Brain Research, 97 (1975) 331-336 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

331

Short C o m m u n i c a t i o n s

Component mechanisms of sensitivity and adaptation in an insect mechanoreceptor

DIANA W. MANN* AND K. M. CHAPMAN Neurosciences Section, Division of Biological and Medical Sciences, Brown University, Providence, R.L 02912 (U.S.A.)

(Accepted July 1st, 1975)

In an adapting sensory receptor, the time-dependent processes that contribute to the decay of discharge frequency with time may be distributed in a variety of ways among the intermediate mechanisms of coupling, transduction and encoding that comprise sensory reception. In mechanoreceptors, time-dependent processes can include visco-elastic stress relaxation of tissues coupling the stimulus to a transducer site of the primary receptor cell, transient behavior of strain-sensitive conductances in the transducer membrane producing the generator current, and accommodation of an axonal encoder determining the impulse frequency. Moreover, each process can make a distinct non-linear contribution to the receptor's behavior. These properties are important in understanding not only sensory phenomena, but fundamental mechanisms of coupling, transduction and encoding as well. We report here an experimental study of components of adaptation and sensitivity in the campaniform sensilla of the cockroach leg. These are mechanoreceptors in the exoskeleton having a proprioceptive function in coordinating posture and locomotion. The experimental approach was based on a technique for measuring the receptor potential of an intact individual sensillum in the amputated but otherwise undissected leg, and on the use of sinusoidally modulated mechanical stimuli to describe the sensitivity and time dependence of accessible intermediate mechanisms by linear transfer functions. Preliminary reports have appeared 6,7. All work was done with large campaniform sensilla in group 6 on the tibia of the metathoracic (hind) leg of the cockroach Blaberus discoidalis 9. Amputated legs were mounted on insect pins which served as afferent impulse recording electrodes, and punctate stimuli were applied to individual sensilla by a fine tungsten probe mounted on a force gauge and electrically controlled piezoelectric bender 1°. Through* Present address: Institute of Marine Biomedical Research, University of North Carolina at Wilmington, N.C., U.S.A.

332 out this study, the probe was applied toward an edge of the cap to avoid its extremely sensitive center 4, and forces of the order of 100 # N were used. The receptor potential was recorded between a 20-30 ¢ m pipet electrode contacting the intact outer cuticular surface of the sensillum through a minute drop of modified cockroach saline, and an indifferent electrode inserted into the hemolymph space through the cut end of the tarsus. Our method was similar to that of Thurm 12 and others1, s, except that we specifically avoided penetrating the cuticle of the sensillum. The drop of modified saline, containing 10 ~ glycerol and 0.01-0.1 ~o alkyl sulfonate detergent (Alkonox), was controlled by a micrometer syringe to cover the sensillum under study, occasionally one or two neighboring ones, and as little else of the surrounding region as possible. Waveforms of applied force, receptor potential and afferent impulses were photographed from an oscilloscope, averaged simultaneously by a PDP-12 digital computer, and subsequently fitted by a least-squares procedure with sinusoids of the form: F (t) = F0 + F1 cos (2~ft .-5 ~r~F) as described elsewhere 3& The stimulus parameters mean force, Fo, peak force, F1, and forcing frequency, f, were selected with a signal generator and oscilloscope during experimentation. Their precise values and that for phase angle ~F were computed by the curve-fitting procedure. Corresponding parameters Vr0, Vrl and ~C,vr were similarly computed for receptor potential Vr, and Y0, yl and ~y for impulse frequency y expressed as the impulse time density histogram. With sinusoidal punctate stimulation, the cap surface developed a smoothly modulated, negative-going receptor potential (Fig. 1). Its magnitude may be taken as a measure of the strength of the generator current, and it thus gives access to the transduction process. The receptor potential reached its negative maximum near the time of maximum force, indicating that the generator current was nearly in phase with force. In fact it usually peaked slightly earlier than force, directly indicating net timedependence in the combined coupling and transduction mechanisms. Action poten-

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1oo msec Fig. 1. Oscilloscope records of the negative-going transcuticular receptor potential (upper trace) and afferent discharge recorded from femoral pin electrodes (lower trace), in response to two successive cycles of sinusoidally modulated punctate force at 5 Hz. Surface negativity indicates the extracellular generator current is directed toward the dendrite of the bipolar neuron; its magnitude reflects the strength of the generator current. Positive-going spikes superimposed on the receptor potential correlate one-for-one with those seen in the afferent nerve. This record was obtained with an extremely small drop of saline contacting the cap cuticle.

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Fig. 2. Computer-averaged waveforms (points) of force, receptor potential and impulse frequency for 10i3 successive cycles of punctate stimulation at 3 Hz, and best-fitting sinusoids (continuous curves). In this example the probe made intermittent contact with the cap and was placed on a relatively insensitive point at its edge. The smaller receptor potential seen here was recorded with a larger saline drop than in Fig. 1.

tials seen superimposed on the transcuticular potential corresponded in perfect oneto-one fashion with the afferent discharge recorded from the femoral nerve. The recorded magnitude of the receptor potential, the transcuticular spike amplitudes and the transcuticular resistance each correlated inversely with drop size. Receptor potentials of the order of 10 mV amplitude with clearly defined spikes were usually recorded with extremely small drops having contact resistances of at least 100 Mr2. Transfer function determinations required averaging many successive cycles of stimulation at several forcing frequencies. These were more easily made with somewhat larger saline drops, and consequently produced smaller receptor potentials. Fig. 2 shows a set of averaged waveforms and best-fitting sinusoids for one such case. In this record, peak force F1 exceeded mean force F0, so that the probe made intermittent contact with the cuticle, lifting clear of it for about a third of the period of the sinusoidal stimulus. During contact, the averaged force, receptor potential and impulse frequency waveforms all showed smooth sinusoidal modulation which was clipped while the probe was out of contact. In such cases, sinusoids were fitted to exclude the clipped portions of the waveforms~. When mean force exceeded peak force, the force and receptor potential waveforms were complete sinusoids, indicating that the combined effect of coupling and transduction was linear for small stimuli. Impulse frequency modulation usually remained clipped, however, reflecting the strong tendency of these and other quiescently silent, phasic receptors to fire in discrete bursts during sinusoidal stimulation (Fig. 1).

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Fig. 3. Bode plots of sensitivity for: (A) the combined coupling and transduction mechanisms; (B) the encoding mechanism, and (C) the throughput of the receptor, at two levels of mean force Fo. Open circles and dashed lines: low mean force (F0 -- - - 5 ± 20/zN). Solid circles and lines: high mean force (F0 -- 170 ± 20/~N). Slopes of the straight lines indicate the extent of time-dependence for each mechanism. The change in sensitivity with mean force in (A) indicates time-independent, non-linear range compression in the coupling and transducer mechanisms. The sharp change in slope in (B) indicates a strong time-dependent non-linear increase in adaptation in the encoder mechanism, with little range compression.

TABLE I POWER

COEFFICIENTS

k

FOR

THE

BODE

Transfer ftmction Coupling-transduction; receptor potential/force Encoding; impulse frequency/ receptor potential Throughput; impulse frequency/force

PLOTS

OF

SENSITIVITY

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3

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High force

Fo= --5 ± 20/tN

Fo=

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0.14 S.E. 0.05

0.29 S.E. 0.15

0.69 S.E. 0.11

0.54 S.E. 0.14

0.83 S.E. 0.10

170 ~ 201~N

335 Linear transfer functions were determined in the conventional way in terms of peak amplitude ratios and phase differences as functions of forcing frequency of the stimulus sinusoids (Fig. 3 and Table I). Forcing frequencies ranged from 0.03 to 100 Hz in this study. The transfer function for combined coupling and transduction was expressed as the sensitivity of receptor potential to applied force, Vrl/F1 (Fig. 3A), and the phase difference (q)Vr--~0F)by which receptor potential modulation leads force modulation. Likewise, that for encoding was expressed as the sensitivity of impulse frequency to receptor potential, yl/Vrl (Fig. 3B), and phase difference (~y--~vr). The 'throughput' transfer function, relating impulse frequency with force, then follows as the product yl/F1 of the sensitivities of the two accessible intermediate mechanisms (Fig. 3C), and the sum (~y--q)f) of their phase differences. In order to identify sources of non-linear sensitivity and adaptation, transfer functions were usually determined with both low, clipping forces and high, non-clipping forces. The sensitivity of each of the intermediate processes varied roughly as the k-th power of the forcing frequency, since the corresponding log-log Bode plots (Fig. 3) could be adequately approximated by straight lines with slopes k < 1. This behavior occurs in many types of adapting sensory receptors and in many other distributed relaxation processes that occur throughout nature~-,5,al. It represents fractional (k-th) order differentiation of the stimulus with respect to time, and predicts a time course of adaptation as t -it following a step stimulus. In the present context the form of this model is not essential to our main conclusions, but it does provide a convenient way of summarizing time dependence: the greater the power coefficient k, the more strongly and more rapidly adapting is the process it describes. Our main findings are illustrated in the data of Fig. 3 and Table I. The couplingtransduction mechanisms showed moderate adaptation, with slopes of 0.25 and 0.14 respectively for the low and high force levels tested. The t -k model predicts that receptor potential should fall to about 20 ~ between 1 msec and 1 sec following a step input ofpunctate force at the low force level, and should adapt evenless, to about 40 ~ , at the higher force. In contrast, the sensitivity of coupling and transduction showed a strong, time-independent non-linear range compression, in that it decreased by a factor of 10 at the higher force level. It is likely that this non-linearity resides in part in the non-linear compliance of the cap cuticle seen at low punctate forces 3. The behavior of the encoding mechanism was strikingly different. At low force the slope of 0.29 indicates that the encoder adapted to the same extent as coupling and transduction, and thus accounted for about half of the 0.54 slope for adaptation of impulse frequency with respect to force. For this the t -k model predicts adaptation to about 2.5 ~ between 1 msec and 1 sec. At the higher force, however, the encoder became much more strongly adapting. Its slope more than doubled to 0.69, increasing the throughput power coefficient to 0.83. The effect predicted by the t -k model is to increase the extent of throughput adaptation of impulse frequency by nearly 10fold, to about 0.3 ~. In contrast, the sensitivity of the encoder to mid-range forcing frequencies was not affected by the force level, so there was no appreciable range compression in encoding. Phase shifts were not investigated in detail in this study, though they also reflect

336 time dependences. The t - k m o d e l predicts t h a t the p h a s e difference for each process should be i n d e p e n d e n t o f forcing frequency, the response leading its stimulus by k × 90 ° . I n each case the average m e a s u r e d p h a s e shift was statistically indistinguishable f r o m the p r e d i c t e d value, a l t h o u g h the scatter in values was large, a c o m m o n finding with sinusoidal s t i m u l a t i o n o f m e c h a n o r e c e p t o r s . I n s u m m a r y , the t r a n s c u t i c u l a r m e a s u r e m e n t o f r e c e p t o r p o t e n t i a l has given access to coupling a n d t r a n s d u c t i o n as distinct f r o m encoding. L i n e a r sinusoidal analysis has identified a t i m e - i n d e p e n d e n t non-linearity in coupling a n d t r a n s d u c t i o n , a n d a t i m e - d e p e n d e n t non-linearity in encoding. W e conclude t h a t coupling a n d t r a n s d u c t i o n c o n t r o l the c a m p a n i f o r m sensillum's t h r o u g h p u t sensitivity, while encoding c o n t r o l s its t h r o u g h p u t a d a p t a t i o n . W e t h a n k Ms. L e n d y L l o y d for p r e p a r i n g illustrations. This w o r k was s u p p o r t ed by G r a n t No. NS-06478 f r o m the U.S. Public H e a l t h Service.

1 BERNARD, J., AND GUILLET, J. C., Changes in the receptor potential under polarizing currents in two insect receptors, J. Insect Physiol., 18 (1972) 2173-2188. 2 BROWN,M. C., AND STEIN, R. B., Quantitative studies on the slowly adapting stretch receptor of the crayfish, Kyberuetik, 3 (1966) 175-185. 3 CHAPMAN,K. M., ANDDUCKROW,R. B., Compliance and sensitivity of a mechanoreceptor of the insect exoskeleton, J. comp. Physiol., 100 (1975) 251-268. 4 CHAPMAN, K. M., DUCKROW,R. B., AND MORAN, D. T., Form and role of deformation in excitation of an insect mechanoreceptor, Nature (Lond.), 244 (1973) 453-454.

5 CHAPMAN,K. M., AND SMITH, R. S., A linear transfer function underlying impulse frequency modulation in a cockroach mechanoreceptor, Nature (Lond.), 197 (1963)699-701. 6 MANN, O. W., The Receptor Potential and 1repulse hdtiation in Cockroach Mechanoreceptors,

Ph.D. Thesis, Brown Univ., 1973, 92 pp. 7 MANN, D. W., AND CHAPMAN, K. M., Trans-cuticular recording of receptor potentials in cockroach mechanoreceptors, Fed. Proc., 30 (1971) 552 Abstract. 8 NICKLAUS, R., Die Erregung einzelner Fadenhaare von Periplaneta americana in Abhfingigkeit yon der Grt~sse und Richtung der Auslenkung, Z. vergl. Physiol., 50 (1965) 331-362. 9 PRINCLE,J. W. S.. Proprioception in insects. II. The action of the campaniform sensilla on the legs, J. exp. Biol., 15 (1938) 114-131. 10 SPINOLA,S. M., AND CHAPMAN,K. M., Proprioceptive indentation of the campaniform sensilla of cockroach legs, J. comp. Physiol., 96 (1975) 257-272. 11 THORSON,J., ANDB[EDERMAN-THORSON,M., Distributed relaxation processes in sensory adaptation, Science, 183 (1974) 161-172. 12 THURM, U., Das Rezeptorpotential einzelner Mechanorezeptorischer Zellen yon Bienen, Z. vergl. Physiol., 48 (1964) 131-156.