second type; the former gave both 'on' and 'off' responses, the (^) latter 'on' responses only. The first type correlates in most respects with the behaviour of the Pacinian corpuscle, which discharges a spike at both 'on' (^) and 'off' of a long pulse (^) (Gray & Malcolm, 1950) and produces depolarizing 'on' and 'off' receptor potentials (Gray & Sato, 1953). It differs in (^) that there is a plateau of (^) supernormality which might be expected to have its counter- part in a depolarization phase persisting during the (^) pulse. Gray & Sato stated, however, that^ in^ the Pacinian^ corpuscle the shape of the receptor potential was the same for the 'on' and 'off' deflexions of a (^) long pulse as for a (^) single pulse. The second type of unit described ('on'-only responder), with a rise (^) of threshold at 'off', would be explained (^) by a (^) receptor potential of the type
described by Katz (1950) in the frog muscle spindle, in which there is a
hyperpolarizing phase at 'off'. The crayfish stretch receptor behaves in a similar way (Eyzaguirre & (^) Kuffler, 1956). Katz also drew a distinction between the (^) dynamic phases of the (^) receptor potential at the 'on' and 'off'
of the stimulus (stretch of the muscle) and the static phase, a low-level
100 W. T. CATTON
102 W. T. CATTON
anodal pulses produced respectively a single spike at 'on' or at 'off' when the strength was increased.
- Cathodal test stimuli simulated the effects of mechanical stimuli.
- The relation of these observations (^) to the possible receptor potential properties of the tactile receptor are discussed in connexion with the known receptor potentials of other mechanoreceptors.
I am indebted to Dr L. E. Molyneux of the Physics Department, (^) King's College, New- castle-upon-Tyne, for the transistor circuit used for (^) driving the (^) moving-coil stimulator.
REFERENCES ALVAREZ-BUYLLA, R. & RAMIREZ DE ARELLANO, J. (1953). Local responses in Pacinian corpuscles. Amer. J. Physiol. 172, 237-244. CATTON, W. T. (1960). The post-excitatory recovery process in frog skin mechanoreceptors. J. Phy8iol. 152, 22P. CATTON, W. T. (1961a). Excitability changes following subliminal stimulation of frog skin mechanoreceptors. J. Phy8iol. 157, 22-23P. CATTON, W. T. (1961b). Threshold, recovery and fatigue of tactile receptors in frog skin. J. (^) Physiol. 158, 333-365. EYZAGUIRRE, C. & KUFFLER, S. W. (1956). Processes of excitation in the dendrites and in the soma of single isolated sensory nerve cells of the lobster and crayfish. J. gen. Physiol. 39, 87-119. GRAY, J. A. B. & (^) MALCOLM, J. L. (1950). The initiation of nerve impulses by mesenteric Pacinian corpuscles. (^) Proc. Roy. Soc. B, 137, 96-114. GRAY, J. A. (^) B. & (^) SATO, M. (1953). Properties of the receptor potential in Pacinian corpuscles. J. (^) Physiol. 122, 610-636. KATZ, B.^ (1950). Depolarization of^ sensory terminals and the initiation of impulses in the muscle spindle. J. (^) Physiol. 111, 261-282. LINDBLOM, U. F.^ (1958). Excitability and^ functional^ organization within a peripheral tactile unit. Acta physiol. scand. (^) 44, Suppl. 153.
