• No results found

Graded Potentials and Spiking in Single Units of the Oval Organ, a Mechanoreceptor in the Lobster Ventilatory System: II Individuality of the Three Afferent Fibres

N/A
N/A
Protected

Academic year: 2020

Share "Graded Potentials and Spiking in Single Units of the Oval Organ, a Mechanoreceptor in the Lobster Ventilatory System: II Individuality of the Three Afferent Fibres"

Copied!
16
0
0

Loading.... (view fulltext now)

Full text

(1)

VENTILATORY SYSTEM

II. INDIVIDUALITY OF THE THREE AFFERENT FIBRES

B Y B . M. H. BUSH

Department of Physiology, University of Bristol, Park Row, Bristol BS15LS

AND V. M. PASZTOR

Department of Biology, McGill University, 1205 Avenue Dr. Penfield, Montreal, Canada H3A 1B1

{Received 14 March 1983-Accepted 24 June 1983)

SUMMARY

1. The peripheral dendritic arborizations of sensory units X, Y and Z of the oval organ have similar branching patterns. All three permeate the whole array of connective tissue strands without apparent regionalization or specialization.

2. The analogue components of sensory responses elicited in fibres X, Y and Z when the connective tissue array is stretched show considerable diver-sity: fibre Z has a higher threshold than X and Y; the dynamic peak values of X and Y saturate at pulls mid-range for Z; X, Y and Z form a spectrum of increasing adaptation.

3. Application of TTX abolishes impulse generation in fibre X earlier than in fibre Y, indicating diversity in spike initiating mechanisms from one fibre type to another.

4. Fibre X only spikes between certain limits of membrane depolariza-tion. Usually the response includes one to five spikes which occur during the dynamic phase of a trapezoidal stretch stimulus.

5. Fibre Y fires throughout the stimulus duration for pulls of moderate amplitude and velocity. Spiking inactivation and a low maximum firing frequency (approximately 80s"') limit the range of length sensitivity in fibre Y.

6. Fibre Z attains higher firing frequencies than either X or Y (approximately 110 s"1). The initial burst frequency (velocity dependent) may equal the firing rate of the dynamic peak.

INTRODUCTION

In the preceding paper (Pasztor & Bush, 1983) we have shown that each of three sensory afferents of the oval organ is capable of transmitting both graded potentials

(2)

452 B. M. H. BUSH AND V. M. PASZTOR

and spikes. Thus each fibre has available two methods of information transfer, effec-tively doubling the number of information channels. Since the oval organ is the onl)| proprioceptor which has so far been identified in the second maxilla as being capable of providing sensory feedback about respiratory beating (Pasztor, 1969), any increase in signalling capacity is likely to be of significance to the animal.

In this paper we investigate the differences which exist between the sensory res-ponses recorded from oval organ afferents X, Y and Z. We will show that the com-pound response of each fibre has distinctive characteristics which are consistent from one preparation to another. This analysis forms the basis for a current study on the functional role of the oval organ in the control of gill ventilation.

MATERIALS AND METHODS

An isolated preparation of the oval organ was dissected from the second maxilla of

Homarus as described in the preceding paper (Pasztor & Bush, 1983). The oval base

of cuticle was anchored to the Sylgard lining of the bath, while the apex of the organ was attached to the puller assembly by which stretch stimuli were presented. Standard trapezoidal (ramp-and-hold) stimuli were presented to the organ at 1-min intervals. Pull amplitude, ramp duration and total stimulus duration were controlled by a function generator. During series of graded amplitude stimuli, ramp duration was held constant and, as a consequence, ramp velocity increased concurrently with amplitude.

Intracellular recordings were taken from pairs of 3 M-KCl-filled micropipettes (15-25 Mfi) inserted into adjacent afferents as close together as possible. Responses were recorded on-line or stored on tape for subsequent analysis, using an Apple-Isaac microprocessor system.

RESULTS

Distribution of sensory terminals

In a previous paper (Pasztor, 1979) the sensory dendrites of the oval organ arboriza-tion were shown to end in naked bulbous terminals, anchored between epidermal cells at the base of an array of connective tissue strands. That ultrastructural study sugges-ted that the terminals of all three fibres, X, Y and Z, formed a single population indistinguishable from one another, and that each fibre gave rise to branches distributed throughout the whole oval organ.

Cobalt backfills of the sensory arborization, through the cut distal stump of the scaphognathite nerve, have confirmed the pervasive nature of dendritic branching. The photograph of one such backfill, Fig. 1, shows closely similar (though not identi-cal) branching of primary dendrites in two stained sensory units. Both penetrate all parts of the oval, and give rise to an even distribution of terminal branches. There is no indication of regionalization or specialization of the arborization of any one fibre.

Differences between X, Y and Z responses attributable to the receptor potential

(3)

Fig. 1. Sensory arborization of the oval organ. Dorsal view of-cobalt backfill through arthrodia] membrane at insertion of connective tissue strands. Two sensory units (sn I and sn2) distribute throughout the oval with very similar branching pattern. Motor axons (mn I and mn2) in the same nerve trunk continue straight through the oval organ. Scale b a r = 100/mi.

[image:3.451.55.383.75.384.2]
(4)
(5)

0-175 mm 0-2 0-25 0-3

0-35 0-45 0-55

I s 25 mV

Fig. 2. Comparison of X and Y fibre responses recorded concurrently during a series of trapezoidal pulls of increasing amplitude (as indicated). Ramp duration, 140 ms; total pull, 1-0 s. Recording sites 5 5 mm from confluence of oval organ dendrites.

0-3 mm 0-5

[image:5.451.49.404.49.280.2]

I s 25 mV

Fig. 3. Comparison of X and Z fibre responses recorded concurrently during a series of pulls of increasing amplitude. Stimulus parameters as in Fig. 2. Recording sites 5'5 mm from oval organ.

(6)

454 B. M. H. BUSH AND V. M. PASZTOR

0 1 0-4 0-5 0-6

[image:6.451.49.405.61.349.2]

Pull amplitude (mm)

Fig. 4. Relationship between graded potential amplitude and pull amplitude for representative X, Y and Z fibre*. X and Y recorded concurrently. Z values taken from another, matched, preparation. Closed symbols: dynamic peak values. Open symbols: static, adapted levels. Recording sites 5-5 mm from oval organ. Stimulus parameters as in Fig. 2.

Y responses were recorded concurrently. The Z responses were from an X-Z pair of a different preparation, chosen because the X responses (not shown) matched in threshold and amplitude the X data used in the graph. This ensures that the two preparations were undergoing comparable stimulation.

(7)

JllUl

1-5

T T X

2-5 3-5 25 mV

Fig. 5. Differential time course of spike abolition by tetrodotoxin (TTX) in X and Y fibres. Samples from a series of responses recorded at 1-min intervals. Times of exposure to 8 X 10"7M-TTX are indicated. Concurrent recordings; upper: fibre X; lower: fibre Y.

O'l mm 0-2

25 mV

[image:7.451.55.399.48.378.2]

Is

Fig. 6. Comparison of X and Y fibre responses to series of pulls of increasing amplitude after spike abolition with tetrodotoxin. Note the similar dynamic peaks of the two, but more pronounced adapta-tion in fibre Y.

(8)

456 B. M. H . B U S H AND V. M. PASZTOR

the brief burst of spikes in X may be a property of the spike generating neural membrane (see below).

Differences between X, Y and Z responses attributable to spiking properties

All three fibres have a peripheral spike initiating zone which is capable of respond-ing to the depolarization of the receptor potential with active regenerative, overshoot-ing spikes. Yet the spikovershoot-ing performances of the three are surprisovershoot-ingly diverse. This can be seen by comparing responses recorded concurrently from pairs of fibres as in Figs 2, 3, H a n d 12.

As described in the preceding paper, the spikes are progressively blocked by 1 0 ~7M - T T X . Of interest in the present context, the rate of development of TTX block is faster in fibre X than in fibre Y (Fig. 5). For example, the (dynamic) impulse response of fibre X in Fig. 5 decreased from three to two spikes within 1 min of TTX application, and was abolished altogether within 2 min, whereas the total number of spikes in the Y fibre response did not change for several minutes, although they began to decline in amplitude fairly soon. This suggests a difference in accessibility to the toxin of their spike-initiating zones, and/or a greater susceptibility of the fast inward current channels of the X fibre membrane to TTX block.

Fibre X

Although it has relatively large receptor potentials, fibre X showed a paucity of spikes. From extracellular evidence alone, one might be tempted to suppose that the X fibre represented a fast-adapting phasic receptor, similar to the Pacinian corpuscle, since it only gave 1—5 impulses per stimulus. Intracellular recordings showed other-wise, since the receptor potential adapted very little, and remained at an almost constant level of depolarization for the duration of the pull stimulus (Figs 2 and 3, also Figs 7 and 9 in the preceding paper). Thus, the brief burst may reflect accommodation

25 mV

25 mV

(9)

at different velocities (Fig. 7B). In each case, spiking started at the same threshold level of depolarization, and then ceased although the membrane continued depolariz-ing. The cessation came at varying times after the initial impulse, but occurred at a particular level of depolarization (in this example 26mV from the resting potential). This suggests that impulse initiation in fibre X is only expressed between lower and upper limits of depolarization. A similar phenomenon has been noted in neurones of the lobster commissural ganglion (Robertson & Moulins, 1981).

Support for an upper spiking limit comes from the two experiments shown in Fig. 8. In Fig. 8A the prevailing membrane potential was manipulated by a combination of conditioning stretches and current injection. At rest length (/0), the first test pull

elicited one spike, but injection of depolarizing current shifted the membrane poten-tial outside the upper spiking limit and the response to the second test pull lacked a spike. Trials at lo + 0-35 mm show the converse situation, where conditioning stretch

depolarized the membrane so that the test pull response was spikeless, then hyper-polarizing current restored the membrane potential to the spiking range, and the test pull again elicited a spike. At 4+0*4 mm, the depolarizing effect of stretch was greater

25 mV

[image:9.451.49.404.355.583.2]

25 mV

(10)

458

B. M. H. BUSH AND V. M. PASZTOR

and the hyperpolarizing effect of current injection was insufficient to counteract it, resulting in a spike-less response to the test pull. In Fig. 8B the resting length of the organ was increased in 01-mm increments (conditioning stretch) and the response was observed to a 03-mm pull (test pull) at each increment. As the membrane depolarized, in response to the conditioning stretches, spiking at the test pull became progressively impaired, and in the last example spikes were absent altogether.

Fibre Y

Under certain stimulus conditions fibre Y also shows limitations in its spiking capacity. In an amplitude series like the example shown in Fig. 9 a blockage of spiking was frequently observed at some critical increment in pull. The stimulus amplitude at which block occurred varied from one preparation to another, and in some fibres it was seen at pulls of 0-3 mm, while in others spiking was only blocked for pulls greater than 0*9 mm, depending partly upon the initial degree of stretch. Spiking always ceased at the dynamic peak of the response when firing frequency had attained 50-60 Hz (see Fig. 11).

Preliminary experiments on this block indicate that it has a complex origin, but mechanical damage, as suggested by the original term 'overstretch', is not an under-lying causal factor. As Eyzaguirre & Kuffier (1955) found with the lobster abdominal MRO, impulse block is not permanent and, under appropriate conditions, spiking will start again later during the static phase, at a firing frequency appropriate to the expected state of adaptation. In several respects the pause in spiking of the Y fibre resembles the 'transition interval' noted by Shepherd & Ottoson (1965) in the frog muscle spindle. The latter, however, rarely exceeded 25 ms, whereas in the oval organ Y fibre pauses of 500 ms and more were not uncommon.

The occurrence of spiking inactivation was governed by the velocity as well as the

0 0 4 mm 0 0 8 012 016

[image:10.451.45.405.420.617.2]

0-5 50 mV

(11)

100 mms"1 25

J

12-5 8-3

50 mV

Fig. 10. Spiking inactivation in a Y fibre elicited by varying velocity in a series of constant amplitude pulls of 1 -0 mm. Recording site 5 5 mm from oval organ.

75

I 50

I

0 125

100

§75

50

25

Fibre Y

Fibre Z

I

0-2mm 0-3 0-4 0-5 0-6 0-71 0-8

T"

0-9 300 ms

(12)

460

B. M. H. BUSH AND V. M. PASZTOR

velocity pulls did not induce block, at least up to the maximum amplitude of stretch applied (1 mm). Unlike the situation with fibre X, it does not seem that level m membrane depolarization is the determining factor. As an alternative hypothesis, one might suppose that rate of depolarization could set limits on spiking ability. The spike initiating mechanism might be susceptible to inactivation by too rapid a change in membrane potential. The evidence of the high amplitude, high velocity pulls which do not produce block, however, suggests that both rate of depolarization and duration of dynamic stretch must reach critical values to bring about spiking inactivation.

Fibre Z

No such inactivation of the spiking mechanism was seen in Z fibre responses no matter what amplitudes or velocities of stretch were tested. As long as the depolariza-tion was supra-threshold, spiking continued. The firing pattern was characteristic and readily distinguishable from that of fibre Y. During the dynamic phase, fibre Z could attain a much higher firing rate than Y, even though the underlying graded potential had a similar magnitude. As shown in the instantaneous frequency plots of Fig. 11, firing rates of over 100 Hz were not uncommon in Z whereas the maximum in Y was

JLJL

0-46 mm 0-44 0-42 0-40

[image:12.451.48.398.307.611.2]

0-28 0-22 0-16 0 0 6 25 mV

(13)

illustrated in Figs 11 and 12. Not only did Y dynamic frequencies show the saturation effect, but spiking inactivation limited the usefulness of fibre Y in coding amplitude information at the upper end of the range. Fibre Z was not so limited and continued to display increments in firing frequency for increases in stimulus. On the other hand, at small amplitude pulls, fibre Y had the greater sensitivity, as shown in the lower four pairs of responses in Fig. 12. Thus the two fibres have different, though overlapping, usable ranges, and can be said to exhibit a certain degree of range fractionation.

DISCUSSION

The lobster second maxilla performs one basic function, gill ventilation, and the beating of its scaphognathite is a relatively stereotyped behaviour. Water is expelled from the exhalant channel of the branchial chamber by the combined levation-depression and antero-posterior rocking of the appendage in a cyclical rhythm. Beat frequency, ranging from 50—120 min"1, is the dominant variable. The legs, by com-parison, are multifunctional and have a wide repertoire of activities including slow postural stances, food gathering, grooming and rapid locomotory rhythms. As a corollary, the legs have a rich proprioceptive system with numerous information channels feeding back to the motor programme generators. (For reviews see Mill, 1976; Bush & Laverack, 1982; Evoy & Ayers, 1982.) The second maxilla has a much simpler array of mechanoreceptors, in which the oval organ predominates. It sends only three afferents to the suboesophageal ganglion, but each can carry two types of signal, graded and impulsive, and each has unique properties.

Distinctive functional properties of fibres X, Y and Z

X fibre responses have the largest graded potentials and the briefest burst of spikes. Since the graded potential is virtually non-adapting, X can be assigned the role of position detector. The analogue signal is continuously variable and well suited to transmitting information about extent of stretch, and hence position of the scaphog-nathite within the beat cycle. As discussed earlier (Pasztor & Bush, 1983), both phases of the cycle, levation and depression, could be encoded in the oscillation of membrane potential of the X unit. The interspike intervals within each burst are too few and too poorly modulated to encode stimulus parameters, but the interburst intervals provide a faithful measure of scaphognathite cycle period. Thus a second function for X is to act as a beat frequency marker.

(14)

462 B. M . H . B U S H AND V. M. PASZTOR

pumping chamber, prolonged stretch could be adequately encoded by firing frequency or membrane depolarization.

During normal gill ventilation the frequency of scaphognathite beating is the parameter showing the greatest variability. Thus, as the beat cycle shortens or lengthens, stretch of the oval organ occurs at higher or lower velocity. As shown in the previous paper, the initial part of the receptor potential waveform is a rapid depolarization where at least the initial phase is velocity dependent. It is this com-ponent which could be expected to show the greatest attenuation during the passive spread of the analogue signal into the ganglion, due to the additive effect of membrane capacity. Indeed such a distortion was seen in the transmission of the visual signal in non-spiking photoreceptors of barnacle (Hudspeth, Poo & Stuart, 1977). In Y and Z fibres the initial depolarization is encoded into an initial burst of impulses so that information about velocity is not lost. This makes both fibres candidates for move-ment detectors. In Z, sensitivity to movemove-ment is accentuated by the greater initial dynamic response, its high frequency firing and the rapidity of adaptive fall. As seen in Fig. 11 the early movement response of Z, the initial burst, sometimes elicits higher firing frequencies than the dynamic peak which, coming at the end of the ramp, signals the maximum extent of stretch.

Is there a morphological basis for physiological differences?

Where individual afferents from a given sense organ have different physiological properties, it might be predicted that they would be differentiated anatomically. Fine structural differences have been demonstrated between S and T fibres of the crab T-C MRO (Whitear, 1965), fast and slow adapting crayfish abdominal MRO fibres (Euteneuer & Winter, 1979; Komuro, 1981), snake muscle spindles (Pallot & Ridge, 1972, 1973; Fukami, 1978, 1982) and primary and secondary endings in mammalian muscle spindles (Banks, Barker & Stacey, 1981). To what extent physiological dif-ferences between fast adapting and slowly adapting units can be ascribed to visco-elastic or other mechanical properties of the receptor organ is still open to discussion, and may differ from one receptor to another (see e.g. Bush & Laverack, 1982). In the crayfish abdomen, for example, the different degrees of adaptation of the two MROs have been attributed largely to differences in spike adaptation (Nakajima & Onodera, 1969). On the other hand, a modelling study on the T-C MRO suggests that the more pronounced differences in the anatomical relationship of the S and T fibres with the receptor muscle could underly their distinctive dynamic responsiveness (Berger & Bush, 1979).

(15)

instance, might be that the X fibre membrane could contain fast outward current channels (probably for K+ ions) similar to those demonstrated in the crab T-C MRO afferents (Mirolli, 1981). The possible effect of such an outward current, by shunting the fast inward Na+ current, could be to limit spike generation in X, though not to suppress spiking altogether as in the T-C MRO.

This work was supported by research grants to BMHB from the Royal Society and the Science Research Council (U.K.) and to VMP from the Natural Sciences and Engineering Research Council of Canada. We thank Alison Walford for technical assistance.

R E F E R E N C E S

BANKS, R. W., BARKER, D. & STACEY, M. J. (1981). Structural aspects of fusimotor effects on spindle sensitivity. In Muscle Receptors and Movement, (eds A. Taylor&A. Prochazka), pp. 5—16. London: Mac-Millan.

BERCER, C. S. & BUSH, B. M. H. (1979). A non-linear mechanical model of a non-spiting muscle receptor. J.

exp. Biol. 83, 339-343.

BUSH, B. M. H. & LAVERACK, M. S. (1982). Mechanoreception. In Neurobiology: Structure and Function, (eds H. L. Atwood & D. C. Sandeman), pp. 399-468, Vol. 3 oiTheBiobgy of Crustacea, (ed. D. E. Bliss). New York & London: Academic Press.

EUTENEUER, U. & WINTER, C. (1979). The abdominal muscle receptor organ in Astacus leptodactylus (Crus-tacea). A fine structural analysis. Cell Tiss. Res. TXH, 41-61.

EVOY, W. H. & AYERS, J. (1982). Locomotion and control of limb movements. In Neural Integration and

Behavior, (eds D. C. Sandeman & H. L. Atwood), pp. 61-105, Vol. 4 of The Biology of Crustacea, (ed. D.

E. Bliss). New York and London: Academic Press.

EYZACUIRRE, C. & KUFFLER, S. W. (1955). 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.

FUKAMI, Y. (1978). Receptor potential and spike initiation in two varieties of snake muscle spindles. J.

Neurophysiol. 41, 1546-1556.

FUKAMI, Y. (1982). Further morphological and electrophysiological studies on snake muscle spindles. J.

Neuwphysiol. 47, 810-826.

HUDSPETH, A. J., Poo, M. M. & STUART, A. E. (1977). Passive signal propagation and membrane properties in median photoreceptors of the giant barnacle. J. Physiol., Land. ZTZ, 25-43.

KOMURO, T. (1981). Fine structural study of the abdominal muscle receptor organs of the crayfish

(Procam-barus clarkii). Sensory endings and synaptic structures. J'. Neurocytol. 10, 27—43.

MILL, P. ] . (1976). Structure and Function of Proprioceptors in the Invertebrates. London: Chapman Hall. MIROLLI, M. (1981). Fast inward and outward current channels in a non-spiking neurone. Nature, Land. 292,

251-253.

NAKAJIMA, S. & ONODERA, K. (1969). Membrane properties of the stretch receptor neurones of crayfish with particular reference to mechanisms of sensory adaptation.^. Physiol., Land. 200, 161—185.

PALLOT, D. J. tc RIDGE, R. M. A. P. (1972). The fine structure of the long-capsule muscle spindles in the snake

Natrix s p . J . Mat. 113, 61-74.

PALLOT, D. J. & RIDGE, R. M. A. P. (1973). The fine structure of the short capsule muscle spindles in snakes of Natrix sp.J.Anat. 114, 13-24.

PASZTOR, V. M. (1969). The neurophysiology of respiration in decapod Crustacea. II. The sensory system. Can.

J.Zool. 47, 435-441.

(16)

464 B. M. H. BUSH AND V. M. PASZTOR

PASZTOR, V. M. & BUSH, B. M. H. (1983). Graded potentials and spiking in single units of the oval organ, a mechanoreceptor in the lobster ventilatory system. I. The characteristics of dual afferent signalling. J. txtA

Biol. 107, 431-449.

ROBERTSON, R. M. & MOULINS, M. (1981). Firing between two spike thresholds: implications for oscillating lobster interneurons. Science, N.Y. 214, 941-943.

SHEPHERD, G. M. & OTTOSON, D. (1965). Response of the isolated muscle spindle to different rates of stretching. Cold Spring Harb. Symp. quant. Biol. 30, 95-103.

WHITEAS, M. (1965). The fine structure of crustacean proprioceptors. II. The thoracico-coxal organs in

Figure

Fig. 1mn
Fig. 3. Comparison of X and Z fibre responses recorded concurrently during a series of pulls ofincreasing amplitude
Fig. 4. Relationship between graded potential amplitude and pull amplitude for representative X, Yand Z fibre*
Fig. 6. Comparison of X and Y fibre responses to series of pulls of increasing amplitude after spikeabolition with tetrodotoxin
+4

References

Related documents

Field experiments were conducted at Ebonyi State University Research Farm during 2009 and 2010 farming seasons to evaluate the effect of intercropping maize with

from a technical mixture with higher degree of chlorination to a technical mixture with lower degree of chlorination over this time period, (2) early, industrial, or combustion

The projected gains over the years 2000 to 2040 in life and active life expectancies, and expected years of dependency at age 65for males and females, for alternatives I, II, and

19% serve a county. Fourteen per cent of the centers provide service for adjoining states in addition to the states in which they are located; usually these adjoining states have

diagnosis of heart disease in children. : The role of the pulmonary vascular. bed in congenital heart disease.. natal structural changes in intrapulmon- ary arteries and arterioles.

National Conference on Technical Vocational Education, Training and Skills Development: A Roadmap for Empowerment (Dec. 2008): Ministry of Human Resource Development, Department

It was decided that with the presence of such significant red flag signs that she should undergo advanced imaging, in this case an MRI, that revealed an underlying malignancy, which

Also, both diabetic groups there were a positive immunoreactivity of the photoreceptor inner segment, and this was also seen among control ani- mals treated with a